ANIMAL BIOTECHNOLOGY (BT2036)

SYLLABUS BT2036 UNIT I ANIMAL BIOTECHNOLOGY ANIMAL CELL CULTURE 12

Introduction to basic tissue culture techniques; chemically defined and serum free media; animal cell cultures, their maintenance and preservation; various types of cultures- suspension cultures, continuous flow cultures, immobilized cultures; somatic cell fusion; cell cultures as a source of valuable products; organ cultures. UNIT II ANIMAL DISEASES AND THEIR DIAGNOSIS 10

Bacterial and viral diseases in animals; monoclonal antibodies and their use in diagnosis; molecular diagnostic techniques like PCR, in-situ hybridization; northern and southern blotting; RFLP. UNIT III THERAPY OF ANIMAL DISEASES 12

Recombinant cytokines and their use in the treatment of animal infections; monoclonal antibodies in therapy; vaccines and their applications in animal infections; gene therapy for animal diseases. UNIT IV MICROMANIPULATION OF EMBRYOS 6

What is micromanipulation technology; equipments used in micromanipulation; enrichment of x and y bearing sperms from semen samples of animals; artificial insemination and germ cell manipulations; in vitro fertilization and embryo transfer; micromanipulation technology and breeding of farm animals. UNIT V TRANSGENIC ANIMALS 5

Concepts of transgenic animal technology; strategies for the production of transgenic animals and their importance in biotechnology; stem cell cultures in the production of transgenic animals. TEXT BOOKS 1. 2. Ranga M.M. Animal Biotechnology. Agrobios India Limited, 2002 Ramadass P, Meera Rani S. Text Book Of Animal Biotechnology. Akshara Printers, 1997.

REFERENCE 1. Masters J.R.W. Animal Cell Culture: Practical Approach. Oxford University Press, 2000

UNIT I PART A 1. Define Cell culture. Cell culture is defined as the technique in which tissue or out growth from an explant is dispersed, most enzymatically into a cell suspension, which may then be cultured as a monolayer or suspension culture. 2. What are the characteristic features of cell culture? Development of a cell line over several generations. Possibility of scale-up Loss of some differentiated characteristics in cells.

3. What are different types of cells in cell culture? (1) Stem cells (2) Pre cursor cells (3) Differentiated cells 4. Define suspension culture. In suspension culture, the cells are dispersed in liquid medium and grow freely and are not attached to any subtract. The medium is agitated so that they do not form sediments. 5. Give some application of animal cell culture. (1) Studies related to cell-cell interaction Example: cell adhesion and motility, metabolic cooperation (2) Evaluation of environmental interactions Example: Cytotoxicity mutagenesis. (3) Studies dealing with genetics Example: genetic analysis. 6. Define culture Media. Culture medium is one single most important factor for culturing cells and tissues. It provides optimum conditions of factors like pH, osmotic pressure etc and the chemical constituents which the cells or tissues are incapable of synthesizing. 7. State the three categories of natural media. (1) Coagula, such as plasma clots (2) Biological fluids, such as serum (3) Tissue extracts of which embryo extract is the most common. 8. Define Cell line. When a primary culture is grown in a medium, as they grow and increase in number, the available medium is used up and there arises a need to subculture it from a heterogeneous primary culture containing many types of cells derived from the original tissues. During sub culturing, a very homogeneous cell line emerges. The culture now called a cell line, can be propagated, characterized and stored. The term cell line implies the presence of several cell lineages either similar or distinct. A cell line may be finite or continuous, depending upon whether it has limited culture life-spans or it is immortal in culture. Finite cell lines grow up to 20-80 population doubling before extinction. 9. Give some important products produced from animal cell culture. (1) (2) (3) (4) (5) Enzymes Example: Pepsin, collagenease Hormones Example: Erythropoietin, luteinizing hormone. Vaccines Example: Vaccines for rabies, influenza Monoclonal antibodies Interferon

10. Define Organ Culture. In this method, the architectural characteristics of an organ are retained and the organ is cultured invitro but cannot be multiplied . Example: Embryo culture.

11. What is the role of serum in cell culture? Some of the major functions of serum are to provide the following (1) (2) (3) (4) (5) Basic nutrients, in solution and bound to proteins. Attachment and spreading factors Binding proteins carrying hormones, vitamins, minerals, lipid pH buffer. Hormones and growth factors stimulating cell growth and specialized functions. PART - B 1. Explain in detail about culture medias. CULTURE MEDIA: The nutrient media used for culture of animal cells and tissues must be able to support their survival as well as growth .i.e., must provide nutritional, hormonal and stromal factors. The various types of media used for tissue culture may be grouped into two broad categories: (1) natural media and (2) artificial media. The choice of medium depends mainly on the type of cells to be cultured (normal, immortalized or transformed), and the objective of culture (growth, survival, differentiation, production of desired proteins). Nontransformed or normal cells (finite life span) and primary cultures from healthy tissues require defined quantities of proteins, growth factors and hormones even in the best media developed so far. But immortalized cells (spontaneously or by trasfection with viral sequences) produce most of these factors, but may still need some of the growth factors control and malignant properties) synthesize their own growth factors; in fact, addition of growth factors may even be detrimental in such cases. But even these cultures may require factors like insulin, transferring, silenite, lipids, etc. Natural Media These media consist solely of naturally occurring biological fluids are of the following three types: (1) cagula or clots, (2) biological fluids and (3) tissue extracts. The natural biological fluids are generally used for organ culture. For cell cultures, artificial media with or without serum are used. Clots. The most commonly used clots are plasma clots, which have been in use for a long time. Plasma is now commercially available either in liquid or lyophilized state. It may be prepared in the laboratory, usually from the blood of male fowl, but blood clotting must be avoided during the preparation. Biological Fluids. Of the various biological fluids used as culture medium, serum is the most widely used. Serum may be obtained from adult human blood, placental cord blood, horse blood or calf blood (foetal calf serum, new born calf serum, and calf serum); of these foetal calf serum is the most commonly used. Serum is the liquid exuded from coagulating blood. Different preparations of serum differ in their properties; they have to be tested for sterility and toxicity before use. Tissue Extracts. Chick embryo extract is the most commonly used tissue extract, but bovine embryo extract is also used. Other tissue extracts that have been used are spleen, liver, bone marrow, etc. extracts. Tissue extracts can often be substituted by a mixture of amino acids and certain other organic compounds. Artificial Media Different ariticial media have been devised to serve one of the following purposes: (1) immediate survival (a balanced salt solution, Table with Table Composition of Earles balanced salt solution (EBSS) and Hanks balanced salt solution (HBSS), composition of Dulbeccos phosphate buffered saline, solution A (PBSA, Ca2+ and Mg2 free) is also given Constituent Inorganic salts CaCl2 (anhyd.) KCl KH2PO4 MgCl26H2O 20(0.18 mM) 40(0.536 mM) --140(1.26 mM) 400(5.36 mM) 60(0.44 mM) 100(0.49 mM) -200(2.68 mM) 200(1.47 mM) -Amount (mg/I) HBSS1,2

EBSS1,2

PBSA3

MgSO4.7H2O NaCl NaHCO3 Na2HPO4.7H2O NaH2PO4. H2O Others D-glucose Phenol red

200(0.82 mM) 6680(114.4 mM) 2220(2.22 mM) --140(0.53mM) 1000(5.55 mM) 10

100(0.41 mM) 8000 (137 mM) 350(0.35 mM) 90(0.34 mM) -1000(5.55 mM) 10

-8000 (137 mM) -2160(8.06 mM) ----

1. EBSS and HBSS are autoclaved below pH 6.5 at 15 psi (pounds per square inch) for 20 min. pH may be adjusted to 7.4 with sterile NaOH prior to use. 2. For use as a general handling, washing and dissection solution. NaHCO 3 and glucose are omitted from EBSS and HBSS. For dissection, add to BSS: (i) penicillin (250 U/ml). (ii) streptomycin (250 g/ml), (iii) Kanamycin (100 g/ml) and (iv) fungizone (2.5 g/l). 3. PBSA is used as a washing solution before disaggregation, and as a diluent for trypsin (trypsin is used at 2.5 g, crude preparation , or 0.1 g, purified by repeated crystallization per litre) pBSA is also used as the base solution for preparing EDTA solution (na2 EDTA.2H2O at 372 mg/I, 1 mM), which is autoclaved before use. Specified pH and osmotic pressure is adequate), (2) prolonged survival (a balanced salt solution supplemented with serum, or with suitable formulation of organic compounds), (3) indefinite growth, and (4) specialized functions. The various artificial media developed for cell cultures may be grouped into the following four classes: (i) serum containing media , (ii) serum-free media, (iii) chemically defined media, and (iv) protein-free media. Serum Containing Media. The various defined media, e.g., Eagles minimum essential medium. Etc., (see, serum-free media), when supplemented with 5-20% serum are good nutrient media for culture of most types of cells. The serum provides various plasma proteins, peptides, lipids, carbohydrates, minerals and some enzyme. Serum serves the following major functions. 1. It provides the basic nutrients for cells: the nutrients are present both in the solution as well as are bound to the proteins. 2. It provides several hormones, e.g., insulin, which is essential for growth of nearly all cells in culture, cortisone, testosterone, prostaglandin, etc. 3. It contains several growth factors, e.g., platelet derived growth factors (PDGF), transforming growth factor (TGF), epidermal growth factor, etc: there are present in concentrations of g/I. Both hormones and growth factors are involved in growth promotion and specialized cell function. A given hormone or growth factors may stimulate growth of one cell type, may have no effect on another and may even be inhibitory to some others. For example, PDGF induces proliferation in fibroblasts, but induces differentiation of some types of epithelia. Further, proliferation of a single cell type may be induced by more than one growth factor, example, fibroblasts respond to PDGF, epidermal growth factor, fibroblast growth factor and somatomidins. 4. A major role of serum is to supply proteins, e.g., fibrobnectin, which promote attachment of cells to the substrate. It also provides spreading factors that help the cells to spread out before they can begin to divide. Although cells do produce these factors, but trypsinized cells are usually unable to attach to the substrate. 5. It provides several binding proteins, e.g., albumin, transferring, etc, which carry other molecules into the cell. For example, albumin carries into cells lipids, vitamins, hormones, etc., Transferring usually carries Fe in a nonbasic form, but binding of transferring to its receptor in cell membrane is believed to be nitrogenic. 6. It increases the viscosity of medium and, thereby protects cells from mechanical damages, e.g., shear forces during agitation of suspension cultures. 7. Protease inhibitors present in the serum protect cells, especially trypsinised cells, from proteolysis. 8. The serum also provide minerals like Na+, K+, Fe2+, Zn2+, etc. 9. it also acts as a buffer. However, there are several disadvantages of using serum in the culture medium; there are summarized below.

1. Serum may inhibit growth of some cell types, e.g, epidermal keratinocytes. 2. Serum may contain some cytotoxic or potentially cytotoxic constituents. For example, foetal calf serum contains the enzyme polyamine oxidase, which converts polyamines like spermidine and spermine (secreted by fast growing cells) into cytotoxic polyaminoaldehydes. 3. There is a large variation in serum quality from one batch to another this requires costly and time consuming testing every time a new batch has to be used. 4. Some growth factors may be inadequate for specific cells types and may need supplementation. 5. It interfere with downstream processing when cell cultures are used for production of biochemicals. 6. The supply of serum is always lower than its demand. Serum-Free Media. In view of the disadvantages due to serum, extensive investigations have been made to develop serum-free formulations of culture media. These efforts were mainly based on the following three approaches: (1) analytical approach based on the analysis of serum constituents, (2) synthetic approach to supplement basal media by various combinations of growth factors, and (3) limiting factor approach consisting of lowering the serum level in the medium till growth stops and then supplementing the medium with vitamins, amino acids, hormones, etc. till growth resumes. These approaches have resulted in several elaborate media formulation in which serum in sought to be replaced by a mixture of amino acids, vitamins, several other organic compounds, etc., hormones growth factors and other proteins are supplemented when required. However, addition of 5-20% of serum even in these media is essential for optimum growth.

The various advantages of serum-free media may be summarized as under. 1. Improved reproducibility of results from different laboratories and over time since variation due to batch change of serum is avoided. 2. Easter downstream processing of products from cultured cells. 3. Toxic effects of serum are avoided. 4. Biassays are free from interference due to serum proteins. 5. There is no danger of degradation of sensitive proteins by serum proteases. 6. They permit selective culture of differentiated and producing cell types from the heterogenous cultures. However, serum-free media suffer from the following disadvantages. 1. Most serum-free media are specific to one cell type. Therefore different media may be required for different cell lines. 2. Reliable serum-free preparations, for most of the media formulations are not available commercially. This necessitates time consuming task of preparing the desired formulations in the laboratory. 3. A greater control of pH, temperature, etc, is necessary as compared to that with serum containing media. 4. Growth rate and the maximum cell density attained are lower than those with serum containing media. 5. Cells tend to become fragile during prolonged agitated cultures unless biopolymers or synthetic polymers are added. Several defined media have been evolved from the Eagles minimal essential medium (MEM) , e.g, Dulbeccos enriched modification (DME), Hams F12, CMRL1066 , RPMII1640, McCoys 5A and Iscoves modified Dulbeccos medium (IMDM); all are commercially available. Often a 1:1 mixture of DME and F12 is used as a serumfree formulation. If needed, purified proteins and/or hormones may be added to the medium. Chemically Defined Media. These media contain contamination free ultra-pure inorganic and organic constituents, and may contain pure protein additives, like insulin, epidermal growth factor, etc., that have been produced in bacteria or yeast by genetic engineering.

Protein-free Media. In contrast, protein-free media do not contain any protein; they only contain non protein constituents necessary for culture of the cells. The formations MEM, DME, RPM -1640, etc, are protein-free where required , protein supplementation is provided. 2. Explain in detail about various types of cultures. Suspension Cultures In scaling up, both chemical (O2,pH, medium constituents and removal of waste) and physical (the configuration of bioreactor and power supplied to the reactor) factors have to be optimized for good results. The medium must be suitably stirred to kept the cells in suspension and to make the culture homogenous; it becomes increasingly difficult with the scaling up. Various types of stirrers range from simple magnetic stirrers, flat blade turbine impellers, to marine impellers, to those using pneumatic energy, e.g., airlift fermenter, and those using hydraulic energy, e.g., medium perfusion. Improved mixing can be obtained by changing the design of stirrer paddle or by sing multiple impellers. The objective of stirring is to achieve good mixing without causing damage to the cells. Vibro-mixer achieves stirring by vertical reciprocating motion of 0.1-3 mm at a frequency of 50 cycle/see of a mixing disc fixed horizontally to the agitator shaft. These stirrers causes random mixing, les foaming and lower shear forces. It is important to supply sufficient O2 without damaging the cells. Mean O2 utilization rate by cells is about 6 mg O2/106 cells/ hour. But O2 is only sparingly soluble in culture medium; the oxygen transfer rate (OTR) from gas phase into medium is about 17 g/cm2/hr. therefore, surface aeration can support about 50 106 cells in 11 culture vessel. Efficient aeration is achieved by bubbling air through the medium (sparging), but this may damage animal cells due to high surface energy of the bubble and on the cell membrane. The damage can be reduced by using larger bubbles, lower gassing rates and by adding non-nutritional supplements like Pluronic F-68 (polyglycol) and sodium carboxmethyl cellulose (these protect cells from damage due to shear forces and bubbles, respectively), Silicone tubing (highly gas permeable) can be arranged inside the culture vessel (2-5 cm tubing of 30 m length for a 1000 1 culture) and air is passed though the tube; however it is inconvenient to use. Aeration may be achieved by medium perfusion, in which medium is continuously taken from culture vessel, passed through an oxygenation chamber and returned to the culture. The cells are removed from the medium taken for perfusion so that the medium can be suitably altered, e.g., for pH control. Perfusion is used with glass bead and, particularly with micro carrier systems. Where considered safe and desirable, O 2 supply in the culture vessel can be enhanced form the normal 21% to a higher value and the air pressure can be increased by 1 atmosphere. This increases the O2 solubility and diffusion rates in the medium but there is risk of O2 toxicity. The reactors used for large scale suspension cultures are of 3 main types: (1) stirred bioreactors, (2) continuous flow reactors, and (3) airlift fermenters. Stirred Bioreactors. These are glass (smaller vessels) or stainless steel (larger volumes) vessels of 1-1000 1 or even 8000 1 (Namalva cells grown for interferon; but in practice , their maximum size is 20 1 since larger vessels are difficult to handle, autoclave and to agitate the culture effectively). These are closed systems with fixed volumes and are usually agitated with motor-driven stirrers with considerable variation in design details, e.g., water jacket in place of heater type temperatue control, curved bottom for better mixing at low speeds, mirror internal finishes to reduce cell damage, etc. Many heteroploid cell lines can be grown in such vessels. The needs for research biochemicals from cells are met from 2-50 1 reactors, while large scale reactors are mainly used for growing hybridoma cells for the production of monoclonal antibodies although their yields from cultured cells is only 1-2% of those obtained by passaging the cells through peritioneal cavity of mice. Continuous-Flow Cultures. These culture systems are either of chemostat or turbidostat type. In both the types, cultures begin as a batch culture. In a chemostat type bioreactor, inoculated cells grow to the maximum density when some nutrient, e.g., a vitamin, becomes growth liming. Fresh medium is added after 24-28 hours of growth, at a constant rate (usually, to support a growth rate lower than the maximum growth rate of culture) and the culture is withdrawn at an equal rate. When the rate of growth equals the rate of cell withdrawl, the cultures are in a steady state, and both the cell density and medium composition remain constant. One of the constituents of the medium is used at a lower concentration to make it growth-limiting. However, chemostat is the least efficient or controllable at the cells maximum growth rate, hence the steady-state growth rates in such a system is much lower than the maximum. In contrast, cells in a turbidostat grow to achieve a predecided density (measured as turbidity using a photoelectric cell). At this point, a fixed volume of culture is withdrawn and the same volume of fresh normal (not having a growth-limiting factor) medium is added; this lowers the cell density or turbidity of the culture. Cells keeps growing, and once the culture reaches the preset density the fixed volume of culture is replaced by fresh medium. This system work really well the growth rate of the culture is close to the maximum for the cell line.

