Transfection

From Wikipedia, the free encyclopedia

Transfection is the process of deliberately introducing nucleic acids into cells. The term is used
notably for non-viral methods [1] in eukaryotic cells. It may also refer to other methods and cell
types, although other terms are preferred: "transformation" is more often used to describe non-
viral DNA transfer in bacteria, non-animal eukaryotic cells and plant cells - a distinctive sense of
transformation refers to spontaneous genetic modifications (mutations to cancerous cells
(Carcinogenesis), or under stress (UV irradiation)). "Transduction" is often used to describe
virus-mediated DNA transfer. The word transfection is a blend of trans- and infection.

Genetic material (such as supercoiled plasmid DNA or siRNA constructs), or even proteins such
as antibodies, may be transfected.

Transfection of animal cells typically involves opening transient pores or "holes" in the cell
membrane, to allow the uptake of material. Transfection can be carried out using calcium
phosphate, by electroporation, or by mixing a cationic lipid with the material to produce
liposomes, which fuse with the cell membrane and deposit their cargo inside.

Transfection can result in unexpected morphologies and abnormalities in target cells.

Contents
[hide]

• 1 Terminology
• 2 Methods
o 2.1 Chemical-based transfection
o 2.2 Non chemical methods
o 2.3 Particle-based methods
o 2.4 Viral methods
o 2.5 Other (and hybrid) methods
• 3 Stable and transient transfection
• 4 RNA transfection
• 5 See also
• 6 References

• 7 External links

[edit] Terminology
The meaning of the term has evolved.[2] The original meaning of transfection was "infection by
transformation", i.e. introduction of DNA (or RNA) from a prokaryote-infecting virus or
bacteriophage into cells, resulting in an infection. Because the term transformation had another
sense in animal cell biology (a genetic change allowing long-term propagation in culture, or
acquisition of properties typical of cancer cells), the term transfection acquired, for animal cells,
its present meaning of a change in cell properties caused by introduction of DNA.

[edit] Methods
There are various methods of introducing foreign DNA into a eukaryotic cell: some rely on
physical treatment (electroporation, nanoparticles, magnetofection), other on chemical materials
or biological particles (viruses) that are used as carriers.

[edit] Chemical-based transfection

Chemical-based transfection can be divided into several kinds: cyclodextrin[3], polymers[4],

liposomes, or nanoparticles [5] (with or without chemical or viral functionalization. See below).

• One of the cheapest methods uses calcium phosphate, originally discovered by F. L.

Graham and A. J. van der Eb in 1973[6] (see also [7]). HEPES-buffered saline solution
(HeBS) containing phosphate ions is combined with a calcium chloride solution
containing the DNA to be transfected. When the two are combined, a fine precipitate of
the positively charged calcium and the negatively charged phosphate will form, binding
the DNA to be transfected on its surface. The suspension of the precipitate is then added
to the cells to be transfected (usually a cell culture grown in a monolayer). By a process
not entirely understood, the cells take up some of the precipitate, and with it, the DNA.

• Other methods use highly branched organic compounds, so-called dendrimers, to bind
the DNA and get it into the cell.

• A very efficient method is the inclusion of the DNA to be transfected in liposomes, i.e.
small, membrane-bounded bodies that are in some ways similar to the structure of a cell
and can actually fuse with the cell membrane, releasing the DNA into the cell. For
eukaryotic cells, transfection is better achieved using cationic liposomes (or mixtures),
because the cells are more sensitive. Popular agents were DOTMA and DOPE , and now
- more effectively - Lipofectamine and UptiFectin [8].

• Another method is the use of cationic polymers such as DEAE-dextran or

polyethylenimine. The negatively charged DNA binds to the polycation and the complex
is taken up by the cell via endocytosis. Popular agents of this type are the Fugene [9] or
LT-1 [10], and JetPEI [11].

• Other proprietary chemical transfection reagents: PromoFectin, GenePORTER, Hilymax

[12]
. Effectene or Altogen's cell line specific reagents.

[edit] Non chemical methods

 • Electroporation is a popular method, although requiring an instrument and affecting the
viability of many cell types, that also creates micro-sized holes transiently in the plasma
membrane of cells under an electric discharge.

• Similarly, transfection applying sonic forces to cells, referred as Sono-poration.

• Optical transfection is a method where a tiny (~1 µm diameter) hole is transiently

generated in the plasma membrane of a cell using a highly focused laser. This technique
was first described in 1984 by Tsukakoshi et al., who used a frequency tripled Nd:YAG
to generate stable and transient transfection of normal rat kidney cells[13]. In this
technique, one cell at a time is treated, making it particularly useful for single cell
analysis.

• Gene electrotransfer is a technique that enables transfer of genetic material into

prokaryotic or eukaryotic cells. It is based on a physical method named electroporation,
where transient increase in the permeability of cell membrane is achieved when
submitted to short and intense electric pulses.

• Impalefection is a method of introducing DNA bound to a surface of a nanofiber that is

inserted into a cell.

[edit] Particle-based methods

• A direct approach to transfection is the gene gun, where the DNA is coupled to a
nanoparticle of an inert solid (commonly gold) which is then "shot" directly into the
target cell's nucleus.

• Magnetofection, or Magnet assisted transfection is a transfection method, which uses

magnetic force to deliver DNA into target cells. Nucleic acids are first associated with
magnetic nanoparticles. Then, application of magnetic force drives the nucleic acid
particle complexes towards and into the target cells, where the cargo is released.[14][15][16]

• Impalefection is carried out by impaling cells by elongated nanostructures such as carbon

nanofibers or silicon nanowires which have been functionalized with plasmid DNA.