The continuous-flow cultures provide a continuous source of cells, and are suitable for product generation, e.g., for the production of viruses and interferons. It is often necessary to use a two-state system in which the first stage supports cell growth, while the second stage promote product generation. Airlift Fermenters. Cultures in such vessels are both aerated and agitated by air bubbles introduced at the bottom of vessels (fig). The vessel has an inner draft tube through which the air bubbles and aerated medium rise since aerated medium is lighter than non-aerated one, this results in mixing of the culture as well as aeration. The air bubbles life to the top of the medium and the air passes out through an outlet. The cells and the medium that lift out of the draft table move down outside the tube and are recirculated. O2 supply is quite efficient but scaling up presents certain problems. Fermenters of 2-90 1 are commercially available, but 2000 1 fermenters are being used for the production of monoclonal antibodies. Immobilized Cultures Cultures based an immobilized cells offer several advantages (1) higher cell densities (50-200 10-6 cells/ml), (20 stability and longevity of cultures, (3) applicability to both suspension and monolayer cultures, (4) protection of the cells from shear forces due to medium flow (in case of many systems), and (5) less dependence of cell at higher densities on external supply of growth factors, which saves culture cost. There are two basic approaches to cell immobilization : (1) immurement and (2) entrapment. Immurement Cultures. In such cultures, cells are confined within a medium permeable barrier. Hollow fibers (section) packed in a cartridge are one such system. The medium is circulated through the fiber, while cells in suspension are present in the cartridge outside the fiber. This I extremely effective for scales up to 11 and gives cell densities of 1-2 108 cells/ml: sophisticated units can yield upto 40g monoclonal antibodies/month. Membranes permitting medium and gas diffusion are also used to develop bioreactors of this type; both small scale and large scale versions of membrane bioreactors are available commercially. The cells may be encapsulated in a polymeric matrix by adsorption, covalent bonding, cross-linking or entrapment; the materials used as matrix are gelatin, polylysine, alginate and agarose. This approach (1) effectively protects cells from mechanical damage in large fermenters and (2) allows production of hormones, antibodies, immunochemicals and enzymes over much longer period than is possible in suspension cultures. (3) The medium diffuses freely into the matrix and into the cells, while cells products move out into the medium. For production of larger molecules like monoclonal antibodies, agarose in a suspension of paraffin oil is preferable to alginate since the latter does not allow diffusion of such products out of the alginate beads. Reactors of up to 3 1 are available commercially. Entrapment Cultures. In this approach, cells are held within an open matrix through which the medium flows freely. An example is the Opticell in which the cells are entrapped within the porous ceramic walls of the unit. Optical units of upto 210 m2 surface are available. Which can yield upto 50g monoclonal antibodies per day. The cells can also be enmeshed in cellulose fibres, e.g., DEAE, TLC, QAE, TEAE. These fibers are autoclaved and washed as prescribed, and added in a spinner/stirred bioreactor at a concentration of 3 g/I. Porous microcarriers are small (170 m- 6000 m) beads of gelatin, collagen, glass or cellulose, which have a network of interconnecting pores. These provide a tremendous enhancement in surface area/volume ratio, permit efficient diffusion of medium and product, are suitable for scaling up, and are equally useful for suspension and monolayer cultures. These can be arranged as fixed bed or fluidized bed reactors or use in stirred bioreactors. It is expected that future development will make the immobilized cell systems the most dominant production systems. 3. Write briefly about Somatic Cell fusion. SOMATIC CELL FUSION: Somatic cells of different types can be fused to obtain hybrid cells. Hybrid cells are useful in a variety of ways, e.g., (i) to study the control of cell division and gene expression, (ii) to investigate malignant transformations. (iii) to obtain viral replication (iv) for gene or chromosome mapping and for (iv) production of monoclonal antibodies by producing hybridoma (hybrid cells between an immortalised cell and an antibody producing lymphocyte), etc. Chromosome mapping through somatic cell hybridization is essentially based on fusion of human and mouse somatic cells. Generally, human fibrocytes or leucocytes are fused with mouse continuous cell lines. When human and mouse cells (or cells of any two mammalian species or of the same species) are mixed, spontaneous cell fusion occurs at a very low rate (10-4). Cell fusion is enhanced 100to 1000 times by the addition of ultraviolet inactivated sendai (parainfluenza) virus on polyethylene glycol (PEG). These agents adhere to the plasma membrane of cell sand later their properties in such a way that facilitates their fusion. Fusion of two cell produces a heterokaryon, i.e., a single hybrid cells with two nuclei, one from each other cells entering fusion. Subsequently, the two nuclei also fuse to yield a hybrid cell with a single nucleus.

A generalized scheme for somatic cell hybridization may be described as follow. Appropriate human and mouse cells are selected and mixed together in the presence of inactivated Sendai virus or PEG to promote cell fusion. After a period time, the cells (a mixture of man, mouse and hybrid cells) are plated on a selective medium, eg., HAT medium (figure) which allows the multiplication of hybrid cells only. Several clones (each derived from a single hybrid cell) of the hybrid cells are thus isolated and subjected to both cytogenetic and appropriate biochemical analyses for the detection of enzyme/protein/trait under investigation. An attempt is now made to correlate the presence and absence of the trait with the presence and absence of a human chromosome int eh hybrid clone. If there is a perfect correlation between the presence and absence of a human chromosome and that of a trait in the hybrid clones, the gene governing the trait it taken to be located in concerned chromosome. The HAT medium is one of the several selective media used for the selection of hybrid cells. This medium is supplemented with hypoxanthine, aminopterin and thymidine, hence the name HAT medium. Antimetabolite aminopterin blocks the cellular biosynthesis of purines and primidines from simple sugars and amino acids. However, normal human and mouse cells can still multiply as they can utilize hypoxanthine and thymidine present in the medium through a salvage pathway, which ordinarily recycles the purines and pyrimidines produces from degradation of nucleic acids. Hypoxanthine is converted into guanine by the enzyme hypoxanthine- guanine phosphor-ribosyltransferase (HGPRT), while thymidine is phosphorylated by thymidine kinase (TK) ; both HGPRT and TK are enzymes of the salvage pathway.

Figure: A schematic representation of somatic cell fusion technique On a HAT medium, only those cells that have active HGPRT (HGPRT+) and TK (TK+) enzymes can proliferate, while those deficient in these enzymes (HGPRT- and /or TK-) can not divide (since they cannot produce purines and pyrimidines due to the aminopterin present in the HAT medium). For using HAT medium as selective agent, man cells used for fusion must be deficient for either the enzyme HGPRT or TK, while mouse cells must be deficient for the other enzyme of this part. Thus, one may fuse HGPRT deficient human cells (designated as TK + HGPRT-) with TK deficient mouse cells (denoted as TK- HGPRT+). Their fusion products (hybrid cells) will be TK+ (due to the human gene) and HGPRT+) (due to the mouse gene)and will multiply on the HAT medium, while the man and mouse cells will fail to do so. Experiments with other selective media can be planned in a similar fashion. 4. Write in detail about cell culture as a source of valuable products. CELL CULTURE PRODUCTS:

Animal cell cultures are used to produce virus vaccines, as well as a variety of useful biochemicals, which are mainly high molecular weight proteins like enzymes, hormones, cellular biochemicals like interferons, and immunobiogical compounds including monoclonal antibodies. Animal cells are also good hosts for the expression of recombinant DNA molecules and a number of commercial products have been/are being developed. Initially, virus vaccines were the dominant commercial products from cell cultures, but at present monoclonal antibody production is the chief commercial activity. It is expected that recombinant proteins would become the prime product from cell cultures in the near future. Transplantable tissues and organs are another very valuable product from cell cultures. Artificial skins are already in use for grafting in burn and other patients , and efforts are focused on developing transplantable cartilage and other tissues. A greater detail of cell culture products is provided under the following heads: (1) vaccines, (2) interferon, (3) monoclonal antibodies, (4) hybrid antibodies and (5) recombinant proteins. Virus Vaccines A vaccine is a preparation containing a pathogen (disease producing organism) either in attenuated or inactivated state. This preparation is introduced into an individual to induce adequate antibody production against the pathogen in question so that the individual becomes protected against infection, to a later date, by that pathogen. The introduction of a vaccine in an individual is called vaccination or immunization as it leads to the development of immunity int eh vaccinated individuals to the concerned pathogen. The immunity is induced by the antigens of pathogen origin present in the vaccine. Any molecule that induces production of antibodies specific to itself when introduced into the body of an animal is called immunogen, while a molecule that interacts with an antibody is known as antigen. Usually, antigents are also immunogeneous. The antigenic function, generally confined to a rather small portion of the antigen molecules : such a region is known as antigenic determinant or epitope. A B-lymphocyte cell becomes predetermined, during differentiation, to produce antibodies of a single specificity. Ordinarily in an animal, the frequency of B-lymphocytes capable of producing antibodies of a given specificity, i.e., against a single epitope, is very low. When an antigen enters into the body of an animal, its antigenic determinant ultimately binds to the specific receptors present on the surface of those B-lymphocytes, which produce antibodies specific to this antigen. This binding stimulates a rapid proliferation of these B-lymphocytes so that the proportion of such cells increases drastically; this is called clonal selection (proposed by N. Jerne). As a consequence, the concentration in the blood serum of antibodies (antibody titre) specific to the concerned antigen also increase dramatically. This phenomenon is the basis of immunization. When an individual is vaccinated, the antigens of pathogen origin stimulate antibody production against themselves. This increase in antibody production takes some time, but since the vaccine does not have virulent pathogens there is not danger of disease development. (If the pathogen present in the vaccine were virulent/alive, vaccination would produce disease before the antibody production could be sufficiently increased). In addition, a population of B memory cell is also produced. When live virulent form of the same pathogen later enters into the system of an immunized animal, the memory B cells quickly produce a high level of antibodies specific to the pathogen; these antibodies inactivate the pathogen and thereby, protect the animal against the disease. The various vaccines can be grouped into two categories: (1) vaccines containing killed or inactivated pathogens, i.e., most bacteria vaccines and some virus vaccines (e.g, influenza virus inactivated by formalin, rabies virus inactivated by phenol or -propiolactone), and (2) those containing live but attenuated pathogens, e.g., most virus vaccines. Attenuation means a drastic reduction in the virulence of a pathogen; this is achieved as follows. 1. Several consecutive passages through an animal, which is not the usual host of the pathogen, e.g., small pox virus in calf. 2. Several passages through cultured cells of the host, e.g., rabies virus in human diploid cell culture, or of a different species, e.g., rabies virus, yellow fever virus in chick embryo cell culture. 3. Selection of les virulent strains of pathogens, e.g., a mutant strain of polio virus. 4. Treatment of the pathogen with some chemicals, e.g., B.C.G. (Bacillus of Calmette Guerien) vaccine produced by culturing the bacteria on a medium containing bile. 5. Culturing pathogens under unfavourable conditions like high temperature, e.g., anthrax vaccine obtained by cultivation of the bacterium (Bacillus anthracis) at 40 -50 C. In general, inactivation of viruses is always coupled with attenuation to minimize the accidental presence in vaccines of active virulent particles which could cause disease in the vaccinated individuals. The different vaccines differ in their composition, efficacy and the duration of effective protective to the vaccinated individuals. Vaccines are one of the earliest examples of biotechnological intervention in human and

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animal health care. (The first case of vaccination was against rabies by Louis Pasteur on July 6, 1885 in case of a young boy bitten by a mad dog. Pasteur used the fluid from spinal cord of another mad dog for vaccination.) Vaccines offer the cheapest and the most effective protection against diseases, and for some diseases, e.g., hepatitis B, AIDS, etc., they are the only means of protection. The effectiveness of vaccines may be highlighted by the success of WHO (World Health Organization) sponsored mass vaccination against small pox in completely wiping out this once ravaging disease from the face of earth. The procedure of virus vaccine production using cell cultures is essentially, and in simple terms, as follows. The cells to be used as host are inoculated in culture vessels and, after suitable growth ahs occurred, the cultures are infected with the virus concerned and incubated. After an appropriate period of virus multiplication and release, the culture medium containing virus particles is collected and suitably processed. In case of killed virus vaccines, the vaccine is usually subjected to a concentration step after virus inactivation. Suitable stabilizers are added to prevent loss of potency and the vaccines are generally stored at low temperature till they are used. A rigorous quality control is maintained throughout the entire operation. A number of vaccines are produced in cultured animal cells; some of these are being produced in India (table). But the procedure has several inherent problems. (1) Rigorous quality control is essential particularly to exclude the possibility of presence o infectious agents in the materials and cells used. In addition, (2) the vaccine may contain a rare virulent and active virus particle, which may cause disease in the vaccinated individuals. Therefore, laterntive approaches of vaccine production and delivery have been/are being developed , viz, (1) production of antigenic proteins or only their antigenic determinants in GEMS (genetically engineered microbes) (2) use of DNA as vaccines and (3) production of antigenic proteins in vegetables/fruits to minimize storage costs and problems. TABLE A list of products generated/likely to be generated from cultured animal cells Product Virus Vaccines Canine distemper Foot and mouth disease (FMD) Hepatitis B Influenza Measles Umps * Polio *Rabies *Rubella (German measles, MMR) Cellular Biochemicals Angiogenic factor -Interferon -and -interferons Immunoregulatory Biochemicals Interleukin -2 Lymphokines Enzymes Collagenase, pepsin, Renin, trypsin Urokinase (Plasminogen activator) Tissue-type plasminogen activator Single chain urokinase type Plasminogen activator Tyrosine hydroxylase Hormones Luteinizing hormone Follicle stimulating hormone Chorionic hormone Erythropoietin Growth hormone ACTH (Adenocroticotrophic hormone). Immunobiologicals Monoclonal antibodies Cell line Dog kidney Cow kidney Human plasma derived Chick embryo Chick embryo Chick embryo Primary kidney 1. Chick embryo 2. Human diploid cells Human diploid cells Human tumour Mouse fibroblast Human leucocytes Lymphoblastoid T-cell line ---Human kidney CHO cell line Human kidney transformed Cell line TCL 98 -------Hybridoma clones Most dominant activity Continuous cell line Remarks

From blood of infected individuals

First vaccine from cell culture

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Inteferons: Interferous are proteins produced by cell infected by a virus, and provide protection to other healthy cells from viruses. Interferon was discovered in 1957 when Issacs and Lindenmann observed that virus free fluid obtained from cultured cells infected with virus protected other cells from virus infection. They called the substance present in these fluids, which interfered with virus infection, Interferon. There are three major types of interferons. (1) Interferon - (INF -; produced by leucocytes or white blood cells), (2) interferon - (INF-: produced by fibroblasts), and (3) interferon - (INF -, produced by stimulated T-lymphocyte; also immune interferon). The mechanism of protection by interferons appears to be as follows. When interferon reacts with the interferon receptors of a cell, the cell enters in a state called interferon-induced antiviral state. In this state, a rapid degradation of mRNA occurs if the cell is infected by an virus. Interferon induces in cells the production of 2,5adenosine polymerase. When such a5-ribonuclease-L. Activated ribonuclease-L degrades all mRNA (of host as well as virus origin) present in the cell bringing the protein synthesis to a halt in such cells. This interference with multiplication of the virus so that virus infection is either stopped or sufficiently slowed down to allow the production of adequate antibodies against all viruses. It is possible that interferons modify ribosomes so that they no longer translate viral mRNAs, although they are fully capable of translating mRNAs of the host origin. Interferons enhance the cytotoxic activitity of natural killer cells(NK cells), which are a type of lymphocytes identifiable as large granular lymphocytes (LGL). NK cells are cytotoxic to some types of tumour cells. Interferons are known to inhibit growth of some types of tumours; most of these tumours are also responsive to chemotherapy. Interferons, therefore, have been employed in treatment of the responsive tumours despite their prohibitive cost (initially, $50 million/g of commercial product.) Interferons are produced from human leucocytes isolated from donor blood and cultured in vitro , and from mouse fibroblast cultures. The production scheme, in simple terms, is a follows. Large scale (1,000 1 to 10,000 1) cell cultures are infected wit Sendai virus, and incubated for 24 hr after which the supernatant (clear fluid) is collected, centrifuged, and used for interferon isolation. The amount of interferon recovered is relatively small (1 g interferon of low purity from leucocytes separated from blood of about 90,000 donors), and the normal leucocytes are difficult to culture preventing up from relatively small inocula. These contributed to the enormous price of the product. In view of the value of and demand for interferons, intensive efforts were made to produce it in genetically engineered organisms, e.g., E.coli yeast, mammalian cell cultures and even in plants. These efforts have drastically reduced the cost (to les than 10% of the initial price) and improved the purity (by several orders of magnitude) of the product. Recombinant Proteins Proteins produced by gene transferred into selected host cells by genetic engineering are called recombinant proteins since they are based on recombinant DNA technology. Recombinant proteins form an important component of biopharmaceuticals. i.e. biotechnology products have pharmaceutical applications. A large number (nearly 2 dozen) of recombinant proteins are being produced in mammalian cell cultures, some of which, viz., human growth hormone (hGH), tissue plasminogen activator (tPA).erythropoietin and blood clotting factor VIII, are already in the rapeutic use. The other products listed in table are in advanced stages of development. The host cells used for large scale production of the various recombinant proteins are Chinese hamster ovary cell line (CHO), baby hamster kidney line BHK, mouse mammary tumour line C127, mouse myeloma cell lines and mammalian cell lines. The use of animal cell lines has been made possible by the following developments in cell culture technology : (1) development of culture systems permitting large scale culture of animal cells at high densities, and (2) development of media, which minimize or even obviate the use of serum (serum interferes with downstream processing, .e. separation and purification of the desired product). These developments have enhanced the yields and reduced the costs of the products to the levels that in many cases they are favourable comparable to the costs of the same products obtained from genetically engineered bacteria and yeast. The use of animal cells for the production of recombinant proteins is preferable to the use of microorganism for the following reasons. 1. The signals for synthesis, processing and secretion of the concerned protein are better recognized in animal cells than in microbes. 2. The protein products are readily secreted into the medium, which makes their recovery much easier. TABLE

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A list of products (recombinant proteins) generated using recombinant DNA technology . The genes are expressed in animal cell culture, and their products are either already in therapeutic use or in advanced stages of development. Product (protein) Currently in Therapeutic Use Human growth hormone (hGH) Tissue plasminogen activator (tPA) Erythropoietin* Blood clotting factor VIII* In Advanced Stages of Development Antithrombin VIII Blood factor VIII Blood factor IX Interleukin (11-2) Human recombinant antibodies Hepatitis-B surface antigen (HBSAg) Interferons (IFN) Plasminogen activator chimeric and modified) CD4 protein Gp160protein P24 protein Interleukin-1 receptor antagonist Atrial natriuretic Ciliary neurotropic factor DNase * Approved for marketing in India 3. The patterns of folding and disulphide bridge formation in the recombinant proteins are similar to those of the natural proteins. 4. The multimeric proteins are also assembled correctly. Hybrid Antibodies When antibody molecules are modified or designed using recombinant DNA technology to suit specific applications, such antibodies are called recombinant or hybrid antibodies, and the approach itself is termed as antibody engineering. The essential steps in antibody engineering are : 1) to develop the antibody design to serve the specific purpose. (2) to bring together the DNA sequences that would generate this antibody molecule into a suitable expression vector, (3) to introduce this recombinant DNA into a myeloma cell line where the recombinant antibody gene expresses itself. 4) to mass culture the transfected (i.e., containing the recombinant antibody gene) clones of myeloma cell line and (5) to harvest the recombinant antibodies produced. It is essential that the myeloma cell line used for transfection is Ig- (immunoglobulin negative), i.e., does not produce any antibody of its own. 1. A recombinant antibody could be constructed so that the constant end of its heavy chain is fused to a polypeptide chain having an enzymatic function (fig). the enzymatic polypeptide chain could replace the C H2 and CH3 regions of the heavy chain and be fused to the CH1 segment, Such an antibody will be extremely useful for ELISA as there will be no need for a second antibody. A gene producing such an antibody can be produced by fusing the heavy chain gene having the appropriate L-V-D-J and 1 sequences (Appendix-4.I) with the sequence coding for the selected enzyme function. 2. The heavy chain gene of an antibody specific to a tumour-specific antigen may be fused with a gene encoding a toxin polypeptide. Such hybrid antibodies will carry the toxin specifically t the tumour cells and thereby, kill them. (including CHO cell line BHK-21 cell line CHO cell line -CHO cell line and Mouse myeloma lines CHO cell line and C-127 cell line. -CHO cell line and Spodoptera Frugiperda Mammalian cell line Mammalian cell line Mammalian cell line Mammalian cell line Mammalian cell line Mammalian cell line Mammalian cell line Anticoagulant Haemophilia Haemophilia-B Cancer therapy Infections, various cancers As vaccine against Hepatitis-B Tumours Thrombolysis AIDS vaccine AIDS vaccine AIDS vaccine Severe sepsis Kidney failure Amytropic selrosis Cystic fibrosis Gene expressed in (host) Mouse mammary tumour cells line Chenese hamster ovary (CHO) cell line CHO cell line CHO cell line Application Dwarfism Thrombolysi Anaemia Haemophilia

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3. Gene segments encoding the variable-region or V-region (involved in antigen-antibody interaction) of an antibody have been fused with the constant-region or C-region of another antibody to yield a hybrid antibody. A hybrid antibody has the antigen specificity of first antibody(which contributed the V-region segments), but its other properties are due to the second antibody (contributing the C-region segments). This approach has been used to produce hybrid antibodies with their antigen-binding portion from a mouse antibody gene, and their C-region from the human antibody gene. Such antibodies are often known as humanized rodent antibodies. This is very useful in cases where it is not possible to either immunize humans or to induce antibody production in cultured human cells.

Figure: A schematic representation of a hybrid antibody and the heavy chain gene producing it. The gene encoding constant region of the heavy chain is fused with a gene encoding the desired enzyme function. The recombinant gene is expressed in a suitable myeloma cell line. 4. The knowledge of three dimensional structures of antigens and antibody regions involved in antigen binding can be used to predict the amino acid sequence of V-region of the antibody specific to the given antigen. This involves a detailed computer modeling using the data on molecular organization of the antigen to predict the corresponding organization of the specific antibody. The gene encoding an antibody having the given amino acid sequence can now be synthesized and expressed in a suitable host cells. The hybrid antibodies are monoclonal in nature since each preparation has antibodies of a single specificity.

UNIT II PART A 1. Define Monoclonal antibody. It is defined as a single type of antibody specific for one antigenic determinant (portion of an antigen that binds to an antibody) that is secreted by a hybridoma clone derived from a single B cell. 2. Define In situ hybridization. It is a powerful method used to locate nucleic acid sequences in histological and cytological preparations of tissues, organelles, cells and chromosomes.

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3. Write short notes on PCR. Polymerase chain reaction (PCR) is a laboratory (in vitro) technique for generating large quantities of a specified DNA. 4. What are the essential requirements of PCR? The essential requirements for PCR are 1. 2. 3. 4. A target DNA (100 35,000 bp in length) 2 primers that are complementary to regions flanking the target DNA 4 deoxyribonucleotides A DNA polymerase that can with stand a temperature up to 95C.

5. What are the 3 stages of PCR? 1. 2. 3. Denaturation Renaturation or annealing Synthesis

6. Give some applications of PCR. 1. In clinical diagnosis 2. In DNA sequencing 3. In gene manipulation & expression studies 4. In Forensic medicine 7. Give some methods to study gene expression. 1. 2. 3. 4. 5. Southern Blot Northern Blot Nuclease SI mapping Nuclease Protection assay Primer extension

8. Define Southern blot. It is a novel technique to detect to detect a known fragment of DNA in the DNA preparation of an organism. This technique is particularly useful to detect the presence of a foreign DNA in genetically modified organisms or to identify the presence and copy number of genes in an organisms genome. 9. Define Northern blot. It specifically detects the size and sequence of the mRNA. The total mRNA is extracted from a cell or tissue suspension and separated by agarose gel electrophoresis and then detected by hybridization. 10. Define RFLP. RFLP Restriction Fragment Length Polymorphisms It represents a stretch of DNA that serves as a marker for mapping a specified gene. RFLPs are located randomly throughout a persons chromosomes and have no apparent function. PART B 1. Explain in detail about Viral & Bacterial diseases. Recombinant vaccines against viral diseases Foot and mouth disease was the first disease against which effective vaccine was produced through gene splicing. The FMD virus genome is a single, positive-sense strand of infectious RNA, made of four viral proteins, viz., VP1, VP2, VP3 and VP4. In this virus, the neutralizing immune response appears to be directed primarily against VP1. VP1 gene carries type and strain specificities, receptor binding sites, T cell epitopes and immunogenic sites. Studies have shown that the peptides corresponding to residues 141-160 and 200-213 of VP 1 are capable of neutralizing

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FMD virus. The neutralization elicited by residues 141-160 is much greater than that of antiserum to residues 200213. These data demonstrate that VP1 of FMDV is an ideal target antigen for the development of a subunit vaccine against FMD. A recombinant live vector vaccine was produced by insertion of eDNA encoding the structural proteins (P1) of foot and mouth disease virus (FMDV) into a replication competent human adenovirus type 5 vaccine strain. (SanzParra et al.,1999). Groups of cattle were immunized twice, by the subcutaneous and/or intranasal routes, with either the Ad5 wt vaccine or with the recombinant FMDV Ad5-P1 vaccine. All animals were challenged by intranasal instillation of FMDV 4 weeks after the second immunization. Significant protection against viral challenge was seen in all of the animals immunized twice by the subcutaneous route with the recombinant vaccine. A tobacco mosaic virus based vector was used to express in plants the complete open reading frame coding for VP1 protein of FMDV (Wigdorovitz et al. 1999). Foliar extracts prepared from infected leaves were injected intraperitoneally into mice and all of the immunized animals developed a specific antibody response to both the complete virus particle and the major immunogenic region. All immunized mice developed a protective immune response against experimental challenge with virulent FMDV. Rhabdoviridae possess an immunogenic surface glycoprotein G (haemagglutinin). Rabies virus subunits or the isolated glycoprotein G alone elicited neutralizing antibody and induced immunity in animals. The glycoprotein % genes of rabies and vesicular stomatitis viruses have been cloned in E. coli. A recombinant vaccinia virus expressing the immunizing rabies G glycoprotein has been developed (Kieny et al., 1984). This vaccine has been successfully used to protect foxes against rabies. The administration of recombinant rabies vaccine to adult foxes, raccoons and skunks elicited high levels of rabies virus neutralizing antibodies and long term protection against rabies. Recombinant vaccines have been produced against rinderpest virus. Dr. T.D. Yilma and his colleagues in USA were the first in the field to construct the Wyeth strain of vaccinia virus recombinants expressing haemagglutinin (H) and fusion (F) genes cloned from the original highly virulent Kabate O strain of rinderpest virus. This vaccine was administered to cattle intradermally in two doses, four weeks apart. All the vaccinated cattle developed neutralizing antibody, but the antibody response in the cattle receiving the recombinant expressing the rinderpest F gene were significantly lower than the response in cattle given the recombinant expressing the rinderpest H gene. Solid immunity with recombinant vaccine require two doses. Dr. T. Barret and his colleagues, working at UK inserted the F-protein gene from Plowrights cell cultured rinderpest vaccine virus into the vaccinia virus. When this vaccine was injected intradermally into cattle and pigs, the antibody response was higher in pigs than in cattle. Two doses were administered. In Japan, Dr. Yamanouchis group inserted the H-protein gene into the tissue culture strain of vaccine virus. The vaccine was given intradermally into rabbits and they produced neutralizing antibodies. Sinnathamby et al. (2001) developed a recombinant baculovirus expressing H protein of Rinderpest virus and studied its protective properties in cattle. Single administration of low doses of purified recombinant extracellular virus with or without adjuvant induced virus neutralizing antibody responses and bovine leukocyte antigen class II restricted helper T cell responses in cattle. Newcastle disease recombinant vaccine had been produced using different virus vectors. The haemagglutinin-neuraminidase (HN) gene and the phosphoprotein (P) gene of Newcastle disease virus (NDV) were inserted into an avian leucosis virus vector. These recombinants were used to immunize four weeks old chicken. Birds receiving the virus containing the HN gene developed low levels of serum haemagglutination inhibition titres. Upon challenge, all birds vaccinated with the HN gene containing virus were protected from the disease. Birds immunized with the P gene containing retrovirus developed more severe clinical signs (Morrison et al., 1990). In another study, a cDNA copy of the RNA encoding the fusion (F) protein of NDV was inserted into a fowl pox virus vector. Ocular or oral administration of recombinant fowl pox virus gave partial protection, whereas both intramuscular or wing web routes of inoculation gave complete protection after a single injection (Taylor et al., 1990). Three glycoproteins (gp 115/110, gp 63 and gp 51) from herpes virus of turkeys and Mareks disease virus would protect chicks against Mareks disease (Ikuta et al., 1984). The surface glycoprotein of infectious bovine rhinotracheitis (Lupton and Reed, 1980) and pseudorabies virions have been reported to induce neutralizing antibodies and protective immunity in cattle and swine, respectively. A -galactoside pseudorabies glycoprotein fusion protein from E. coli is reported to protect mice from challenge with pseudorabies virus. The genome of blue tongue virus is divided into 10 RNA segments. VP2 proteins of type 10 blue tongue virus induce both neutralizing antibodies and protective immunity in sheep. RNA segment 2 which represents the VP2 neutralizing antigen, has been inserted into an insect baculovirus (Autographa californica, AcNPV). Infection of Spodoptera frugiperda cell lines (which supports the baculovirus to grow in high titre) by the recombinant virus

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produced VP2 protein that was precipitated with BTV-10 and to a lesser extent BTV-serotype 11 and 17 viruses (Roy and Inumam, 1989). Canine distemper virus (CDV) and measles virus (MV) are members of the Morbillivirus genus of the family Paramyxoviridae and contain a non-segmented single-stranded RNA genome. These viruses have membrane glycoprotein haemagglutinin (HA) which is responsible for haemagglutination and attachment of the virus to the host cell and the fusion glycoprotein (F) which causes membrane fusion between the virus and the infected adjacent cells. Vaccinia virus-MV, HA and F recombinants were produced and inoculated into dogs. Of these two recombinants, vaccinia virus-MVHA gave better neutralizing antibody production than VV-MVF protein (Taylor and Paolettic 1991). VP2 of canine parvovirus which is a major component of the capsid protein was expressed in baculovirus. This recombinant virus was able to elicit good protective response (Lopez et al., 1992). Dogs immunized with the inactivated recombinant plant virus, displaying a peptide derived from the CP2 capsid protein of canine parvovirus (CPMV-PARVO1 elicited high titres of peptide-specific antibody (Langeveld et al., 2001). Since plant virus-derived vaccines have the potential for cost-effective manufacture and are not known to replicate in mammalian cells, they represent a viable alternative to current replicating vaccine vectors for development of both human and veterinary vaccines. Recombinant vaccines against bactorial diseases The first work of rDNA bacterial protein vaccines involved the cloning of somatic pili of enteroxigenic E. coli (ETEC) for use in preventing diarrhea in livestock. The ETEC have somatic pili composed of 14 to 22 KDa protein molecules that adhere to the mucosal surface of the small intestines, initiating colonization by the bacteria. Pili isolated from ETEC have been used as vaccines to prevent diarrhoeal diseases in both humans and animals. An alternate method for protecting domestic animals against ETEC has been achieved through genetic engineering. Monoclonal antibody to K99 pilus antigen produced in hybridomas and amplified in mice significantly reduced the severity of scours and the mortality of calves so created (Sherman et al. 1983). This product was successfully tested in Canada and had since been approved by the USDA for use in the US. 2. Explain in detail monoclonal antibodies production, advantage & disadvantage. Polyclonal Antiserum Vs. Monoclonal Antibody Animals respond to an antigenic stimulus by producing a large variety of antibody structures directed against the immunogen. Even a single antigenic determinant is likely to be recognized by a variety of different antibody structures. Thus, heterologous mixtures of antibody structures are produced in a polyclonal response, and the populations of antibodies continuously change within the same animal, since each antibody is secreted by all such individual cells into a common blood pool. The composition of polyclonal antibodies will change from day to day and from animal to animal. Thus, polyclonal antisera, even when prepared employing well standardized procedures tend to differ from batch to batch. But MAbs produced by a single clone of cells which react with a single antigenic determinant are the most specific biological probes available making possible finer and more sophisticated differences. In 1975, Kohler and Milstein gave the concept of one lymphocyte-one antibody in the form of hybridomas. Hybridomas are hybrids between myeloma tumour cells and antigen stimulated lymphocytes which can be cloned, grown in large quantities and for indefinite periods of time, and secrete high concentration of monoclonal and hence monospecific antibodies. Each lymphocyte produces only one type of antibody, although the isotype may change. The homogeneity, reproducibility and permanent availability of MAbs are the attributes arousing the greatest interest in this quickly developing field. Advantages of Monoclonal Antibodies 1. 2. 3. 4. 5. 6. 7. 8. Single MAbs are chemically defined, single-specificity molecules, and can be used easily for standardization of specific assays. MAbs provide a perpetual source of well defined homogeneous reagents. All the antibodies produced (100%) are active antibodies. Therefore, high specific activity of labeling is possible in Radioimmuno assay. Enzyme linked immunosorbent assay and Fluorescent immunoassay. MAbs could be advantageously used as immunosorbents for antigen purification using immunosorbent column chromatography. Large amounts of MAbs (1-20 mg/ml of ascetic fluid) could be obtained with modest investments. MAbs specific for a particular target antigen can be obtained even without prior purification of antigens. MAbs are easily manipulatable. For example, bispecific antibodies can be prepared from 2 MAbs and used in novel assays, antigenic targeting in Electron microscopy and cytological studies. Distinct antigenic cross reactivities can be easily defined and hence most useful in diagnosis.