[edit] Viral methods

DNA can also be introduced into cells using viruses as a carrier. In such cases, the technique is
called viral transduction, and the cells are said to be transduced. This can be done using insect
cells.

[edit] Other (and hybrid) methods

Other methods of transfection include nucleofection, heat shock.

[edit] Stable and transient transfection
For most applications of transfection, it is sufficient if the transfected genetic material is only
transiently expressed. Since the DNA introduced in the transfection process is usually not
integrated into the nuclear genome, the foreign DNA will be diluted through mitosis or degraded.

If it is desired that the transfected gene actually remains in the genome of the cell and its
daughter cells, a stable transfection must occur. To accomplish this, a marker gene is co-
transfected, which gives the cell some selectable advantage, such as resistance towards a certain
toxin. Some (very few) of the transfected cells will, by chance, have integrated the foreign
genetic material into their genome. If the toxin is then added to the cell culture, only those few
cells with the marker gene integrated into their genomes will be able to proliferate, while other
cells will die. After applying this selective stress (selection pressure) for some time, only the
cells with a stable transfection remain and can be cultivated further.

A common agent for selecting stable transfection is Geneticin, also known as G418, which is a
toxin that can be neutralized by the product of the neomycin resistant gene.

[edit] RNA transfection

Main article: RNA transfection

RNA can also be transfected into cells to transiently express its coded protein, or to study RNA
decay kinetics. The later application is referred as siRNA transfection or RNA silencing, and has
become a major application in research (to replace the "knock-down" experiments, to study the
expression of proteins, i.e. of Endothelin-1 [17]) with potential applications in gene-therapy.

A limitation of the silencing approach rely on the toxicity of the transfection for cells, and its
suspected effect on the expression of other genes/proteins.

Gene knockout
From Wikipedia, the free encyclopedia

A gene knockout (abbreviation: KO) is a genetic technique in which an organism is engineered

to carry genes that have been made inoperative (have been "knocked out" of the organism). Also
known as knockout organisms or simply knockouts, they are used in learning about a gene that
has been sequenced, but which has an unknown or incompletely known function. Researchers
draw inferences from the difference between the knockout organism and normal individuals.

The term also refers to the process of creating such an organism, as in "knocking out" a gene.
The technique is essentially the opposite of a Gene Knock-in. Knockout is often abbreviated as
KO. Knocking out two genes simultaneously in an organism is known as a double knockout
(DKO). Similarly the terms triple knockout (TKO) and quadruple knockouts (QKO) are used
to describe 3 or 4 knocked out genes, respectively.

Contents
[hide]

• 1 Method
• 2 See also
• 3 References

• 4 External links

[edit] Method

A laboratory mouse in which a gene affecting hair growth has been knocked out (left), is shown
next to a normal lab mouse.

Knockout is accomplished through a combination of techniques, beginning in the test tube with a
plasmid, a bacterial artificial chromosome or other DNA construct, and proceeding to cell
culture. Individual cells are genetically transformed with the DNA construct. Often the goal is to
create a transgenic animal that has the altered gene. If so, embryonic stem cells are genetically
transformed and inserted into early embryos. Resulting animals with the genetic change in their
germline cells can then often pass the gene knockout to future generations.

To create knockout moss, transfection of protoplasts is the preferred method. Such transformed
Physcomitrella-protoplasts directly regenerate into fertile moss plants. Already eight weeks after
transfection the plants can be screened for gene targeting via PCR.[1]
Wild-type Physcomitrella and knockout-mosses: Deviating phenotypes induced in gene-
disruption library transformants. Physcomitrella wild-type and transformed plants were grown on
minimal Knop medium to induce differentiation and development of gametophores. For each
plant, an overview (upper row, scale bar corresponds to 1 mm) and a close-up (bottom row, scale
bar equals 0.5 mm) is shown. A, Haploid wild-type moss plant completely covered with leafy
gametophores and close-up of wild-type leaf. B-D, Different Mutants.[2]

The construct is engineered to recombine with the target gene, which is accomplished by
incorporating sequences from the gene itself into the construct. Recombination then occurs in the
region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt
the gene. With its sequence interrupted, the altered gene in most cases will be translated into a
nonfunctional protein, if it is translated at all.

A knockout mouse (left) that is a model of obesity, compared with a normal mouse.

A conditional knockout allows gene deletion in a tissue or time specific manner. This is done by
introducing short sequences called loxP sites around the gene. These sequences will be
introduced into the germ-line via the same mechanism as a knock-in. This germ-line can then be
crossed to another germline containing Cre-recombinase which is a bacterial enzyme that can
recognize these sequences, recombines them and deletes the gene flanked by these sites.

Because the desired type of DNA recombination is a rare event in the case of most cells and most
constructs, the foreign sequence chosen for insertion usually includes a reporter. This enables
easy selection of cells or individuals in which knockout was successful. Sometimes the DNA
construct inserts into a chromosome without the desired homologous recombination with the
target gene. To eliminate such cells, the DNA construct often contains a second region of DNA
that allows such cells to be identified and discarded.

In diploid organisms, which contain two alleles for most genes, and may as well contain several
related genes that collaborate in the same role, additional rounds of transformation and selection
are performed until every targeted gene is knocked out. Selective breeding may be required to
produce homozygous knockout animals.

Knock-in is similar to knock-out, but instead it replaces a gene with another instead of deleting
it.

Genetically modified organism

From Wikipedia, the free encyclopedia
"GMO" redirects here. For other uses, see GMO (disambiguation).

GloFish, the first genetically modified animal to be sold as a pet

A genetically modified organism (GMO) or genetically engineered organism (GEO) is an

organism whose genetic material has been altered using genetic engineering techniques. These
techniques, generally known as recombinant DNA technology, use DNA molecules from
different sources, which are combined into one molecule to create a new set of genes. This DNA
is then transferred into an organism, giving it modified or novel genes. Transgenic organisms, a
subset of GMOs, are organisms which have inserted DNA that originated in a different species.

Contents
[hide]

• 1 Production
• 2 History
• 3 Uses
o 3.1 Detection
o 3.2 Transgenic microbes
o 3.3 Transgenic animals
 3.3.1 Fruit flies
 3.3.2 Mammals
 3.3.3 Cnidarians
 3.3.4 Fish
o 3.4 Gene therapy
o 3.5 Transgenic plants
o 3.6 Cisgenic plants
• 4 Controversy
o 4.1 Biological process
o 4.2 Foodchain
o 4.3 Trade in Europe and Africa
o 4.4 Agricultural surpluses
o 4.5 Labeling
o 4.6 Testing
o 4.7 Impoverished nations
o 4.8 Private investments
 o 4.9 Transgenic organisms
o 4.10 "Terminator" and "traitor"
o 4.11 Governmental support and opposition
 4.11.1 Australia
 4.11.2 Canada
 4.11.3 Japan
 4.11.4 Pakistan
 4.11.5 New Zealand
 4.11.6 United States
 4.11.7 Zambia
 4.11.8 Other Africa
 4.11.9 France
 4.11.10 Germany
 4.11.11 Other European Countries
• 5 See also
• 6 References
• 7 External links
o 7.1 General
o 7.2 Transgenic animals

o 7.3 Transgenic plants

[edit] Production
Further information: Genetic Engineering, Horizontal Gene Transfer and
Transformation (genetics)

Genetic modification involves the insertion or deletion of genes. When genes are inserted, they
usually come from a different species, which is a form of horizontal gene transfer. In nature this
can occur when exogenous DNA penetrates the cell membrane for any reason. To do this
artificially may require attaching the genes to a virus or just physically inserting the extra DNA
into the nucleus of the intended host with a very small syringe, or with very small particles fired
from a gene gun.[1] However, other methods exploit natural forms of gene transfer, such as the
ability of Agrobacterium to transfer genetic material to plants,[2] or the ability of lentiviruses to
transfer genes to animal cells.[3]

[edit] History
This section requires
expansion.

The general principle of producing a GMO is to add new genetic material into an organism's
genome. This is called genetic engineering and was made possible through the discovery of
DNA and the creation of the first recombinant bacteria in 1973; an existing bacterium E. coli
expressing an exogenic Salmonella gene.[4] This led to concerns in the scientific community
about potential risks from genetic engineering, which were thoroughly discussed at the Asilomar
Conference. One of the main recommendations from this meeting was that government oversight
of recombinant DNA research should be established until the technology was deemed safe.[5][6]
Herbert Boyer then founded the first company to use recombinant DNA technology, Genentech,
and in 1978 the company announced creation of an E. coli strain producing the human protein
insulin.[7]

In 1986, field tests of bacteria genetically engineered to protect plants from frost damage (ice-
minus bacteria) at a small biotechnology company called Advanced Genetic Sciences of
Oakland, California, were repeatedly delayed by opponents of biotechnology. In the same year, a
proposed field test of a microbe genetically engineered for a pest resistance protein by Monsanto
Company was dropped.

[edit] Uses
GMOs are used in biological and medical research, production of pharmaceutical drugs,
experimental medicine (e.g. gene therapy), and agriculture (e.g. golden rice). The term
"genetically modified organism" does not always imply, but can include, targeted insertions of
genes from one species into another. For example, a gene from a jellyfish, encoding a fluorescent
protein called GFP, can be physically linked and thus co-expressed with mammalian genes to
identify the location of the protein encoded by the GFP-tagged gene in the mammalian cell. Such
methods are useful tools for biologists in many areas of research, including those who study the
mechanisms of human and other diseases or fundamental biological processes in eukaryotic or
prokaryotic cells.

To date the broadest and most controversial application of GMO technology is patent-protected
food crops which are resistant to commercial herbicides or are able to produce pesticidal proteins
from within the plant, or stacked trait seeds, which do both. The largest share of the GMO crops
planted globally are owned by the US firm Monsanto.[8] In 2007, Monsanto's trait technologies
were planted on 246 million acres (1,000,000 km2) throughout the world, a growth of 13 percent
from 2006.

In the corn market, Monsanto's triple-stack corn—which combines Roundup Ready 2 weed
control technology with YieldGard Corn Borer and YieldGard Rootworm insect control—is the
market leader in the United States. U.S. corn farmers planted more than 32 million acres
(130,000 km2) of triple-stack corn in 2008,[9] and it is estimated the product could be planted on
56 million acres (230,000 km2) in 2014–2015. In the cotton market, Bollgard II with Roundup
Ready Flex was planted on approximately 5 million acres (20,000 km2) of U.S. cotton in 2008.[10]

According to the International Service for the Acquisition of Agri-Biotech Applications

(ISAAA), of the approximately 14 million farmers who grew biotech crops in 2009, some 90%
were resource-poor farmers in developing countries. These include some 7 million farmers in the
cotton-growing areas of China, an estimated 5.6 million small farmers in India (Bt cotton),
250,000 in the Philippines, South Africa (biotech cotton, maize and soybeans often grown by
subsistence women farmers) and the other twelve developing countries which grew biotech crops
in 2009.[11] 10 million more small and resource-poor farmers may have been secondary
beneficiaries of Bt cotton in China.