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Disadvantages of Monoclonal Antibodies 1. 2. 3. 4. MAbs are too specific. Limited MAb might miss important cross reactive determinants. This could relate to the animals immune response to the antigenic stimulus. Polyclonal sera may offer advantage of broad reactivity. Biological effects of MAbs on binding do not represent situation in animals. Single MAbs is a single chemical, and physical or chemical treatment that affects one molecule will affect all the MAbs in that population. Treatments like freeze thawing, proteolytic enzyme contamination and ammonium sulphate precipitation may destroy all MAb activity. It is time consuming to produce MAbs. The entire process of producing MAbs takes 3-4 months for each fusion experiment.

Monoclonal Antibody Production Strategy The one cell one antibody theory provided the conceptual basis for the practical advantage offered by the MAb technique. Each clone of lymphocytes produce a single antibody specificity. Nevertheless, the immune responses to most antigens are polyclonal; it involves hundreds of clones of lymphocytes, each secreting a different antibody. These antibodies are markedly heterogeneous. They usually consist of antibodies directed at different antigenic determinants. MAb production involves the creation of hybrid cells derived from 2 parent cell types: normal antibody producing cells and neoplastic myeloma cells. The hybrids are formed by fusing the normal and neoplastic cells. The hybrids are formed by fusing the normal and neoplastic cells. Hybrids have characters of both parents, viz., the infinite life of the meyloma cells and antibody secreting functions of primed cells. Hybridization of antibody forming cells with malignant myeloma cells results in hybridomas in which virtues of specific antibody secretion and continuous growth are combined. Further, selection and cloning of the hybrid cells allow the derivation of monoclonal antibodies of predefined specificity and desired activity. The method of establishing permanent cell lines capable of producing antibodies directed to predefined immunogen is based on the fusion of immune lymphocytes (usually spleen cells from mice previously immunized with antigen) with myeloma cells adapted for growth in tissue culture conditions. The most common fusion agent is polyethylene glycol (PEG). The selection for growth of hybrid cells is based on the facts that (a) spleen cells die in the tissue culture medium and (b) the myeloma lines that serve for fusions are mutants lacking either the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT) (azaguanine resistant) or thymidine kinase (bromodeoxyuridine resistant). Such mutants die in the presenceof aminopterin which blocks the main pathway of DNA synthesis because they cannot use the salvage pathway. Hypoxanthine, aminopterin and thymidine (HAT) allows the selective growth of hybrids. The parental myeloma cells that have not fused will not grow in the presence of HAT medium. Normal lymphocyte die after a short period in culture. Therefore, only the hybrid cells between the spleen cells and the meyloma cells will survive in the presence of aminopterin, but for their growth, they require hypoxanthine and thymidine that are utilized by salvage pathway. The hybrid cells growing in HAT are than screened for their ability to produce and secrete the desired antibody. The positive hybridomas obtained after cloning are the source for monoclonal antibodies. Such cells can be cryopreserved and for the production of large amounts of MAbs, they can be grown in mass cultures, where they secrete 10-50 g antibody/ml or propagated as ascetic tumour, where they usually produce 1-20 mg/ml of MAbs into the ascetic fluid.

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Fig. Production of monoclonal antibodies Though the methodology involved in the production of MAbs appears simple, there are some crucial steps, especially in the screening and selection of positive hybridomas, that require preplanned, sensitive, simple and fast assays. In addition, several precautions and devotion have to be practiced with regard to the tissue culture conditions, especially during the first phase of the hybridoma establishment. 3. Describe Monoclonal antibodies use in diagnoses. Uses of Monoclonal Antibodies There are three main areas in which MAbs are used in human and veterinary medicine. These areas are: (1) immuno diagnostic reagents, either for detection of the causative agents directly in tissues or body fluids or as a reagent used in indirect diagnosis such as serological detection of antibodies to the causative agent; (2) for experimental purposes ranging from molecular dissection of antigenic epitopes to monoclonal anti-idiotype antibody utilized as a vaccine to induce protective immunity and (3) immunoprophylaxis or immunotherapeutics applied to infectious diseases or as a vehicle for delivering toxic substances to, for example, tumours or as a tool to identify and locate target tumours. Immuno diagnostic reagents Antigen detection With the development of MAbs of various specificites, advances have been made in the detection of infectious agents (or their antigens) directly in tissue or body fluid specimens. More amount of work have been done on viruses because of the difficulties of viral culture. MAbs have been developed against a large number of viruses including rabies virus (Koprowski and Wiktor, 1980), foot and mouth disease virus (Russel and Alexander, 1983), feline leukemia virus (Jochen et al., 1983), bovine leukemia virus (Portetelle et al., 1984) bovine enteric coronavirus (Crouch et al., 1982) and rotavirus (Sona et al. 1983). MAbs have been produced against various parasites, including Trichinella spiralis (Gamble and Graham, 1983), Babesia bovis (Wright et al., 1983, Dirofilaria immitis (Scott, 1983) and Trypanosoma cruzi (Araccio et al., 1982), MAbs have also been raised against bacterial species, including Mycobacterium spp. (Kolk et al., 1964, Morris

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and Ivanyi, 1985 & Young et al, 1985) Brucella spp. (Holman et al, 1983) and Vibrio cholerae (Gustafsson et al., 1982).

For detection of antigen, the following are the four commonly used methods using MAbs. 1. MAbs are attached to particles like latex or erythrocytes, which then detect the presence of an antigen in fluids by visible agglutination, usually in minutes. Disadvantage of this technique is the inability of small antigens to cause agglutination. Direct demonstration of antigen in tissue sections, cultured cells or smears by the use of a MAb conjugated to a fluorochrome, enzyme (used in conjunction with a substrate that has an insoluble cleavage product) or radio-isotope as a detecting agent. A competitive-binding type of assay in which MAb is attached to a solid matrix such as the well of a polystyrene microtitre plate. Antigen conjugated with a fluorochrome, enzyme or radio-isotope is added only in sufficient quantity to bind all the available antigen-binding sites of the antibody, then premixed with the known sample to be tested for the presence of an antigen. Decrease in binding of the known antigen indicates the presence and quantity of antigenic material in the unknown sample. Antibody immobilized in polystyrene wells can trap antigen in fluid preparations. The trapped antigen may then be detected using another antibody to the antigen. This second antibody may be conjugated with a fluorescent, radioactive or enzymic agent for easy detection. Alternatively, layers of anti-antibody can be added to increase sensitivity or an amplified system may be used. For this type of assay, MAbs may be used for both the trapping and detection, but they must have different specificities for the test to function properly unless the antigen is a single repeating unit such as the o-chain of some bacteria.

2.

3.

4.

Although MAbs are very useful in the detection of antigens or their components, caution should be exercised in that some MAbs may be too specific for general diagnostic use. Van Zaane (1983) reported that two MAbs were capable of distinguishing some strains of hog cholera virus from bovine viral diarrhoea virus; however, both antibody preparations failed to react with some strains of hog cholera virus by an immunofluorescence test. It is therefore essential to ascertain the reactivity of the MAb with an antigenic determinant present on all antigens to be tested for, before diagnostic use. On the other hand, the exquisite specificity of MAbs allows the differentiation of antigens previously thought to be indistinguishable. For example, using a panel of MAbs, it is possible to differentiate various street strains (i.e. non-laboratory strains isolated from natural sources) and two vaccine strains of rabies virus using immunofluorescence technique (Webster et al., 1986). Similarly, MAb to Mareks disease tumour associated antigen can be used to identify Mareks disease in chicken. As this antibody does not cross react with antigen expressed by lymphoid leucosis virus, a differential diagnosis is possible (Lee et. Al.1983). Because specific MAbs could be produced to rare and minor antigens, they have the potential to distinguish closely related strains of viruses, which cannot be differentiated by conventional serological methods. For example, MAbs that distinguish canine parvovirus from feline panleukopenia virus have recently been produced (Parish and Carmichael, 1983). MAbs were produced against 42 KDa major structural proteins of rotavirus, which could be used for preliminary serotyping of strains from different animal species (Greensberg et al., 1983). Anderson (1984) described an enzyme immunoassay in which a blocking MAb was used to detect groupspecific antibodies against blue tongue virus. This assay was capable of detecting antibodies against all 22 serotypes of blue tongue virus. The use of MAbs against bovine diarrhoea virus in immunofluorescent antibody techniques has aided preliminary serological characterization of strains (Peters et al., 1986) and both indirect and competitive ELISA techniques utilizing MAbs have been used to detect antibodies against the virus in bovine sera. Accurate and rapid diagnosis of infectious agents is of considerable importance in veterinary medicine, particularly when dealing with highly infectious agents such as foot and mouth disease virus. MAbs have an important role in the diagnostic laboratory; however, if the antibody is not carefully selected, erroneous diagnosis may result. The following criteria should be followed for selection of MAbs for diagnostic purpose. 1. 2. 3. Antibody must have sufficient affinity for the antigen to permit efficient combination with low antigen concentrations and to displace efficiently host antibody already attached to the antigen. The antibody should ideally be directed against an epitope of the antigen not recognized by host antibodies and therefore not masked by them. The antibody should be directed against a repeating epitope which is readily accessible such as lipopolysaccharides of Gram-negative bacteria or virus coat proteins.

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4.

Multivalency of the antibody may increase test sensitivity. For example, IgM may be more effective than IgG.

In addition to these criteria, the antibody should be stable, should accept conjugation procedures without loss of antigen binding capacity and should be of the appropriate specificity for its purpose. Care should be taken that MAb is not directed against a host cell or contaminant present in virus prepared from tissue culture. MAb in Cancer diagnosis Although polyclonal antiserum (produced in animals) have been used in diagnosis of cancers, they are not very reliable, since they cross react with antigenic determinants of normal tissues. MAbs have helped to overcome these problems. They are used in three different ways in cancer diagnosis. (a) to detect tumour-related antigens or tumour markers in body fluids or on cells. (b) to demonstrate tumour antigens in histological section and (c) to serve as imaging agents. Tumour markers Blood levels of certain tumour products are used as tumour markers in the diagnosis and prognosis of cancers. When the tumour load is reduced, the blood level of the tumour marker is also reduced considerably. Hence, this can be used to monitor the tumour burden and judge the efficiency of therapy. Carcino-embryonic antigen is a tumour marker elevated in 91-100% of persons with colorectal cancer. CA-125, another marker, is elevated in 82% of women with ovarian cancer. Prostrate-specific antigen is a marker for late stage prostrate cancer. MAbs raised against a variety of tumours have helped in the definition and characterization of tumour specific antigens, thus correct typing and diagnosis. Accurate diagnosis of the precise tumour type is essential since subsequent therapy depends on the type of tumour. MAbs raised against a variety of tumours have helped in the definition and characterization of tumour specific antigens, thus correct typing and diagnosis. Accurate diagnosis of the precise tumour type is essential since subsequent therapy depends on the type of tumour. Diagnostic tumour pathology The study of pathological change has been performed by microscopical examination of tissue section stained with MAbs using immuno histochemical techniques. A two step immunoperoxidase procedure, which gives a colour reaction at the end, is commonly used to demonstrate location of epitopes in a cell. MAbs are widely used in diagnosis of haematopoietic malignancies. MAbs to lymphoid cell surface markers are extensively used to assess the level of immunodeficiency in primary and acquired immunodeficiency syndromes (AIDS). Immunoscintigraphy MAbs with specificities to many types of human cancers are now available. These can be tagged withradioisotopes such as 131I or 99Technetium and injected intravenously into the patients; the antibody localize in the tumour, which can then be detected by imaging the radioactivity. This technique of imaging is known as immunoscintigraphy. Although imaging techniques like CT scanning, ultrasound scanning and magnetic resonance imaging can detect small lesions, they are unable to differentiate between cancerous and non-cancerous growth. Being tumour specific, antibody imaging has certain advantages. Small metastatic lesions which remain undetected by CT scan can be detected successfully in many cases with radiolabeled MAbs. MAbs as therapeutic agents Mouse MAbs directed against the particular organism have been found to protect chimpanzees against challenge with hepatitis B virus of rodents against infections with bacteria Haemophilus influenzae, E. coli, Proteus sp. And Streptococcus sp. MAbs against antigens present on the surface of cancer cells can exert site specific cytotoxic effect brought about the mechanisms such as complement mediated cytotoxic antibody dependent cell mediated cytotoxicity, phagocytosis of antibody coated cells by cells of reticulo-endothelial systems. In attempts to overcome rejection by cell mediated immune response, patients undergo immunosuppressive therapy, following organ transplantation. This therapy is in the form of immunodepressive drugs such as cyclosporine plus prednisone or patients are generally treated with glucocorticoid hormones in higher doses for short periods. MAbs specific for human T cell surface antigens (and for specific subpopulation of T cells) are now available. These MAbs can be used selectively to eliminate T cell populations responsible for allograft (transplant) rejection; for example, a MAb reacting with mature intravascular T cells has been found to be useful in subjects who have undergone renal transplantation.

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Rabies virus neutralizing human MAbs were evaluated in vitro and in a Syrian hamster model as a potential future alternative to rabies immunoglobulins (RIG) which is used for post exposure prophylaxis (Hanlon et al., 2001). Seven MAbs neutralized representative rabies virus. In hamsters, one MAb resulted in protection that was comparable to human RIG. One of the best example of use of MAbs in therapy is MAb OKT3, which is directed against CD3 is an antigen on the surface of T lymphocytes and CD3-activated T cells can play crucial role in allograft (organ transplant) rejection. OKT3 can be used to prevent acute renal allograft rejection in humans. For effective immune therapy against HIV infection, genetic engineering has been used to link the Fc portion of mouse MAb to human CD4 complex. CD4 is a cell surface receptor on T helper cells and one which has the receptor site for HIV. HIV attaches to T cells prior to infecting them by specifically attaching to CD4 receptors using the gp-120 receptors on its own surface. After infection of a T cell, gp-120 antigens appear on the surface of that cell. If Fc-CD4 molecules are given to the sufferers, these bind to HIV infected cells (displaying g-120) and the exposed mouse Fc fragment induces cell mediated destruction of the infected cell. This strategy may prove useful in preventing the spread of HIV throughout the T cell population. Drug targeting MAbs can be used as vehicles for the delivery of drugs in vivo. Conjugates of antibody wit drugs are able to bind to an appropriate target cell and the antibody-linked drug is then introduced directly into that cell. This allows higher concentration of the drug to reach the desired site and minimize systemic toxicity. Drugs such as daunamycin, vindesin, methotrexate and adriamycin can be coupled to MAbs. Better survival rates can be observed in experimental mice with lymphomas given daunamycin attached to antilymphoma MAbs compared to those receiving the drug alone. A recombinant chimeric MAb against fibrin coupled to tissue plasminogen activator (tPA) has been used in animal models. Because of its affinity for fibrin, the recombinant protein becomes concentrated in a clot, thereby locally raising the concentration of tPA. This helps in promoting an enhanced lysis of the clot. Such thrombolytic agent would have a crucial role to play in cardiovascular diseases. Antibody detection The second use of MAbs in disease diagnosis is in the detection of antibody most commonly used as an antiglobulin reagent conjugated with a detection system (fluorochrome, enzyme or isotope) in a primary binding assay. The most commonly used method is an indirect assay in which the antigen is passively adsorbed to a plastic (polystyrene) surface by hydrophobic interaction. The sample-serum or other body fluid suitably diluted is applied to the immobilized antigen. After an incubation period, the monoclonal anti-species antibody conjugate is added. Each step is followed by a thorough washing cycle. These are essential steps in which measures are usually taken to eliminate non-specifically bound proteins to reduce background activity in negative samples. In the final step if an enzyme conjugate is used, then a chromogenic substrate is added followed by spectroscopy, while radio-isotope or fluorochrome conjugate binding may be assessed directly. Primary binding assays are currently employed only to a small extent in veterinary medicine. However, with suitable apparatus becoming more common, primary binding assays and, in particular, enzyme immunoassay will replace current serological test (complement fixation test, serum neutralization test, precipitation test and agglutination test). Standardized reagents are essential for conduct of these assays for obtaining consistent results. In this respect, MAb has an advantage that antibody of identical specificity can be prepared at will and the enzyme conjugation procedure results in no discernible differences from batch to batch.

Anti-idiotype antibody Anti-idiotype antibody is an antibody that recognizes the antigen combining sites of other antibodies. Antiidiotype antibody (Ab2) has antibody activity to the antigen combing amino acid epitope sequence (of the folded molecule) of the hypervariable region of the antibody (Ab1) molecule. Anti-idiotype antibody can be prepared by immunization of a heterologous species or a homologous species with affinity purified Ab1. The resulting Ab2 should be able to compete with the original antigen for the antigen binding sites of Ab1. If a polyclonal Ab2 is made, even in a homologous species, it generally has to be absorbed extensively to remove anti-species or anti-allotype activity. If Ab2 is monoclonal, it would have to be tested exhaustively with immunoglobulins. The monoclonal Ab2 can be used in a primary binding assay as a capture agent for Ab1. If Ab2 was raised in a homologous species. Fab and Ab2 should be used for capture, while an anti-Fc reagent conjugate should be used for detection. An alternative approach is to attach Ab2 chemically to particles such as erythrocytes or latex beads and passive agglutination should take place in the presence of Ab1. By either method, detection of antibody may be accomplished in the absence of an antigen, a technique which may prove useful to serology that otherwise involves antigens that may be very expensive, toxic or difficult to obtain. Anti-idiotype antibodies have been produced against Staphylococcus spp. And Streptococcus spp.

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(Arulanandam et al., 1985) and Brucella abortus (Nielsen et al., unpublished work). 4. Explain in detail about Southern & Northern blotting & In-Situ hybridization. Southern blots The technique of Southern blotting is named after its inventor, Ed Southern. DNA that has been digested with restriction enzymes is applied to a gel and separated according to size on electrophoresis. The gel is treated with alkali to denature the DNA and a filter is placed over the gel. The DNA is transferred to the filter by capillary diffusion figure. The pattern of fragments of DNA on the filter is a replica of that in the gel. The DNA is firmly fixed to the filter and hybridized. The position of the band (autoradiographic signal) is a measure of its size whereas the intensity of the band is a measure of the number of copies of sequences that hybridize.

Figure Northern blots Northern blots are similar to Southern blots except that the nucleic acid being analyzed is RNA instead of DNA. The RNA is denatured before electrophoresis, so that gel does not need to be pretreated before transferring the RNA to the filter. The probes are often cloned genes, so that information is obtained on expression of genes and on the size and relative amounts of transcripts. Dot/Slot blots In dot blots, DNA or RNA in solution is spotted onto filters and allowed to dry. For slot, blots, the procedure is same except that the nucleic acid is applied through a slot-shaped template. The nucleic acid is denatured and bound firmly to the filters. Hybridization is carried out with labeled probes. The autoradiograph shows which dots contain sequences complementary to the probe. Dot blots are faster to set up than Southern or Northern blots because gels do not have to run and the transfer of nucleic acid to the filter is far quicker. However, dot blots give no information on the size and number of different sequences contributing to the hybridization signal. Dot blots are well suited to analysis of many samples at once and have the added advantage that it is easy to prepare replicate filters. In situ hybridization In situ hybridization is a powerful method used to locate nucleic acid sequences in histological and cytological preparations of tissues, organelles, cells and chromosomes. Before hybridization, samples are pretreated in such a way that, ideally:

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The morphological features of the tissue/cell/chromosome are retained; The nucleic acid is neither extracted nor modified; The localization of the nucleic acid is unchanged. The probes are detection agents gain access to the nucleic acid.

Applications include identifying the location of genes on normal and aberrant chromosomes; identifying the sites of gene expression; determining levels of transcription and how these change with development and detecting viral pathogens. In situ hybridization can be used to detect two or more target nucleic acids simultaneously and can detect both a particular transcript and the protein encoded by it. The method is particularly suited to detecting a sequence that is present in only a few cells within a large population, for example chromosomal translocations in residual disease. For many applications such as detecting genes on chromosomes, the use of sophisticated microscope is required and may be beyond the resources of many laboratories. 5. Explain in detail about PCR. Gene Amplification Through Polymerase Chain Reaction The polymerase chain reaction (PCR) technique, developed by Kary Mullis in 1985, is extremely powerful. It generates microgram (g) quantities of DNA copies (upto billion copies) of the desired DNA (or RNA) segment, present even as a single copy in the initial preparation, in a matter of few hours. The PCR process has been completely automated and compact thermal cyclers are available in the market. The PCR utilize the following: (1) a DNA preparation containing the desired segment to be amplified, (2) two nucleotide primers (about 20 bases long) specific, i.e., complementary, to the two 3-borders the sequences present at the 3 ends of the two strands) of the desired segment, (3) the four deoxycyctidine triphosphate, viz. TTP (thymidine triphosphate), dCTP (deoxycytidine triphosphate), dATP (deoxyadenosine triphosphate) and dGTP (deoxyguanosine triphosphate), and (a) a heat stable DNA polymerase, e.g. Taq (isolated from the bacterium Thermus acquaticus), Pfu (from Pyrococcus furiosus) and Vent (from Thermococcus litoralis) polymerases. Pfu and Vent polymerases are more efficient than the Taq polymerase. Procedure of PCR: At the start of PCR, the DNA from which a segment is to be amplified, and excess of the two primer molecules, thefour deoxyriboside triphosphates and the DNA polymerase a mixed together in the reaction mixture that has appropriate quantities of Mg2+. The following operations are now performed sequentially. Denaturation: The reaction mixture is first heated to a temperature between 90 980 C (commonly 940 C) that ensures DNA denaturation. This is the denaturation step. The duration of this step in the first cycle of PCR is usually 2 min at 940 C. Annealing: The mixture is now cooled to a temperature (generally, between 40 600 C) that permits annealing of the primer to the complementary sequences in the DNA. As a rule, these sequences are located at the 3 ends of the two strands of the segment to be amplified. This step is called annealing. The duration of annealing step is usually 1 min during the first as well as the subsequent cycles of PCR. Since the primer concentration is kept very high relative to that of the template DNA, primer template hybrid formation is greatly favoured over reannealing of the template strands. Primer Extension: The temperature is now so adjusted that the DNA polymerase synthesizes the complementary strands by utilizing the 3 OH of the primers; this reaction is the same as that occurs in vivo during replication of the leading strand of a DNA duplex. The primers are extended towards each other so that the DNA segment lying between the two primers is copied; this is ensured by employing primers complementary to the 3 ends of the segment to be amplified. The duration of primer extension is usually 2 min at 72 0 C. Taq polymerase usually amplifies DNA fragments of up to 2Kb; special reaction conditions are necessary for the amplification of longer segments. The completion of the extension step completes the first cycle of amplification; each cycle may take few (ordinarily 4 5) minutes. It should be noted that extension of the primer continues till the strands are separated during the denaturation step of the next PCR cycle. Therefore, the products of this extension step, and indeed of every cycle based on the original template DNA, is of indefinite length: this PCR product is usually called the long product. At the second cycle of PCR primers will anneal to the long product much before its 3- end , where sequences complementary to them are located. Extension step in the cycle will produce a product that will be much shorter than the long product; this is the correct PCR product and represents the target sequence. The original template sequence

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Figure: A schematic representation of the three steps performed during PCR and their consequences. Note that the two primers used are complementary to the 3-end sequences of the DNA segment to be amplified. The product of the first cycle is the long product

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Figure: During second and subsequent cycle, the long product accumulates linearly, while the correct copy of the target sequence increases exponentially. At the end of 4th cycle, there are only 8 long product strands, as compared to 22 strands of the correct product. Will also be copied during the second and all subsequent cycles to generate the long product. Therefore, the long product continues to increase linearly, while the correct PCR product will multiply exponentially. The next cycle of amplification is initiated by denaturation (Step 1), which separates the newly synthesized DNA strands from the old DNA strands. This step is usually of one minute as against 2 min in the first cycle. Annealing allows the primers to base pair with both the new and old strands, the total number of strands being twice their original number. Synthesis of new strands takes place, which doubles the number of copies of the desired DNA segment present at the end of step 1. This completes the second cycle. Thus at each cycle, both new and old strands anneal to the primers and serve as templates for DNA synthesis. As a result, at the end of each cycle, the number of copies of the desired becomes twice the number present at the end of the previous cycle. Thus at the end of n cycles 2 copies of the segment are expected; the real values are quite close to but lower than this expectation. Usually, 30 -45 cycles are carried out in most PCR experiments. In case of automated PCR machines, called thermal cyclers, the researchers has to only specify the number and duration of cycles, etc., after placing the complete reaction mixture for incubation, and the machine performs the entire programme of operations precisely. After PCR cycles, the amplified DNA segment is purified by gel electrophoresis and can be used for cloning. DNA sequencing, etc. But Taq polymerase does not have proof reading function. Therefore, PCR product have much higher mutations (base substitutions) than DNA obtained by in vivo replication. This aspect has to be guarded against while using PCR products. Variations of PCR. PCR is a highly versatile technique; it has been modified in a variety of ways to suit specific situations and applications. Some important variations of PCR are as follows.

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1.

PCR can be used to amplify the sequences flanking (located on either side of) a segment the border sequences of which are known. This is done by using as primers oligonucleotides complementary to the 5 ends of the segment in question. This orients the free 3- OH of the primers outward of the sequences, as a result, the newly synthesized chain grows away from the borders of the concerned segment. This is called inverse PCR. The target DNA is cut with a restriction enzyme that produces sticky ends cuts at unknown sites on either side to the known region. The restriction fragment is allowed to circularize: is ligated, and then opened with an enzyme that cuts only within the known region. The resulting linear fragment now has the known region on both its ends and it used for PCR. When sequence of only one end of the desired segment or gene is known, the primer complementary to the 3 end of this strand is used to produce several copies of only one strand of the gene. Now a poly G (or any other homopolymer) tail is added to the 3- ends of the single strand DNA copies produced by PCR. This allows the use of complementary homopolymer, poly C , to be used as primer for copying the DNA single strands generated by PCR, and gives rise to the complete DNA duplex that can be amplified normally. This approach has been termed as anchored PCR. The above variation can be used to amplify RNA sequences into DNA duplexes. The mRNA is first copied into a single cDNA strand using a short poly T as primer, which pairs with the 3 poly A tail of the mRNA strand. A poly G tail is now added to the 3 end of cDNA single strand by the enzyme terminal nucleotide transferase. The cDNA single strand now has a poly T sequence at one end (5 end) and a poly G sequence all the other (3 end). We now use an oligo C as an primer to copy the single strand cDNA into cDNA duplex, which is amplified using oligo T and oligo C as primers. This procedure is called RT PCR (reverse transcriptase PCR) In another variation the recognition sites for a given restriction endonuclease are added to the 5 ends of the two primers used in PCR for amplification of the desired DNA segment. The recognition sequences would remain as single strand tails when the primers are annealed to the original template DNA. But the amplified copies of the desired segment acquire built in known terminal endonuclease sites; this is very useful during subsequent cloning of the amplified segment. PCR can be used to generate single strand copies of a DNA sequence which can be directly used for DNA sequencing (Appendix 2.IV). To achieve this objective, the quantities of the two primers for the 3 borders of the segment are so adjusted in the reaction mixture that one of them is exhausted about 10 or so cycles before the PCR is terminated (The two primers may be used in, say, a 100: 1 ratio). As a result, in the terminal 10 or so cycles only a single strand of DNA segment is copied; these single strand copies are the ideal starting materials for DNA sequencing. This variation is known as asymmetric PCR.

2.

3.

4.

5.

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Figure: Use of PCR to produce a large number of cDNA copies of an Mrna; it is expected that the mRNA preparation is of high purity. This procedure is called RT PCR. 6. PCR can be used to induce desired mutations at specified sites in a gene (site directed mutagenesis). For this, sequence of the side (for a stretch of 20 bp or more) where mutation is to be induced must be known. This sequence is used to construct the primers used for PCR; mutations are induced by making the primers with desired changes in their base sequence. These primers with modified base sequences are used for PCR amplification of the desired gene; the new copies of the gene therefore contain at the appropriate sites the modifications induced in the base sequences of the primers used for PCR. Therefore, it is easiest to induce mutations in the ends of the gene sequences of which are used as primers. But modifications can also be induced within internal sites of a gene using the overlap extension variation of PCR.

Applications of PCR. PCR has many exciting and varied applications. Some of these are briefly outlined below. 1. PCR can be used to amplify a specific gene present in different individuals of a species and even in different somatic cells or gametes, say, human sperms. These copies can be used for cloning. Alternatively, they can be sequenced to obtain information on the mutational changes in the genes of different individuals, cells or gametes. Such data can be used in disease diagnosis, population genetics, estimation of recombination frequencies etc. PCR has been used to study DNA polymorphism in the genome using known sequences as primers. Synthetic nucleotides of any sequence can be used as random primers to amplify polymorphic DNAs having sequences specific to the primers used. Such an application of PCR generates random amplified polymorphic DNA (RAPD), pronounced as rapid), which is detected as bands after electrophoresis. RAPD bands of different strains or species can be compared. They can be used to construct RAPD maps, similar to RFLP map (Appendix 4. III) PCR can be used to detect the presence of a gene transferred into an organism (transgene) by using the end sequences of the transgene for amplification of DNA from the putative transgenic organism. Amplification will occur only when the trangene is present in the organism; the amplified DNA is detected as a band on the electrophoretic gel. Microdissected segments of chromosomes, e.g., of salivary gland chromosomes of Drasophila, can be used for PCR amplification to determine the physical location of specific genes in chromosomes.

2.

3.

4. 5.

PCR can be used to determine the sex of embryos. Thus the sex of in vitro fertilized cattle embryos could be determined using Y chromosome specific primers before their implantation in the uterus. PCR Versus Gene Cloning: The PCR is a revolutionary technology. It is more efficient (as it needs much less amount of the desired DNA; a single copy is enough) (2) does not require difficult to store and costly restriction enzymes, ligase, and vector DNA, which reduces the cost drastically; (3) needs for less work, time and skill; (4) and has many more applications than gene cloning. Typically, gene cloning experiments take 2 4 days, while PCR takes up to 4 -5 hours. In addition (5) PCR is fully automated, while gene cloning is not. But for PCR one does need sequence information for construction of primers, and a thermal cycler (cost, around Rs. 2 lac) or PCR machine. It is expected that PCR will eventually take over most of the applications of gene cloning and will find many novel applications as well.

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UNIT III PART A 1. Define vaccine. An agent that is used to promote an antibody response that protects the organism against infection; confers immunity to disease. Examples include protein DNA, living or non living cirrus or micro organism. 2. Define gene therapy. Gene therapies are aimed at treating or eliminating the causes of disease, where as most current drugs treat the symptoms. The use of genes to correct a genetic or acquired disorder. 3. What are the techniques is gene therapy? 1. 2. Somatic cell gene therapy Germ line gene therapy

4. What are the basic steps is gene therapy? 1. 2. 3. 4. The faculty gene that causes a specific disease must be identified. The location of the affected cells must be pinpointed. A healthy version of the gene must be available The healthy gene has to be delivered to the cell.

5. What are the types of vaccines? 1. 2. 3. Dead bacteria or inactivated viruses Live non virulent or weakened (attenuated) bacteria / or viruses Viral fragments or bacterial molecules (subunit vaccines).

6. What are the steps involved is production of antibodies? 1. Immunization 2. Cell fusion 3. Selection of hybridisms 4. Screening the products 5. Cloning & propagation 6. Characterization & storage 7. How the therapeutic use of monoclonal antibodies classified? 1. 2. Direct use of monoclonal antibodies as therapeutic agents Monoclonal antibodies as targeting agents.

8. Give 2 gene transfer vectors with its advantages and disadvantages. 1. Adenovirus Advantage 1. Very high transfection efficiency exvivor and in vior 2. Transfection proliferating and non proliferating cells 1. Fairly prolonged expression 2. High transfect ion efficiency exviur. Disadvantage 1. Repeat dosing ineffective ouring to strong immune response. 2. Insert size limit of 7.5 kb 1. Low transfection efficiencies inviur 2. Insert size limit of 8 kb.

2. Retrovirus

9. What is the use of radio labelled monoclonal antibody? They have been developed for delivery of a cytotoxic effecter to target cells and for radioimaging. The advantage of using radiolabelled antibodies is that they can kill cells from a distance and thus can also kill cells adjacent to antigen expressing cells. 10. Define cytokines. Extra cellular signaling proteins or peptides produced by the immune system and other tissues that are involved in cell cell communication, includes interferons colony stimulating factors, interleukins and tumor neurosis factors.