The global commercial value of biotech crops grown in 2008 was estimated to be US$130
billion.[11]

In the United States, the United States Department of Agriculture (USDA) reports on the total
area of GMO varieties planted.[12] According to National Agricultural Statistics Service, the
states published in these tables represent 81–86 percent of all corn planted area, 88–90 percent of
all soybean planted area, and 81–93 percent of all upland cotton planted area (depending on the
year).

USDA does not collect data for global area. Estimates are produced by the International Service
for the Acquisition of Agri-biotech Applications (ISAAA) and can be found in the report,
"Global Status of Commercialized Transgenic Crops: 2007".[13]

Transgenic animals are also becoming useful commercially. On February 6, 2009 the U.S. Food
and Drug Administration approved the first human biological drug produced from such an
animal, a goat. The drug, ATryn, is an anticoagulant which reduces the probability of blood clots
during surgery or childbirth. It is extracted from the goat's milk.[14]

[edit] Detection

Testing on GMOs in food and feed is routinely done by molecular techniques like DNA
microarrays or qPCR. The test can be based on screening elements (like p35S, tNos, pat, or bar)
or event-specific markers for the official GMOs (like Mon810, Bt11, or GT73). The array-based
method combines multiplex PCR and array technology to screen samples for different potential
GMOs,[15] combining different approaches (screening elements, plant-specific markers, and
event-specific markers). The qPCR is used to detect specific GMO events by usage of specific
primers for screening elements or event-specific markers.

To avoid any kind of false positive or false negative testing outcome, comprehensive controls for
every step of the process is mandatory. A CaMV check is important to avoid false positive
outcomes based on virus contamination of the sample.

[edit] Transgenic microbes

Bacteria were the first organisms to be modified in the laboratory, due to their simple genetics.[16]
These organisms are now used for several purposes, and are particularly important in producing
large amounts of pure human proteins for use in medicine.[17]

Genetically modified bacteria are used to produce the protein insulin to treat diabetes.[18] Similar
bacteria have been used to produce clotting factors to treat haemophilia,[19] and human growth
hormone to treat various forms of dwarfism.[20][21]

[edit] Transgenic animals

Some chimeras, like the blotched mouse shown, are created through genetic
modification techniques like gene targeting.

Transgenic animals are used as experimental models to perform phenotypic and for testing in
biomedical research.[22] Other applications include the production of human hormones such as
insulin.

[edit] Fruit flies

In biological research, transgenic fruit flies (Drosophila melanogaster) are model organisms
used to study the effects of genetic changes on development.[23] Fruit flies are often preferred
over other animals due to their short life cycle, low maintenance requirements, and relatively
simple genome compared to many vertebrates.

[edit] Mammals

Genetically modified mammals are an important category of genetically modified organisms.

Transgenic mice are often used to study cellular and tissue-specific responses to disease.

In 1999, scientists at the University of Guelph in Ontario, Canada created the genetically
engineered Enviropig. The Enviropig excretes from 30 to 70.7% less phosphorus in manure
depending upon the age and diet.[24] In February 2010, Environment Canada determined that
Enviropigs are in compliance with the Canadian Environmental Protection Act and can be
produced outside of the research context in controlled facilities where they are segregated from
other animals.[25]

In 2009, scientists in Japan announced that they had successfully transferred a gene into a
primate species (marmosets) and produced a stable line of breeding transgenic primates for the
first time.
[edit] Cnidarians

Cnidarians such as Hydra and the sea anemone Nematostella vectensis have become attractive
model organisms to study the evolution of immunity and certain developmental processes. An
important technical breakthrough was the development of procedures for generation of stably
transgenic hydras and sea anemones by embryo microinjection.[26]

[edit] Fish

Genetically modified fish have promoters driving an over-production of "all fish" growth
hormone. This resulted in dramatic growth enhancement in several species, including salmonids,
[27]
carps[28] and tilapias.[29]

[edit] Gene therapy

Gene therapy,[30] uses genetically modified viruses to deliver genes that can cure disease into
human cells. Although gene therapy is still relatively new, it has had some successes. It has been
used to treat genetic disorders such as severe combined immunodeficiency,[31] and treatments are
being developed for a range of other currently incurable diseases, such as cystic fibrosis,[32] sickle
cell anemia,[33] and muscular dystrophy.[34] Current gene therapy technology only targets the non-
reproductive cells meaning that any changes introduced by the treatment can not be transmitted
to the next generation. Gene therapy targeting the reproductive cells—so-called "Germ line Gene
Therapy"—is very controversial and is unlikely to be developed in the near future.

[edit] Transgenic plants

Kenyans examining insect-resistant transgenic Bt corn

Transgenic plants have been engineered to possess several desirable traits, including resistance to
pests, herbicides, or harsh environmental conditions; improved product shelf life, and increased
nutritional value. Since the first commercial cultivation of genetically modified plants in 1996,
they have been modified to be tolerant to the herbicides glufosinate and glyphosate, to be
resistant to virus damage as in Ringspot virus resistant GM papaya, grown in Hawaii, and to
produce the Bt toxin, a potent insecticide. Most of transgenic varieties grown today are known as
first generation transgenics, because the transgenic trait provides benefits to farmers. Plants of
the second generation should directly benefit the consumer with nutritional enhancement, taste,
texture, etc. Transgenic plants of the second generation are being developed by both public
research institutions and private companies. Currently there is no such transgenic variety on the
market. Genetically modified sweet potatoes have been enhanced with protein and other
nutrients, while golden rice, developed by the International Rice Research Institute, has been
discussed as a possible cure for Vitamin A deficiency. In January 2008, scientists altered a carrot
so that it would produce calcium and become a possible cure for osteoporosis; however, people
would need to eat 1.5 kilograms of carrots per day to reach the required amount of calcium.

The coexistence of GM plants with conventional and organic crops has raised significant concern
in many European countries. Since there is separate legislation for GM crops and a high demand
from consumers for the freedom of choice between GM and non-GM foods, measures are
required to separate foods and feed produced from GMO plants from conventional and organic
foods. European research programs such as Co-Extra, Transcontainer, and SIGMEA are
investigating appropriate tools and rules. At the field level, biological containment methods
include isolation distances and pollen barriers.

[edit] Cisgenic plants

Cisgenesis, sometimes also called Intragenesis, is a product designation for a category of

genetically engineered plants. A variety of classification schemes have been proposed,[35] that
order genetically modified organisms based on the nature of introduced genotypical changes
rather than the process of genetic engineering.

While some genetically modified plants are developed by the introduction of a gene originating
from distant, sexually incompatible species into the host genome, cisgenic plants contain genes
which have been isolated either directly from the host species or from sexually compatible
species. The new genes are introduced using recombinant DNA methods and gene transfer.
Some scientists hope that the approval process of cisgenic plants might be simpler than that of
proper transgenics,[36] but it remains to be seen.[37]

[edit] Controversy
See also: Genetically modified food controversies

The examples and perspective in this article may not represent a

worldwide view of the subject. Please improve this article and discuss the
issue on the talk page. (March 2010)

[edit] Biological process

The use of genetically modified organisms has sparked significant controversy in many areas.[38]
Some groups or individuals see the generation and use of GMO as intolerable meddling with
biological states or processes that have naturally evolved over long periods of time, while others
are concerned about the limitations of modern science to fully comprehend all of the potential
negative ramifications of genetic manipulation.

[edit] Foodchain

The safety of GMOs in the foodchain has been questioned by some environmental groups, with
concerns such as the possibilities that GMOs could introduce new allergens into foods, or
contribute to the spread of antibiotic resistance.[39] All studies published to date have shown no
adverse health effects resulting from humans eating genetically modified foods,[40] environmental
groups still discourage consumption in many countries, claiming that GM foods are unnatural
and therefore unsafe.[41] Such concerns have led to the adoption of laws and regulations that
require safety testing of any new organism produced for human consumption.[42]

GMOs' proponents note that because of the safety testing requirements imposed on GM foods,
the risk of introducing a plant variety with a new allergene or toxin using genetic modification is
much smaller than using traditional breeding processes. An example of an allergenic plant
created using traditional breeding is the kiwi.[43] One article calculated that the marketing of GM
salmon could reduce the cost of salmon by half, thus increasing salmon consumption and
preventing 1,400 deaths from heart attack a year in the United States.[44]

[edit] Trade in Europe and Africa

In response to negative public opinion, Monsanto announced its decision to remove their seed
cereal business from Europe, and environmentalists crashed a World Trade Organization
conference in Cancun that promoted GM foods and was sponsored by Committee for a
Constructive Tomorrow (CFACT). Some African nations have refused emergency food aid from
developed countries, fearing that the food is unsafe. During a conference in the Ethiopian capital
of Addis Ababa, Kingsley Amoako, Executive Secretary of the United Nations Economic
Commission for Africa (UNECA), encouraged African nations to accept genetically modified
food and expressed dissatisfaction in the public’s negative opinion of biotechnology.[41]

[edit] Agricultural surpluses

Patrick Mulvany, Chairman of the UK Food Group, accused some governments, especially the
Bush administration, of using GM food aid as a way to dispose of unwanted agricultural
surpluses. The UN blamed food companies and accused them of violating human rights, calling
on governments to regulate these profit-driven firms. It is widely believed that the acceptance of
biotechnology and genetically modified foods will also benefit rich research companies and
could possibly benefit them more than consumers in underdeveloped nations.[41]

[edit] Labeling

While some groups advocate the complete prohibition of GMOs, others call for mandatory
labeling of genetically modified food or other products. Other controversies include the
definition of patent and property pertaining to products of genetic engineering. According to the
documentary Food, Inc. efforts to introduce labeling of GMOs has repetedly met resistance from
lobbyists and politicians affiliated with companies like Monsanto.

[edit] Testing

Bruce Stutz's article, “Wanted: GM Seeds for Study,” highlights a story of two dozen scientist
who spoke out against the research restrictions put forth by companies producing genetically
modified (GM) seeds such as DuPont, Monsanto, and Syngenta. In February 2009, after scientist
warned the U.S. Environmental protection Agency (EPA) “that industry influence had made
independent analyses of transgenic crops impossible,” the American Seed Trade Association
(ASTA) agreed that they “would allow researchers greater freedom to study the effects of GM
food crops.” This agreement left many scientist optimistic about the future, but there is little
optimism as to whether this agreement has the ability to “alter what has been a research
environment rife with obstruction and suspicion.”[45]

[edit] Impoverished nations

Some groups believe that impoverished nations will not reap the benefits of biotechnology
because they do not have easy access to these developments, cannot afford modern agricultural
equipment, and certain aspects of the system revolving around intellectual property rights are
unfair to "undeveloped countries". For example, The CGIAR (Consultative Group of
International Agricultural Research) is an aid and research organization that has been working to
achieve sustainable food security and decrease poverty in undeveloped countries since its
formation in 1971. In an evaluation of CGIAR, the World Bank praised its efforts but suggested
a shift to genetics research and productivity enhancement. This plan has several obstacles such as
patents, commercial licenses, and the difficulty that third world countries have in accessing the
international collection of genetic resources and other intellectual property rights that would
educate them about modern technology. The International Treaty on Plant Genetic Resources for
Food and Agriculture has attempted to remedy this problem, but results have been inconsistent.
As a result, "orphan crops", such as teff, millets, cowpeas, and indigenous plants, are important
in the countries where they are grown, but receive little investment.[46]