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PART B 1. Explain about monoclonal antibodies in therapy. Therapeutic Applications: Such an application involves the use of Mabs for either treatment of or protection from a disease. Some important developments in this area are briefly listed below. 1. Antibodies specific to a cell type, say, tumour cells, can be linked with a toxin polypeptide to yield a conjugate molecule called immunotoxin. The antibody component of immunotoxin will ensure its binding specifically and only to the target cells and the attached toxin will kill such cells. Immunotoxins having Ricin have been prepared and evaluated for killing of tumour cells with considerable success. Ricin is a natural toxin found in the endosperm of castor (Ricinus communis). It has two polypeptides cells A (toxin peptide) and B (a cell binding polypeptide, lectin). Ricin A polypeptide enxymatically and irreversibly modifies the larger subunit of ribosomes (in fact, their EF2 binding site) making them incapable of protein synthesis. This toxin is effective against both dividing and nondividing cells as it inhibits protein synthesis. Antibody Ricin A conjugate has been shown to reduce protein synthesis in mouse B cell tumours; the antibody used in the conjugate was specific to the antigen molecules present ont eh surface of target tumour cells. It is noteworthy that this immunotoxin did not bind to either other tumour cells or the normal cells. The same principle has been used to deliver radioactivity specifically to target tumour cells. In such cases, radioactivity due to131 I (iodine), 90Y (yitrium), 67Cu, 212Pb, etc is incorporated into the tumour specific antibody in the place of toxin. Radio labelled antibodies have been used in patients having hepatoma, HTLV 1 (human T Cell leukaemia / Iymphoma virus 1), ATL (adult T cell leukaemia), etc. 2. B-lymphocyte proliferation, maturation and antibody secretion are dependent on interleukin w produced by activated T lymphocytes. Further, Tc cells mediate graft rejection. Thus an effective strategy to minimize the refection of grafts from other individuals would be to eliminate the T cells from their bone marrow / circulatory system (blood stream) by using T cell specific Mabs. T cells exhibit several antigens of which CD3, CD4, CD8, etc., have been the preferred targets for Mab development. In bone marrow transplantation, the bone marrow cells of the recipient are inactivated by appropriate irradiation. The donor bone marrow cells are treated with T cell specific antibodies to destroy the T cells present in them; the remaining cells are then transplanted into the recipient. Experiments with mice have shown remarkable success. In order to minimize tissue graft refection, the T cells present in circulatory system of the recipient are eliminated prior to the transplant by an administration of T cell specific Mabs. This treatment abolishes, though temporarily, the ability of recipient to mount immune response against any foreign antigen, including those present in the graft tissue. Mab OKT3 is the most widely used for treatment of acute cases of rejection of kidney transplants.

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Figure: A schematic representation of the principle of action of an immunotoxin containing the A polypeptide (toxin polypeptide) or Ricin, a seed protein from castor (Ricinus communis) 3. Mabs can be administered to provide passive immunity against diseases. in case of active immunity the immunized individual itself produces the antibodies against the concerned pathogen. But in case of passive immunity, antibodies produced elsewhere are introduced into the body of an individual to provide immunity against a pathogen. Mabs are very useful in the purification of antigens specific to pathogens; these purified antigens are used as vaccines.

4.

2. Explain vaccines and their application is animal infections. Vaccines: Prevention of diseases is the most desirable, most convenient and highly effective approach to health; this is achieved by vaccination or immunization using biological preparations called vaccines. A vaccine is a preparation containing a pathogen (disease producing organism) either in attenuated or inactivated state. This preparation is introduced into an individual to induce adequate antibody production against the pathogen, in particular, so that the individual becomes protected against infection, at a later date, by that pathogen. The introduction of a vaccine in an individual is called vaccination / immunization as it leads to the development of immunity in the vaccinated individuals to the concerned pathogen. Any molecule that induces production of antibodies specific to itself when introduced in the body of an animal is called antigen. Usually antigenic function is confined to a rather small porting of the antigen molecules known as antigenic determinant or epitope. When an individual is vaccinated, the antigens of pathogen origin stimulate antibody production against themselves. This increase in antibody production takes some time, but since the vaccine does not have virulent pathogens there is no danger of disease development. When live virulent form of the same pathogen later enters into the system of an immunized animal, the high level of antibodies specific to the pathogen inactivate the pathogen, and thereby protect the animal against the disease. The various vaccines can be grouped into 2 categories: 1. 2. Vaccines containing killed or inactivated pathogens. Those containing live but attenuated pathogens.

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Attenuation means a drastic reduction in the virulence of a pathogen; this is achieved in one of the following ways. Several consecutive passages through an animal which is not the usual host of the pathogen, e.g., smallpox virus in calf. Several passages through cultured cells of the host, e.g. rabies virus in human diploid cell culture, or of a different species; yellow fever viruses in chick embryo cell culture. Selection of less virulent strains of pathogens, e.g. a mutant strain of polio virus. Treatment of the pathogen with some chemicals, e.g., BGG vaccines produced by culturing the bacteria on a medium containing bile. Culturing pathogens under unfavourable conditions like high temperature, e.g. anthrax vaccine obtained by cultivation of the bacterium, Bacillus anthracis, at 40 500 C. In general, inactivation of viruses is always coupled with attenuation to minimize the accidental presence in vaccines of active virulent particles, which could cause disease in the vaccinated individuals. The different vaccines differ in their composition, efficacy and duration of effective protection to the earliest examples of biotechnological intervention in human and animal health care. Vaccines offer the cheapest and most effective protection against diseases, and for some diseases, e.g. hepatitis B, AIDS, etc., they are the only means of protection. The effectiveness of vaccines may be highlighted by the success of WHO sponsored mass vaccination against smallpox in completely wiping out this once ravaging disease from the face of earth. Recombinant vaccines: A recombinant vaccine contains either a protein or a gene encoding protein of pathogen origin that is immunogenic and critical to the pathogen function; the vaccine is produced using recombinant DNA technology. The vaccines based on recombinant proteins (= proteins produced by recombinant DNA technology) are also called subunit vaccines. Genes that encode desired antigen in the immunization process are introduced into the non pathogenic organism for a mass production of the proteins. The concerned proteins are then purified and mixed with suitable stabilizers and adjuvants, if required, and used for immunization. A subunit vaccine has no potential for reverting to a pathogenic organism because only limited number of components from the original pathogen is present in the vaccine. A subunit vaccine should also have fewer side effects, as it does not have any infectious agents. Steps in subunit vaccine production: Identify protective proteins or epitopes on the proteins. Once this is done, an individual can either produce a subunit vaccine by rDNA technology or by synthetic peptide technology. Identify gene coding for the protein Clone the gene coding for the specific protein and express it in a suitable expression system. Purify the protective protein to homogeneity using bovine herpes virus 1. BHV 1 has four glycoproteins GVPI, GVPII, GVPIII and GVPIV. Immunosorbent columns with monoclonal antibodies are prepared and used for purification of large quantities of the BHV I glycoproteins. These glycoproteins are then mixed with the adjuvant avidine and used to immunize animals against BHV I virus.

The host organism used for expression of immunogenic proteins to be used as vaccines may be a genetically engineered micro organism. Hepatitis B virus core protein is planted in E. coli, yeast, Bacillus, etc. AIDS viral coat protein is engineered in vaccinia virus; foot and mouth disease (FMD) core protein is planted in E. coli. Malarial parasites and syphilis organism which are difficult to be grown has been planted in E. coli. Myrin et al., have developed a more effective and less toxic cholera vaccine. It gives immunity equivalent to infection with natural bacterium. It was genetically altered Vibrio cholerae cells of EIT / Incaba strain. Side effects like diarrhea can be eliminated by scaling the does. Malarial vaccine from Hoffmann La Roche of Switzerland is on the horizon. Surface antigen in sporozoite stage has been identified and isolated to give protection. Antigens and concerned genes from organisms in other parts of parasitic life cycle are under search, for the preparation of a multiple vaccine, which may give a broader spectrum protection against malaria. DNA of spirochaete (Treponema pallidum, the causative agent of syphilis) has been isolated from infected rabbit testicles and is cloned in E. coli using vector coliphase. This has enabled development of a more specific diagnostic screening test for syphilis and manufacturing of an effective vaccine. Need for such vaccine is due to the difficulty of extraction and purification of antigen by conventional techniques, on the one hand, and Treponema pallidum becoming resistant to antibiotics in current use and, therefore, difficulty faced in the treatment of syphilis,

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on the other. Foot and mouth disease (FMD), a highly contagious disease in cattle, sheep and swine worldwide, is caused by the virus with a single stranded RNA. German scientists have successfully cloned double stranded DNA copies of viral RNA into plasmid pBR 322 and grown in E. coli. It was found that more than 100 molecules of viral proteins were synthesized per bacterial cell with a positive serological test. FMD virus is having a capsid with 60 copies of 4 polypeptides GVP I, GVP II, GVP III and GVP IV. Treatment with trypsin results in the cleavage VP I and considerable decrease in infectivity and loss of immunizing activity. Synthesis of VPI protein precursor is being attempted to give long lasting immunity. Rebies is a zoonosis endemic in animals and humans in several countries of Africa, South America and Asia and is increasing in Europe. Virus propagation is difficult, so the cost of vaccine production is high. The virus has to be propagated in human cells. Rabies virus protein encoding gene is now cloned in E. coli, thus producing cheaper vaccine will be possible. Hepatitis B virus antigen producing gene can be cloned in E. coli to produce antigen and the vaccine. But with E. coli, complications can be there due to the risk of contamination by endotoxins. Hardy et al (1981) have described the production of Bacillus subtilis with both core antigen of hepatitis B virus and major antigen (VP I) of FMD virus. Gene coding for a hepatitis B virus surface antigen is introduced into bakers yeast also. This antigen can provide a strong immune response. Yeast cells are then cultured in large fermentors several days during which time the cells rupture. A vital part of the process is proprietary chemical process, which assembles the antigenic protein into a circular particle and resembles the natural molecule closely. About 12 million people suffer from leprosy. In India, 300,000 new cases are added each year. To eliminate the disease, an effective vaccine in large quantity will be required. Mycobacterium leprae, the causative agent of leprosy s difficult for cultivation in laboratory and so difficult is vaccine production. Scientists are on the way to identify important antigens of M. leprae and introduce them in E. coli by genetic engineering. Three leprosy vaccines are already on trial in India. There has been significant progress on four of the five vaccines against agents that cause diarrhea rotavirus, Salmonella sp., cholera organisms and shigella dysentriae. Other diseases for which vaccines based on antigens produced through genetic engineering are being attempted and are in the various stages of development and / or trial, and AIDS, influenza, meningitis, herpes, rabies, pertussis, measles and poliomyelitis, tuberculosis, rheumatic fever, pneumococcal infections. Vaccines designed to control fertility, based on genetically engineered products such as beta chain of the hormone human chorionic gonadotrophin (HCG), are also under investigation. The antibodies produced inactivate the hormone and the uterus does not accept the fertilized egg. Birth control vaccine developed in India is proved to the safe and devoid of any side effects. HCG currently used for vaccine production is from the trimester urine of a pregnant woman through a procedure that is difficult and expensive. Cheap vaccine for schistosomiasis, which affects about 250 million people in the tropical regions of the world every year, is on the horizon. There is also progress in making a genetically engineered vaccine for dengue fever. Dengue fever is a mosquito borne viral disease endemic in Asia, South Africa and South and Central America. The discovery of a unique HB antigen called Australia antigen (HBs Ag) in 1965 in the blood of infected persons has lead to the development of two effective vaccines against this dreaded disease: a plasma derived vaccine in 1981, and a more acceptable recombinant DNA technology based yeast derived vaccine in 1986. Dr. Baruch Blumberg received Nobel Prize in 1976 for his pioneering contributions to HB. The HBs Ag is present in the serum of infected people as small particles of 22 nm size. These nucleic acid free particles have no infectious properties. The blood serum taken from an acute case of HB contained enough HBsAg to accord at least this partial protection. In 1981, Through this concept, HB vaccine was made available by Merck and Co., USA and Institute Pasture Production, Paris as Hepatavax B. The second generation of HB vaccine has been developed by employing the techniques of genetic engineering. The fragments of DNA are jointed to the DNA of a suitable vector. The vector may be a plasmid, phage or a cosmid. This is done by the use of restriction endonucleases. Artificial linkers containing restriction sites can be attached to the foreign DNA fragments to allow insertion into the vector. Once joined, the composite DNA is introduced into the bacteria or eukaryotic cell system either by transformation or transfection. After successful integration of the composite genome into that of the host cell, it affects the metabolism of the host cell. This further directs the translation of additional genetic information packed in vector DNA, and consequently leads to the synthesis of foreign proteins by the host cells. In this way, the host cell acts as a mini factory for the production of specific protein which is foreign to it.

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3. Explain about gene therapy in animal diseases. Results of Animal Gene Therapy: There have been experiments on animal models as a prerequisite to experiments on humans. There have been several animal systems for testing human gene therapy made. Retroviruses have been successfully used to target genes into animal cells for several years (Thomas, 1986) and foreign genes have been expressed in mice using treated cells (Bernstein et al., 1986; Keller and Wagner, 1986). Larger animal models are under study to determine the persistence of infected bone marrow cells in transplanted animals (Anderson et al., 1986, Kwork et al., 1986). Various types of retrovirus vectors have been developed. Human bone marrow cells have been infected and have expressed inserted drug resistance genes (Miller et al., 1986). There was expression of the human ADA gene in murine cells in vivo (Williams et al., 1986). One group of scientists placed the human gene for the production of ADA into the bone marrow into the rhesus and macaque monkeys and foetal sheep in preliminary experiments (Anderson et al., 1986). Several experiments using SCID mice and human ADA deficient lymphocytes expressing as inserted ADA gene have been successful, as described. Other groups continued to work on procedures to infect haematopoietic stem cells, for diseases such as ADA deficiency (Cournoyer et al., 1990). These are the reasons for optimism with the approved gene therapy trial; it is based on the years of preliminary work by many groups. The human globin gene has been expressed in mice that were treated with retrovirus infected stem cells, so that the expression problems in stem cells may be soon overcome (Dzierzak et al., 1988) another experiment in mice was to test a model for treating retinoblastoma in tumour development. Both positive acting tumour suppressor genes need to be mutated. Both alleles of a suppressor gene need to be turned off before cancer can proceed. In gene transfer studies the suppressor gene function for retinoblastoma could be reinserted and tumor cell formation repressed preventing the tumour formation (Freidmann, 1990 b). This work may be extended to humans. There is a contrast between diseases that affect the lymphoid cells and diseases that affect the haematopoietic system. The haematopoietic cells have a short life and so cannot be useful targets of gene therapy. One of these diseases is Gauchers disease. Human cells have expressed the genes for glucocerebrosidases needed for the correction of Gauchers disease (Choudary et al., 1986; Sorge et al., 1986) it is another disease with no known cure, which affects 0.2% of people born (2% of Jewish population). The absence of glucocerebrosidase results in the accumulation of non degraded membrane lipoprotein within the reticuloendothelial cells. An appropriate in vivomodel for the detection and measurement of pluripotent haematopoietic setm cells does not yet exist as further experiments are needed. But there is some progress (Parkman and Kohn, 1990). One of the positive outcomes of extensive animal trials has been the development of effective and stable retroviral vectors for gene transfer. There are alternatives to retroviral vectors being developed, such as methods of targeted modification of human genes, and these will soon be possible. Promising alternatives to retroviral vectors are being developed, such as the use of laser micropuncture of the cell membrane to facilitate direct gene transfer. This technique has been used on cultured human cells with an efficiency 100 fold more than the standard calcium phosphate method of DNA transfer in which cells are passively incubated in the DNA solution. The efficiency of the physical methods is about 1% of the target cells that incorporate the genes. If there are no naturally occurring animal models for some human genetic diseases, targeted gene mutagenesis can be used to create diseased animals. Mice have been genetically engineered using embryonic stem cells to be HPRT deficient mice. These were hoped to provide mouse equivalents of humans suffering from Lesch Nyhan disease. Genetic therapy has been tried on them, as preliminary experiments for human gene theraphy, correcting the gene deficiency. There have been a variety of trials in animals. There is some progress on gene transfer methods. Lipoprotein vesicles using the substance lipofectin has been used to insert genes into the neuroepithelium of frog embryos, as well as into skeletal muscle cells of young mice. The biolistic approach used in plants for gene transfer is being applied to animals, and will also be used in the gene therapy trial on ADA gene insertions in humans, which has been approved. After conception, the genotype may be normal, without a genetic disease, or abnormal, with a genetic disease. There are several stages at which therapy could occur, germ line gene therapy must occur in the very early embryo. After this stage, somatic cell therapy can be performed, before or after birth. Symptomatic therapy usually is given after birth. But with modern medicine, symptomatic therapy may also be given before birth in some diseases. The knowledge of the biochemical basis of genetic diseases is often essential in order to develop a reasonable therapy. In the majority of genetic diseases, there is some therapy available and in some cases the therapy can effectively restore normal health in spite of the continued presence of the abnormal genotype.

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Protein replacement Symptomatic therapy involves using the normal gene product as a substitute for the defective gene product. One of the first diseases to the treated this way was diabetes mellitus. Diabetes is due to inadequate production of the hormone insulin. The treatment varies from just a diet which is low in carbohydrate, to taking regular amounts of the hormone insulin to ensure a sufficient level in the body for normal function. Insulin is a relatively simple molecule so it has been produced in large amounts for some time. It can be obtained from animal tissue extraction, usually pigs, or the exact human protein can now be produced from genetically engineered bacteria. Dietary treatment substitutional therapy is not successful in every case, as we may not be able to administer the defective gene product or enzyme from outside the body. In phenylketonuria (PKU), the defective enzyme is localized in the liver and a substitute can be inserted. However, the disease can be successfully treated by a dietary treatment involving as reduction in the intake of phenylalanine. This is possible because the disease only affects people due to the accumulation of an abnormal toxic product derived from a specific substance in the diet, so these people can live otherwise normal lives if they omit this substance from their diet.

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UNIT IV PART A 1. Define micromanipulation. It involves in vitro micro surgically assisted fertilization producers. This is required when the sperms are unable to penetrate the zone apothecia of acolyte and fertilize. Micromanipulations are usually done in sever cases of male factor infertility.

2. What are approaches for the artificial breeding of animals? 1) 2) 3) 4) Artificial insemination Embryo Transfer In vitro fertilization Embryo cloning.

3. Define artificial insemination. As the male produces millions of sperms daily, the semen can be used to produce several off springs. This is made possible by artificial insemination (AI) of females. Artificial insemination is done on standing animals through a technique, know a rectal palpation. 4. Define Embryos transfer. The process of implantation of embryos from a do mar animal, or developed by in vitro fertilization into the interes of a recipient animal. 5. Define In vitro fertilization. The fertilization of eggs of a female in the laboratory conditions and transferring the fertilized eggs (zygotes) in to the inters a few days later. 6. What are the limitations of IVF? 1) 2) 3) 4) 5) Genetic defects in oocytes Genetic defect in fertilizing sperms Environmental mutagenesis Inadequate supply of nutrients and hormones Exposure to toxic agents and free radicals

7. What are the limitations of embryo transfer? The supply of embryo from super ovulated donors is limited. Freezing and thawing of care to keep them functionally intact. Embryo transfer requires technical skill, besides high cost factor.

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8. Define Germ cell. A reproductive cell which on maturation can be fertilized to produce the organism. 9. What are the steps in IVF? 1) 2) 3) 4) 5) Induction of super ovulation Monitoring of ovarian response Oocyte retrieval Fertilization invitro Embryo transfer.

10. Define super ovulation. Super ovulation means induction of multiple ovulation by application of exogenous hormones in the early follicular or in the luteal phase of the estrus cycle, in order to collect a large number of fertilized eggs. PART B 1. Explain in detail about the steps involved in Embryo transfer technology. Embryo Transfer Technology The first successful transfer of fertilized rabbit eggs was reported in 1931 by Walter-Heape at Cambridge (UK). In farm animals embryo transfer was successfully done for the first time in 1934 in sheep and goats (Warwick et al., 1934). The first offspring after transfer of embryo in cattle and swine were reported in 1951 (Willet et al., 1951). Embryo transfer includes a sequence of various steps which are (a) selection of donors, (b) induction of super ovulation, (c) embryos collection, (d) evaluation of embryos, (e) selection of recipients and (f) transfer of embryos. Until now, embryo transfer techniques have been extensively used in cattle and more sporadically in sheep, goats and pigs. Selection of donor Regularly cycling cows or heifers can successfully respond to superovulatory treatment and be used for embryo transfer. The following selection criteria for donors will ensure a good probability of producing embryos of high quality. 1. 2. 3. 4. 5. 6. Between the ages of 3 and 10 years Free of genetic diseases and conformational abnormalities Exhibit regular oestrus cycle Shows superior production traits of economic importance Free from infectious diseases Previous sound reproductive performances including no more than two inseminations per conception.

Induction of super ovulation Super ovulation means induction of multiple ovulation by application of exogenous hormones (PMSGpregnant mare serum gonadotrophin; FSH follicle stimulating hormone; HMG-human menopausal gonadotrophin) in the early follicular or in the luteal phase of the oestrus cycle, in order to collect a large number of fertilized eggs. Most frequently PMSG or FSH is used. Only a single injection of PMSG (2000 to 3000 IU) is sufficient as the substance has a long half life time. In contrast, multiple injections of FSH (35-50 mg) are required to induce superovulatory responses (twice daily injections over four days). HMG (1000-1500 IU) also requires multiple injections; however, the drug is more expensive and only rarely used. Superovulatory response, embryo quality and fertility after treatment with different gonadotrophins in native Mertolengo cattle were studied (Lopes da Costa et al., 2001). In one experiment, superovulatory response (SR), embryo quality and plasma progesterone (P4) levels between donors treated with eCG treated donors. Fertilization rates were significantly higher in bred than in inseminated donors. Plasma P4 levels were only significantly different (higher) between responder and non-responder donors on the day of embryo recovery. Forty eight to sixty hrs after beginning the gonadotrphin treatment, prostaglandins (PGF 2) are administered to induce oestrus. Inseminations are performed at oestrus and the embryos are recovered 6-8 days

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after insemination. On an average, only one third of the super ovulated bovine donors show a good ovarian response. Embryo collection In bovines, embryos are recovered by non-surgical methods. Special catheters are introduced via the cervix into the uterine horn and embryos are flushed with 250 to 300 ml of flushing medium (phosphate buffered solution with 1-2% fetal calf serum). In the cow, the embryos usually enter the uterus on day four after oestrus. Non-surgical techniques avoid the potential damage of the reproductive tract by adhesions, are repeatable and do not require elaborate facilities. Embryos are collected by non-surgical methods 6-8 days after the onset of oestrus. Prior to day 5, the embryos may be in the oviduct and after day eight they may be hatched (escape from the zona pellucida), difficult to visualize and fragile. At day 7, bovine embryos are about 150-190 in diameter, are still within the zona pellucida and at late morulla or blastocyst stage of development. In case of other farm animals like sheep, pigs and goats, embryo recovery is performed by surgical methods. The adomen of the donors is opened by midline cut and embryos are recovered by flushing oviduct and/or uterine horns. Embryos are collected 4-5 days after insemination or mating. Evaluation of embryos After recovering ova and embryos from the flushing medium, their further development capacity has to be evaluated. Morphological criteria in embryo evaluation are shape, colour, number and compactness of cells, size of the perivitelline space, number and size of viscles and the zona pellucida. Embryos are classified into 4 groups: (a) (b) (c) (d) Excellent embryos embryos in the appropriate developmental stage with a perfect morphology. Good embryos embryos in the appropriate stage of development with slight morophological deviations. Degenerated and/or retared embryons. Unfertilized ova.

Selection of recipients The normal physiology and health conditions, the reproductive status, lack of any reproductive disorders, compatibility of the donor with respect to the size of the foetus and oestrus synchronization are of considerable importance. In bovine, it has been proved that heifers and young cows are best suited as recipients. The oestrus of donors and recipients should be synchronized within 24 hrs. Spell et al. (2001) studied the effects of corpus luteum characteristics, progesterone concentration, donorrecipient synchrony, embryo quality, type and development stage on pregnancy rates after embryo transfer. Of the 526 recipients presented for embryo transfer, 122 received a fresh embryo and 326 received a frozen embryo. Pregnancy rates were greater with fresh embryos (83%) than frozen-thawed embryos (69%). Pregnancy rates were not affected by embryo grade, embryo stage, donor-recipient synchrony of the palpated integrity of the CL. There was a significant, positive simple correlation between CL diameter ort luteal tissue volume and plasma progesterone concentration. Transfer of embryos Special catheters are available to perform non-surgical transfer in cattle. It is important to transfer the embryos into the tip of the uterine horn without damaging the endometrium. In cattle, after superovulatory treatment 8-15 follicles are detected at oestrus and about 6-11 ovulations on the day of embryo recovery. The average number of embryos recovered is 5-10 representing 60-70% of the number of corpora lutea. Pregnancy rate after transfer of freshly collected embryos is 55-65%, and of frozen/thawed embryos recovered in dairy cattle. About 60-70 of these embryos are considered to be capable of further development. Statistically 25 to 3 calves per successful flushing can be produced. The oestrus cycle of the donor and recipient should be closely synchronized if transferred embryos are to survive. Pregnancy rates are generally reduced unless the embryo is placed in the lumen of the uterine horn on the same side of the corpus luteum.

2. Explain in detail about Invitro fertilization & Embryo transfer in animals. In vitro fertilization in farm animals

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Among farm animals, in vitro fertilization has been achieved in the cow, pig and goat; but the number of newborn animals were limited. The occytes are recovered from ovarian follicles or from oviducts near the time of in vitro ovulation following treatment of donors with PMSG and PG. For in vitro cap citation, semen is incubated, treated with high ionic strength medium and subsequently incubated in defined medium prior to insemination of the oocytes. Lambert et al. (1986) produced six calves from a combination of in vitro fertilization and incubation in the rabbit oviduct before transfer. Laparoscopy was used to recover in vivo matured follicular oocytes from super ovulated heifers. The oocytes enclosed in the expanded cumulus were then mixed in vitro for fertilization with fresh semen in a single medium and used at a concentration of 1 x 10 6 sperms/ml. Following the fertilization period (1620 hrs), the embryos were cultured in vitro upto 2 or 8 cell stage. Following in vitro development, the 2 or 8-cell embryos were either transformed into the cow oviduct or the rabbit oviduct. The embryo (8-cell to blastocyts) obtained after incubation in the rabbit were transformed to the uterus of virgin heifers either surgically or nonsurgically. Number et al. (2000) evaluated the difference in birth weight and gestation length between Japanese Black calves obtained from transfer of bovine embryos produced in vitro (IVP) and those developed in vivo (IVD). Both the IVD and IVP embryos were transferred non-surgically to Holstein recipients on day 7 1 of estrus cycle. Results indicated that IVP calves had heavier birth weights than IVD calves, but that the average gestation length of IVP calves was not always longer than that of IVD calves. Further, the birth rate of heavier calves and the incidence of stillbirth and perinatal mortality up to 48 hr post partum in IVP calves were greater than those in IVD calves. The transfer of in vitro fertilized embryos is now practicable, but the laboratory methods of fertilization still need development. Embryo Transfer in Cattle Young embryos of cattle of superior genotype are collected prior to their implantation in uterus, and are implanted in the uterus of other females of inferior genotype where they complete development; this is called embryo transfer. The chief objective of embryo transfer is to obtain several progeny per year from a single female of superior genotype. In a country like India, most cattle are of inferior genotype with rather low productivity, while superior genotype females are limited in number and of high price. Therefore, a programme of artificial insemination (AI) is that superior genes (50%) inferior genes. In contrast, in embryo transfer technique, the inferior females used as surrogated or substitute mothers do not contributed any genes to the progeny; they only serve as extremely sophisticated natural incubators for the normal development of young embryos. As a result, the progeny obtained by embryo transfer are of superior genotype. The technology of embryo transfer in cattle may be briefly summarized as follows. 1. A genetically superior and high productivity female serves as the donor of embryos to be transferred. 2. Healthy, young females of inferior genotype are selected to be the recipients of embryos to be transferred; these females are called surrogate or substitute mothers. 3. The donor females are treated with appropriate doses of the selected gonadotrophin. E.g., follicle stimulating hormone (FSH) or luteinising hormone (LH), to increase the number ova released at the time of ovulation; this is called super ovulation. Under optimum treatment conditions, a single female can provide up to 15 embryos in a single cycle. The chief objective of super ovulation is to greatly increase the number of embryos recovered per female in a single cycle. 4. When the donor female is in heat, it is artificially inseminated using semen from a genetically superior bull of top pedigree. 5. The fertilized eggs/young embryos are collected by flushing the uterus of donor females with a special nutrient solution 7 days after the insemination. The embryos are examine under a stereoscopic microscope and normal looking healthy embryos are selected. 6. The selected embryos are incubated in special nutrient medium at 37oC till their transfer into the surrogate mothers. Alternatively, they may be frozen and stored in liquid nitrogen for future use. 7. A single embryo is transferred into the uterus of each surrogate mother. It is important that the oestrus cycles of donor and surrogate mothers are synchronized by administering prostaglandins to provide the optimum uterine environment for survival, establishment and normal development of the young embryos. Applications of Embryo Transfer Technology 1. This technology achieves a surprisingly rapid rate of multiplication of animals of the selected superior genotype. In natural course, a single female will produce a single progeny in about one year. But using super ovulation and embryo transfer technology, it is feasible to collect around 36 embryos from one female in one year. Assuming an average success rate of 50% in the embryo transfer, an average of 18 progeny can be derived from one superior female in one year. Each young embryo can be split into 2-4 parts, each of which would develop into a separate progeny; this is called embryo splitting. By combining embryo splitting with superovulation, the rate of multiplication can be further increased.

2.

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The young embryos can be frozen and stored in liquid nitrogen (at -190oC; cryopreservation) for up to 10 years or more and used at a subsequent date. The frozen embryos are far more easier to transport, and present negligible quarantine problems as compared to the animals themselves. 4. Superior cows that are unfit to carry the foetus for full term, can server as donors of the young embryos. Limitations 1. 2. 3. A high degree of expertise is required for an efficient and successful embryo transfer operation. The cost of producing each progeny is several-fold higher than that from the natural process. The donor females are removed from production for the period they are used as donors of young embryos.

3.

3. Explain about Embryo splitting and Embryo sexing. Embryo Splitting Mammalian eggs are more tough and can sustain extensive damage and still develop normally. Early cleavage-stage embryos are favoured for manipulation to produce identicals. Embryos from the late morullae upto late blastocyst stage can be collected easily by non-surgical procedures and after splitting into two halves, they can be returned directly to recipients non-surgically. Embryos to be bisected are collected non-surgically 6-7 days after oestrus. At this time embryos are compact morullae or blastocysts. After collection, the embryos are washed in modified PBS supplemented with 20% heat-inactivated FCS and classified morphologically. Only embryos of good quality should be selected for splitting. The embryo of good quality is fixed on the holding pipette and the zona pellucid a is cut by a vertical motion of the micro knife. The crack in the zona pellucid a is opened by moving the glass needle and the hook, the embryo is either split inside the zona or after moving it out of the zona. Bisection can be done by cutting with the knife or by separating the two halves with the glass needle. One half remains in and will be put back into the original zona. The second half is transferred to a surrogate zona (made by preparing a zona of a degenerated embryo). Bisection of bovine embryos result in two genetically identical demi-embryos. The demi-embryos can be cultured for several hours or transferred immediately after splitting. The can be done in one of the following ways: (a) each half in one recipient into the ipsilateral horn (unilateral transfer); (b) both halves in one recipient into both horns (bilateral transfer) and (c) both halves in one recipient into the ipsilateral horn. Alternatives (b) and (c) are similar or identical to naturally arising twin pregnancies. Alternative (a) will be used in commercial programmes, because it will result in the highest number of calves per collected embryos. Improvement on embryo micromanipulation techniques led to the use of embryo bisection technology in commercial embryo transfer programmes, and made possible the direct genetic analysis of preimplantation bovine embryos by biopsy. For example, aspiration and micro section, allow bovine embryos sexing by detection of malespecific Y-chromosome in a sample of embryonic cells. Pregnancy rates achieved with fresh bisected or biopsied embryos (50-60%) were similar to the fresh intact embryos (55-61%). The PCR protocol used for embryo sexing showed 92% to 94% of efficiency and 90 and 100% of accuracy (Lopes et al., 2001). These results demonstrate that these procedures are suitable for use in field conditions. Cry preservation of micro surgically split bovine embryos allows the production of identical twice of different ages. Survival rates are significantly lower, only about 25% of frozen/thawed embryos will survive. Embryo Sexing The sex of an individual is determined at fertilization and depends on whether the X-bearing ovum is fertilized by a X or Y-bearing spermatozoa. There are several methods for diagnosing the sex of embryos. (a) (b) (c) (d) Sex chromosome identification Demonstration of H-Y antigen Assay of sex-linked enzyme Blotting DNA probes

Park et al (001) developed a rapid and reliable PCR method for the sexing of 8 to 16-cell stage bovine embryos. The BOV97M and bovine 1.715 satellite DNA sequences were selected for amplification of male and bovine-specific DNA, respectively. In consecutive and multiplex PCR, the first 10 PCR cycles were done with malespecific primer followed by an additional o23 cycles with bovine-specific primer. This PCR method was applied successfully using groups of 8, 4, 2 and 1 blastomeres dissociated from the sexing efficiency was 100, 96.3, 94.3 and 92.1%, respectively. This proved to be a rapid (within 2 hrs) and effective PCR method for the sexing of 8 to 16-cell stage bovine embryos using a single blast mere.

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UNIT V PART A 1. Define a) Transgene 2) Transgenic. a) Transgene: The target gene responsible for the development of transgenic organisms. b) Transgenic An organism that carrier a foreign DNA (transgene). 2. What are the methods of introducing a foreign gene? 1. 2. 3. Retroviral vector method Micro injection method Embryonic stem cell method.

3. Define micro injection. The delivery of DNA or other compounds into eukaryotic cells using a fine microscopic needle. 4. Give two examples of transgenic animal, protein product and its importance. Transgenic animal 1. Cow 2. Pig 5. What are transgenic animals? A transgenic animal is one that carrier a foreign gene that has been deliberately inverted into its genome. 6. Why are transgenic animals produced? 1. Produced for specific economic traits Eg: Transgenic cattle were created to produced milk containing particular human an proteins which may help in treatment of human emphysema. 2. Some are produced as disease models. Eg: Ones mouse. 7. How are transgenic animals produced? 1. 2. 3. 4. A cloned gene is injected in to the nucleus of a fertilized egg. The inoculated fertilized eggs are implanted in a receptive female Some of the offspring derived from the implanted eggs carry the cloned gene in all of their cells. Animal with the cloned gene integrated in their germ line cells are bred to establish new genetic cells. Protein product Interferon Hemoglobin Biological Importance Provides resistance against viral infections Blood transfusion

8.. Define transformation / transfection. It specifies the introduction of a DNA segment, either naked or integrated into a vector, in to an animal cell. It is also known as transformation.