[edit] Private investments

The development and implementation of policies designed to encourage private investments in

research and marketing biotechnology that will meet the needs of poverty-stricken nations,
increased research on other problems faced by poor nations, and joint efforts by the public and
private sectors to ensure the efficient use of technology developed by industrialized nations have
been suggested. In addition, industrialized nations have not tested GM technology on tropical
plants, focusing on those that grow in temperate climates, even though undeveloped nations and
the people that need the extra food live primarily in tropical climates.[41] Some European
scientists are concerned that political factors and ideology prevent unbiased assessment of GM
technology in some EU countries, with a negative effect on the whole community.[47]

[edit] Transgenic organisms

Another important controversy is the possibility of unforeseen local and global effects as a result
of transgenic organisms proliferating. The basic ethical issues involved in genetic research are
discussed in the article on genetic engineering.

Some critics have raised the concern that conventionally-bred crop plants can be cross-pollinated
(bred) from the pollen of modified plants. Pollen can be dispersed over large areas by wind,
animals, and insects. In 2007, the U.S. Department of Agriculture fined Scotts Miracle-Gro
$500,000 when modified genetic material from creeping bentgrass, a new golf-course grass
Scotts had been testing, was found within close relatives of the same genus (Agrostis)[48] as well
as in native grasses up to 21 km (13 miles) away from the test sites, released when freshly cut
grass was blown by the wind.[49]

GM proponents point out that outcrossing, as this process is known, is not new. The same thing
happens with any new open-pollinated crop variety—newly introduced traits can potentially
cross out into neighboring crop plants of the same species and, in some cases, to closely related
wild relatives. Defenders of GM technology point out that each GM crop is assessed on a case-
by-case basis to determine if there is any risk associated with the outcrossing of the GM trait into
wild plant populations. The fact that a GM plant may outcross with a related wild relative is not,
in itself, a risk unless such an occurrence has negative consequences. If, for example, an
herbicide resistance trait was to cross into a wild relative of a crop plant it can be predicted that
this would not have any consequences except in areas where herbicides are sprayed, such as a
farm. In such a setting the farmer can manage this risk by rotating herbicides.

The European Union funds research programs such as Co-Extra, that investigate options and
technologies on the coexistence of GM and conventional farming. This also includes research on
biological containment strategies and other measures to prevent outcrossing and enable the
implementation of coexistence.

If patented genes are outcrossed, even accidentally, to other commercial fields and a person
deliberately selects the outcrossed plants for subsequent planting then the patent holder has the
right to control the use of those crops. This was supported in Canadian law in the case of
Monsanto Canada Inc. v. Schmeiser.

[edit] "Terminator" and "traitor"

An often cited controversy is a "Technology Protection" technology dubbed 'Terminator'.[50] This

uncommercialized technology would allow the production of first generation crops that would
not generate seeds in the second generation because the plants yield sterile seeds. The patent for
this so-called "terminator" gene technology is owned by Delta and Pine Land Company and the
United States Department of Agriculture. Delta and Pine Land was bought by Monsanto
Company in August 2006. Similarly, the hypothetical trait-specific Genetic Use Restriction
Technology, also known as 'Traitor' or 'T-gut', requires application of a chemical to genetically
modified crops to reactivate engineered traits.[50][51] This technology is intended both to limit the
spread of genetically engineered plants, and to require farmers to pay yearly to reactivate the
genetically engineered traits of their crops. Genetic Use Restriction Technology is under
development by companies including Monsanto and AstraZeneca.
In addition to the commercial protection of proprietary technology in self-pollinating crops such
as soybean (a generally contentious issue), another purpose of the terminator gene is to prevent
the escape of genetically modified traits from cross-pollinating crops into wild-type species by
sterilizing any resultant hybrids. Some environmentalist groups, while considering outcrossing of
GM plants dangerous, felt the technology would prevent re-use of seed by farmers growing such
terminator varieties in the developing world and was ostensibly a means to exercise patent
claims.[citation needed] However other environmental groups welcomed the terminator gene as a means
of preventing GM crops from mixing with natural crops.[citation needed]

Hybrid seeds were commonly used in the developed countries long before the introduction of
GM crops. Hybrid seeds cannot be saved, so purchasing new seed every year is already a
standard agricultural practice.

There are technologies evolving which contain the transgene by biological means and still can
provide fertile seeds using fertility restorer functions. Such methods are being developed by
several EU research programs, among them Transcontainer and Co-Extra.

Animal and Plant Transformation: The Application

of Transgenic Organisms in Agriculture

Matthew B. Wheeler, Stephen K. Farrand, and Jack M. Widholm

A transgenic organism carries in all its cells a foreign gene that was inserted by laboratory
techniques. Each transgenic organism is produced by introducing cloned genes, composed of
deoxyribonucleic acid (DNA) from microbes, animals, or plants, into plant and animal cells.
Transgenic technology affords methods that allow the transfer of genes between different
species.

Animal Transformation

Through transgenic animal transformation, new genetic information is introduced into an animal
in one generation without compromising or limiting the overall pool of genetic information.
Transgenic animals are produced by inserting genes into embryos prior to birth. Each transferred
gene is assimilated by the genetic material or chromosomes of the embryo and subsequently can
be expressed in all tissues of the resulting animal. The objective is to produce animals which
possess the transferred gene in their germ cells (sperm or ova). Such animals are able to act as
"founder" stock to produce many offspring that carry a desirable gene or genes.

Transgenic animals have been produced by three methods: microinjection of cloned gene(s) into
the pronucleus of a fertilized ovum, injection of embryonic stem cells into embryos, and
exposure to retroviruses. The third method is not discussed in this article.

The first method is the one that is most widely and successfully used for producing transgenic
mice. After microinjection, the recently fertilized single cell embryos are removed from the
animal. Micromanipulators on a specially equipped microscope are used to grasp each embryo.
A glass pipette drawn or pulled to a fine point immobilizes the embryo on one side, as shown in
the photos to the right. On the opposite side, the foreign DNA is injected into the embryo's
pronucleus--either of two nuclei (male or female) containing half the chromosomes of a fertilized
ovum--with a second finely drawn injection needle. After the injection, the embryos are
transferred back into the hormonally prepared or pseudopregnant recipient females or foster
mothers. The recipients follow normal pregnancy and deliver full-term young. This method is
presently the most efficient for generating transgenic animal lines: about 1 to 4 percent of the
injected embryos result in a transgenic offspring.

The second method involves microinjection of embryonic stem (ES) cells derived from the inner
cell mass of blastocyst-stage embryos (about 7 days postfertilization) into embryos to produce
"hybrid" embryos of two or more distinct cell types. The ES cells are able to produce all tissues
of an individual. Once isolated, ES cells may be grown in the lab for many generations to
produce an unlimited number of identical cells capable of developing into fully formed adults.
These cells may then be altered genetically before being used to produce embryos. When these
transformed cells participate in the formation of sperm and eggs, the offspring that are produced
will be transgenic. Results have shown this method to be promising for producing transgenic
mice. Studies are presently under way at the University of Illinois Department of Animal
Sciences to develop ES cell lines for livestock species such as swine, cattle, and sheep.
To produce transgenic mice, Matthew B. Wheeler microinjects DNA into the pronucleus of one-
cell embryos.

Injection of cloned DNA into embryos. One-cell embryo is positioned for micro-injection into
the pronucleus (left). The plasma membrane has been pierced, and the tip of the needle remains
inside the pronucleus, while DNA is expelled from the needle, causing the pronucleus to swell
visibly.

These methods, which enable the insertion of foreign genes into embryos, have provided the
tools for producing new strains or breeds of animals that carry new, beneficial genetic
information. These technologies do not produce new species but work within the established
genetic framework of existing species to improve them. Some new strains developed include
leaner, more feed-efficient, faster-growing swine containing additional copies of the growth
hormone gene, and mice containing the regulatory elements of the human immunodeficiency
virus (HIV) genome. The latter are used as a noninfectious animal model for the study of AIDS.

The scope of the information acquired from transgenic animal technology is pertinent to virtually
all areas of modern agriculture and biomedical science--cancer research; immunology;
developmental biology; gene expression and regulation; and models for human genetic diseases
such as muscular dystrophy, Lou Gehring's disease, and sickle cell anemia. Potential applications
for transgenic animals include manipulation of milk composition, growth, disease resistance,
reproductive performance, and production of pharmaceutical proteins by livestock.

Plant Transformation

There has been much excitement in the last few years about our ability to genetically engineer
plants using the new techniques of gene isolation and insertion. Paired with standard
methodologies of plant tissue culture and plant regeneration, these new techniques allow us to
construct transgenic plants that contain and express a single, well-defined gene from any source -
microbe, animal, or other plant species. The transgenic plants, usually normal in appearance and
character, differ from the parent only with respect to the function and influence of the inserted
gene.

This directed genetic engineering of plants requires that genes of interest are available, that the
gene be introduced into plant cells capable of regenerating into intact plants, and that the gene
carries with it a selectable marker so that the transformed plant cells can be isolated from a large
population of untransformed, normal cells. Finally, the transformed plant cell must retain its
capacity to regenerate. Certain species such as tobacco and petunia regenerate plants quite easily,
making transgenic plants readily obtainable. Although corn, soybean, and wheat--the primary
agricultural crops of Illinois and the Midwest--are more recalcitrant to these manipulations,
progress is being made toward routine transformation and regeneration of transgenic progeny of
these species.

Several techniques can introduce genes into plant cells. Perhaps the most successful method
involves the pathogenic bacterium Agrobacterium tumefaciens, which has the innate ability to
transfer DNA to plant cells. In nature, this transfer results in formation of plant tumors (crown
galls) at the infection site. Molecular biologists, however, have disarmed this bacterium and
constructed domesticated strains that no longer cause tumors but transfer any DNA of interest to
plant cells. The major disadvantage of the highly efficient Agrobacterium system is that it does
not work with all plant species, most notably the cereals.

Other techniques use physical or chemical agents to transfer DNA into plant cells. Protoplasts,
plant cells that have been stripped of their protective cell walls, will take up pure DNA when
treated with certain membrane-active agents or with electroporation, a rapid pulse of high-
voltage direct current. Once inside the cell, the DNA is integrated and the foreign gene will
express. These two techniques largely depend upon the development of protoplast systems that
retain the capacity to regenerate intact plants. Transgenic corn, rice, and soybean have been
produced with these techniques, especially electroporation. Success rates, however, are low, and
the techniques not very reproducible.

DNA can also be microinjected into target plant cells using very thin glass needles in a method
similar to that used with animals. Microinjection, however, has produced only a few transgenic
plants. The technique is laborious, technically difficult, and limited to the number of cells
actually injected.
Biolistics, a new method, involves accelerating very small particles of tungsten or gold coated
with DNA into cells using an electrostatic pulse, air pressure, or gunpowder percussion. As the
particles pass through the cell, the DNA dissolves and becomes free to integrate into the plant-
cell genome. This improbable technique actually works quite well and has become, along with
electroporation, one of the methodologies of choice. Biolistics has the advantage of being
applicable to whole cells in suspension or to intact or sliced plant tissues. For example, plant
meristems or tissues capable of regeneration can be targeted directly. Unlike transformation or
electroporation, the technique does not require protoplasts or even single-cell isolations. Using
biolistics, transgenic corn and soybean plants have been produced that contain heritable copies of
the inserted gene.