9. What are the steps in development of transgenic cattle?

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Collection of oocytes Invitro maturation Invitro fertilization Centrifugation of eggs DNA micro injection in to mate

In Vitro embryonic development to blastocyst Screening of off spring for transgene embryo implantation
10. Give some transfection methods. (i) (ii) (iii) (iv) (v) (vi) Calcium phosphate precipitation Direct micro injection Retrovirus injection Lipofection Particle gun delivery Electroporation.

PART B 1. Explain in detail about embryonic stem cell method.

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Embryonic stem cell method Cells from the inner cell mass of the blastocyst stage of a developing mouse embryo can proliferate in cell culture. These cells, referred to as pluripotent embryonic stem (ES) cells, are capable of differentiating into other types of cells (including germ line cells) when transferred to another blastocryst embryo. The embryonic stem cell technology basically involves the introduction of a foreign DNA into ES cell Embryonic stem cells in culture can be subjected to genetic manipulations without changing their plutipotency. Foreign DNA can be introduced into ES cells by electroporation or micro injection. The desired genetically engineered cells with transgene can be identified by a selection procedure using a marker gene or PCR analysis (described below). The transfected cells can be cultured, introduced (by micro injection) into blastocyst and then implanted into foster mother (i.e., pseudopregnant female mouse). By this way, transgenic founder mice are produced. Transgenic line can be established by suitable breeding strategies of the founder mice. Selection of transgene containing cells Several strategies have been developed for the selection of transgene containing cells. the important ones are briefly described. Selection by use of marker gene coding for thymidine kinase: It is worthwhile to know the role of thymidine kinase to understand its utility as a marker gene. There are two pathways for the synthesis of deoxribonucleotides (dATP, dGTP, dCTP and dTTP), the basic units of DNA structure. One is the salvage pathway that recycles the degraded nitrogenous bases formed form DNA. The other alternate pathway is an endogenous synthetic pathway from different precursors (glycine, aspirate, glutamine, methyl tetrahydrofolate etc.). Fig. Embryonic stem cell (ES) method to produce transgenic mice. The enzyme thymidine kinase (TK) is involved in the salvage pathway. TK phosphorylates thymidien to produce thymidine monophosphate (dTMP) which is finally converted to thymidine triphosphate (dTTP). The gene that encodes the enzyme tymidine kinase can be used as a marker to determine whether the transgene has been inserted. This is illustrated in fig. The mammalian cells are capable of synthesizing dTTP by salvage pathway and endogenous synthetic pathway. The cells lacking TK gene cannot produce dTTP. If such cells are cultured in a HAT containing hypoxanthine (H), aminopterine (A) and thymidine (T), they cannot grow and therefore die. This is because thymidine cannot be utilized in the salvage pathway due to lack the enzyme thymidine kinase. Further, aminopterine blocks the endogenous pathway (by inhibiting the enzyme dihydrofolate reductase, required for one carbon metabolism). If a transgene is joined to a TK gene, inserted into a mammalian cell (TK -) and then the cell can grow in HAT medium. This is possible only if the TK gene is corporated into the mammalian cells. and logically, the cells that survive in HAT medium carry the transfene. In this fashion, thymidine kinase can be effectively used as a marker gene. There are other enzymes that serve as markers for identifying transgene insertion. These include

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dihyrofolate reductase (resistant to methotrexate) and neomycin phsosphotransferase (resistant to antibiotic G418) and PCR analysis for selecting transgene containing cells. The last one is a more direct and recent method, and is successfully used for detecting transgene containing cells. Promoter sequence to facilitate transgenesis In the early experiments on transgenesis, the mouse metallothionein (MMT) gene promoter was used. MMT gene encodes for a metal binding protein that is involved in metal homeostasis. The foreign gene (i.e., a rat growth hormone gene) can be linked to a promoter sequence of MMT. By doing so, the promoter switches on the growth hormone gene when the MMT is activated by a metal in the environment (e.g., cadmium).

Fig. Thymidine kinase (TK) marker gene for selecting transgene containing cells (X-represents the genes for synthesizing dTTP by endogenous synthetic pathway). Thus, the metal inducer (Cd) can stimulate the promoter (MMT promoter) to facilitate the transgene (growth hormone gene) to express. Therefore, addition of cadmium triggers the growth hormone production. 2. Briefly explain the concepts of transgenic animal technology. What are the Transgenic Animals? The nuclei of all the cells in every living organism contain genes made up of DNA. These genes store information that regulates how our bodies form and function. Genes can be altered artificially, so that some characteristics of an animal are changed. For example, an embryo can have an extra, functioning gene from another source artificially introduced into it, or a gene can be introduced which can knock out the functioning of another particular gene in the embryo. Animals that have had their DNA manipulated in this way are known as transgenic animals, Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, although over 95% of all existing transgenic animals are mice. A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. Why are these animals being produced? The two most common reasons are: 1. 2. Some transgenic animals are produced for specific economic traits. For example, transgenic cattle were created to produce milk containing particular human proteins, which may help in the treatment of human emphysema. Other transgenic animals are produced as disease models (animals genetically manipulated to exhibit disease symptoms so that effective treatment can be studied). For example, Harvard scientists made a major scientific breakthrough when they received a U.S. patent for a genetically engineered mouse, called Onco Mouse or the Harvard mouse, carrying a gene that promotes the development of various human cancers.

How are Transgenic Animals Produced? Until recently, selective breeding was the only way to enhance the genetic feature of domesticated animals. However the combination of the successful transfer of genes into mammalian cells and the possibility of creating genetically identical animals by transplanting nucleifrom embroyonic tissue into enucleated eggs (nuclear transfer, nuclear cloning) led researchers to consider putting single, functional genes or gene clusters into the chromosomal DNA of higher organisms. Conceptually, the strategy used to achieve this end is simple.

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 A cloned gene is injected into the nucleus of a fertilizer egg. The inoculated fertilized eggs are implanted into a receptive female (because successful completion of mammalian embryonic development is not possible outside of a female). Some of the off spring derived form the implanted eggs carry the cloned gene in all of their cells. Animals with the cloned gene integrated in their germ line cells are bred to establish new genetic lines. The genetic improvement of multicellular organisms by the introduction of relevant transgenes is only slowly being realized. However, transgenesis has become a powerful technique for studying fundamental problems of mammalian gene expression and development, for establishing animal model systems for human diseases, and for using the mammary gland to produce pharmaceutically important proteins in milk. With this application in mind, the term pharming was coined to convey the idea that milk from transgenic farm (Pharm) animals can be source of authentic human protein drugs or pharmaceuticals. The basic methods of producing transgenic animals were described in chapter. The ease with which transgenic animals can be produced and the transfection techniques to be employed depend chiefly on the reproductive biology, husbandry requirements and responses to various experimental procedures of the concerned animal. The following features greatly facilitate gene transfer efforts. 1. 2. 3. 4. 5. 6. Production of larger number of eggs either naturally, e.g. in fish, or in response to hormone regimens used for superovulation, e,g. in m ice, pigs, etc. Short breeding cycle, i.e., time taken from birth to reaching the reproductive age, greatly facilitates analysis of the transgenic individuals produce. It is desirable that ovulation occurs throughout the year so that ova are readily available for experimentation. The size and the structure of eggs should be amenable to micro injection, the only tarnsfection technique successful with almost all animal species. In many animal species, e.g. fish, body size may be an important factor to allow sufficient tissue samples to be taken for the detection of transgene integration and function without sacrificing the individual. In the case of mammals where transfected ova / embryos must be transferred into surrogate mothers, the following features are critical: Synchronization of females used for superovulation and as surrogate mother by hormone administration. In vitro fertilization and culture. Transfer of the mbryos into surrogate mother and completion of the mbroyo development in them. Production and maintenance of embroyonic stem (ES) cell lines capable of giving rise to germ cells, which is essential for the application of ES cell transfer technology.

3. Explain in detail about transfection method. Transfection Methods Several approaches have been used for the introduction of DNA into animal cells/embryos, which are listed as follows: (i) calcium phosphate precipitation, (ii) direct microinjection, (iii) retrovirus infection, (iv) lipofection, (v) particle gun delivery and (vi) electroporation. In addition, transgenic mice are readily produced by the technique of embryonic stem (ES) cell transfer; this is the only approach, which permits targeted gene transfers. Calcium Phosphate Precipitation In this approach, the DNA preparation to be used for transfection is first dissolved in a phosphate buffer. Calcium chloride solution is then added to dissolved in a phosphate buffer. Calcium chloride solution is then added to the DNA solution; this leads to the formation of insoluble calcium phosphate, which co-precipitates with the DNA. The calcium phosphate DNA precipitate is added to the cells to be transfected. The precipitate particles are taken in by the cells by phagocytosis Initially, 1-2% of the cells were transfected by this approach. But the procedure has now been modified to obtain transfection of up to 20% of the cells.

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Fig. A schematic representation of transfection by calcium phosphate precipitation In a small proportion of the transfected cells, the DNA becomes integrated into the cell genome producing stable or permanent transfectino. This approach can be applied to virtually all mammalian cells, but many cell lines do not like the calcium phosphate precipitate adhering to their surfaces or to their substrate. Lipofection The delivery of DNA into cells using liposomes is called lipofectino. Liposomes are small vesicles prepared from a suitable lipid. Initially, nonionic lipids were used for preparing liposomes so that DNA had to be introduce with in the vesicles following specific encapsidation procedures. The use of cationic lipids for the construction of liposomes is a distinct advantage as DNA spontaneously and efficiently complexes with these liposomes making encapsidatin procedures unnecessary. The cationic liposomes have a single lipid bilayer membrane (unilamellar), and hey bind to the cells efficiently. Probably the fuse with the plasma membrane and, thereby, deliver the DNA (complexed with them) into the cells, which brings about transfection. Considerable work has been done on lipofection due to its potential application for targeting genes to specific human tissues for gene therapy. Usually, liposomes are prepared by dispersion of a phospholipids like phosphatidyl choline (PC) in water by mechanical methods, like sonication, which tend to destroy DNA. DNA of upto 1 kb has been incorporated into small sonicated liposomes. However, some other techniques allow the entrapment of large DNA sequences into liposomes. Liposomes prepared by the above approaches are pahsgocytosed by the cell. The phagocytosis vehicles thus produced ordinarily fuse with lysosomes leading to DNA degradation and low transfection frequencies. The use of cationic liposomes to which DNA binds on the outside by electrostatic attraction has been quite successful. These liposomes cause perturbations in phasma membrane due to which they fuse and the DNA enters into the cytoplasm. Cationic liposomes are available commercially (marked as Lipofection by GibcoBRL). Lipofectino is the method of choice for transfection of mammalian cells in vitro. It has also been used to deliver DNA into live animals by direct injection or intravenous injection. Cationic liposomes have been used in

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intravenous or intratracheal injection in mice for the expression of marker genes in lungs. Targeted delivery has also been demonstrated by incorporating specific ligan proteins into the liposome membranes. The mechanism of movement of the DNA from cell cytoplasm into the nucleus is not known. Electroporation In this approach, transfection mixture containing cells and DNA is exposed for a very brief period (few milliseconds) to a very high voltage gradient (e.g., 4000-8000 V/em). This induces transient pores in the cell membranes through which DNA seems to enter the cells. Treatment of cells with colcemid before electroportio increases the frequency of transfection. Linearized DNA is far more efficient in transfection than circular supercoiled DNA. Electroportion technique has a general applicability, and many animal cell types the could not be transfected by other approaches were successfully transfected by this approach. Retroviral Infection Recombinant retrovirus produce virions, which are used to infect animal cells and mice embryos. When embryos beyond the 4-celled stage are used, often all the cells in an embryo may not be infected by the retrovirus, resulting in chimaeric mice. A chimaera is an individual, which has in its body cells of two or more genotypes. The recombinant retrovirus RNA genome is copied by reverse transcriptase to yield a DNA copy (reverse transcription), which becomes integrated into the cells gemone. The reverse transcriptase is encoded by the retrovirus, and is produced immediately after infection however, the reverse transcription can occur only in those cells, which go through S phase, i.e., are miotically active. The DNA copy of the recombinant retrovirus integrates into the cellular genome at random sites, and usually is not accompanied with diletions or rearrangements. Micro injection In this method, DNA solution is injected directly into the nucleus of a cell or into the male pronucleus of a fertilized one to two cell ovum. Typically, a microinjection assembly consists of a low power stereoscopic dissecting microscope ( to view the ovum and the entire process) and two micro manipulators, one for a glass micropipette to hold the ovaum by partial suction and the other for a glass injection needle to introduce the DNA into the male pronucleus. The male pronucleus is much larger than the female pronucleus of fertilized mammalian ova. However in fish ova, the DNA is injected into the egg cytoplasm. The general procedure for microinjection is as follows Donor females are induced to superovulate using appropriate hormone treatments. The superovulated females are then mated with fertile males, and large numbers of fertilized one to two cell ova / embroyos are collected surgically. Alternatively, unfertilized ova are collected from superovulated females; the ova are then fertilized in vitro. The transgene construct is prepared in a buffer solution and is injected in to the male pronuclei of fertilized eggs using a micro injection assembly. Typically, 2; (ocp;otre = 10-12 1-10-9 ml) of the DNA solution is injected in a pronucleus. But in case of fish, 20 nl (nanolitre = 10-9 1- 10-6 ml) DNA solution, containing 106-108 linearized transgene constructs, is injected into the cytoplasm of a single ovum. In mice, the microinjected embryos are cultured in vitro upto the morula or blastocyst stage. The surviving embryos are then transferred into the uterus of surrogate mothers, i.e., females, which have been made receptive by hormone treatments; these embroyos develop to full term and give rise to progeny mice (fig.) A proportion of the progeny so produced will be transgenic in that all their cells will contain the transfene stably integrated into their genomes.

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Fig. A schematic representation of the micro injection technique of transfection for producing transgenic mice. In mice, an average of about 3-6% of the progeny derived from microinjected embryos are transgenic; the frequency is much lower in other animals, e.g., <1% in sheep and pigs. In case of fish about 35-80% of the embryos survive microinjection, of which 10-70% may be transgenic. The transgenic animals contain the transgene in their germ cells and, as a consequence, pass it on to their progeny; transgenes show typical Mendelian inheritance. The transgene integration occurs at random sites in the genome, but in a given cell or embroyo usually only a single chromosomal site is involved. However, there is generally a wide variation in the number of copies integrated ranging from the common one copy to several hundred copies. The multiple copies are integrated at a single site in a head to tail arrangement. Consequently, the site of integration of a transgene in different transgenic animals differs greatly and may involve different locations of the same chromosome or different chromosomes. The transgene integration occurs at an early stage of embryo are involved. But often the integration may be delayed, and the transgene remains in the extrachromosomal state during this period. Subsequently, transgene integration occurs only in some cells of the embryo; this results in chimaeric progeny. Pure transgenic individuals can be recovered through suitable breeding schemes from such chimaeric animals in whose germ cells the transgene has become suitably integrated. All the transfectino techniques are applicable to cultured animal cells. but microinjection is ordinarily not used due to the tediousness of the technique and the limited number of cells that can be handled. For transfectino of mice embryos, the preferred techniques are microinjection, retroviral infection and embroyonic stem cell technology, micro injection being the most commonly used. Embryos of other animals are generally transfected by micro injection technique only. In case of fish embryos, microinjection and electro portion are routinely employed.

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BT 2036 - Animal Biotechnology (Nov Dec 2010) Part A (102=20 marks) 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Indicate the role of serum in animal cell culture. Why are mammalian cells cultured in CO2 incubator? What is in-situ hybridization? Give its application in disease diagnosis. Outline the use of PCR in disease diagnosis with example Enlist the steps involved in northern hybridization. How do you evaluate the embryo sex? Brief any two methods used to manipulate reproduction in animals? How do you enrich Y- bearing sperms from semen samples? What is FACS? How this technique could be used to enrich stem cells? What are the advantages of continuous flow culture?

Part- B(516=80 marks) 1 (a) (i) Describe the strategies for scaling uo anchorage dependent animal cells in culture. (ii) what is trypsinisation? Explain its uses in animal cell culture. Or (b) (i) Give the list of therapeutic proteins produced using animal cell culture. Write a note on biopharming. (ii) How do you characterize the efficacy of cytotoxi drugs using cell cultures? Explain with an example. 12 (a) Explain the different molecular techniques used in diagnosis of animal infection. Or (b) You have been given the task of developing a simple, sensitive and reproducible diagnostic procedure for a double stranded DNA virus that is devastating a local cattle population. Briefly explain how would you proceed. 13 (a) What are the different types of vaccines available from recombinant DNA technology? Outline the production of DNA vaccines and their application in animal application. Or (b) Explain the procedures and selection criteria for making a hybridoma and an EBV transformed cell line. How to you increase the monoclonal antibody titre in a culture. 14 (a) Explain the process of gene targeting in animal cells. How do you select a homogeneous recombinant after gene transfection in animal cells. Or (b) write a short notes on (i) Micromanipulation technology in breeding of farm animals (ii) Embryo splitting and embryo sexing 15 (a) Explain the production of transgenic animals? Write about the research opportunities and application of transgenic animals. Or (b) (i) Explain cytokines and their application in animal diseases (ii) Discuss the construction of viral vectors for gene therapy.

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