Only a few genes of agronomic importance have been inserted into plants: genes conferring
resistance to certain insects and viruses and also those conferring tolerance to broad-spectrum
herbicides. The latter result in increased herbicide specificity, allowing the farmer to use more
effective, environmentally safe chemical agents. More recently, a gene has been introduced into
tomato that delays overripening and prolongs shelf life of the fruit.

Other traits of interest include those associated with grain quality. Genes to increase the content
of amino acids such as lysine, methionine, and tryptophan in seed will increase nutritional value,
thereby decreasing the need for amending grains with costly feed supplements.

All traits discussed here are associated with expression of single genes. But many important
agronomic traits such as yield and lodging are not well understood and are controlled by many
genes. Manipulating such polygenic traits by genetic engineering will require further research
and the development of techniques for isolating, reconstructing, and transferring complex blocks
of genes. Extensive and promising research is being conducted about additive disease resistance
and stress tolerance, important polygenic traits. Plant genetic engineering is thus moving slowly
but steadily from the laboratory bench into the field.

Matthew B. Wheeler, assistant professor of animal sciences; Stephen K. Farrand, professor of

plant pathology and microbiology; and Jack M. Widholm, professor of plant physiology,
Department of Agronomy
Methods of creation of transgenic animals

For practical reasons, i.e., their small size and low cost of housing in comparison to that for
larger vertebrates, their short generation time, and their fairly well defined genetics, mice have
become the main species used in the field of transgenics.

The three principal methods used for the creation of transgenic animals are DNA microinjection,
embryonic stem cell-mediated gene transfer and retrovirus-mediated gene transfer.

a) DNA microinjection.

This method involves the direct microinjection of a chosen gene construct (a single gene or a
combination of genes) from another member of the same species or from a different species, into
the pronucleus of a fertilized ovum. It is one of the first methods that proved to be effective in
mammals (Gordon and Ruddle, 1981). The introduced DNA may lead to the over- or under-
expression of certain genes or to the expression of genes entirely new to the animal species. The
insertion of DNA is, however, a random process, and there is a high probability that the
introduced gene will not insert itself into a site on the host DNA that will permit its expression.
The manipulated fertilized ovum is transferred into the oviduct of a recipient female, or foster
mother that has been induced to act as a recipient by mating with a vasectomized male.

A major advantage of this method is its applicability to a wide variety of species.

b) Embryonic stem cell-mediated gene transfer.

This method involves prior insertion of the desired DNA sequence by homologous
recombination into an in vitro culture of embryonic stem (ES) cells. Stem cells are
undifferentiated cells that have the potential to differentiate into any type of cell (somatic and
germ cells) and therefore to give rise to a complete organism. These cells are then incorporated
into an embryo at the blastocyst stage of development. The result is a chimeric animal. ES cell-
mediated gene transfer is the method of choice for gene inactivation, the so-called knock-out
method.
This technique is of particular importance for the study of the genetic control of developmental
processes. This technique works particularly well in mice. It has the advantage of allowing
precise targeting of defined mutations in the gene via homologous recombination.

c) Retrovirus-mediated gene transfer.

To increase the probability of expression, gene transfer is mediated by means of a carrier or

vector, generally a virus or a plasmid. Retroviruses are commonly used as vectors to transfer
genetic material into the cell, taking advantage of their ability to infect host cells in this way.
Offspring derived from this method are chimeric, i.e., not all cells carry the retrovirus.
Transmission of the transgene is possible only if the retrovirus integrates into some of the germ
cells.

For any of these techniques the success rate in terms of live birth of animals containing the
transgene is extremely low. Providing that the genetic manipulation does not lead to abortion, the
result is a first generation (F1) of animals that need to be tested for the expression of the
transgene. Depending on the technique used, the F1 generation may result in chimeras. When the
transgene has integrated into the germ cells, the so-called germ line chimeras are then inbred for
10 to 20 generations until homozygous transgenic animals are obtained and the transgene is
present in every cell. At this stage embryos carrying the transgene can be frozen and stored for
subsequent implantation.

Transgenic Animals as Biotechnology

Transgenic animals are just one in a series of developments in the area of biotechnology.
Biotechnology has transformed the way in which we understand processes such as engineering
and manufacturing. These terms now include the use of living organisms or their parts to make
or modify products, to change the characteristics of plants or animals, or to develop micro-
organisms for specific uses. The novel uses of biological techniques such as recombinant DNA
techniques, cell fusion techniques, mono and polyclonal antibody technology and biological
processes for commercial production have altered traditional distinctions and methods (US
Congress, Office of Technology Assessment, 1989). Genetic manipulations at the level of DNA
have also changed long held views as to what is considered to be animal, plant and human. In
turn, these changes have made it more difficult to evaluate the ways in which animals are used
and have obscured distinctions between pure and applied research.
Consideration of the acceptability of creating specific transgenic animal strains or genetic
manipulation involving interchanging DNA between species and kingdoms could be a simple
animal care issue or a societal decision. The following is an attempt to show what the ability to
create transgenic animals or engage in other forms of DNA manipulation means in terms of
traditional ACC functions, not forgetting that this impacts on wider considerations of human
responsibility for the welfare of other life forms.

The creation of transgenic animals is resulting in a shift from the use of higher order species to
lower order species, and is also affecting the numbers of animals used. This shift in the patterns
of animal use is being monitored by the CCAC through the use of the Animal Use Data Form.

An example of the replacement of higher species by lower species is the possibility to develop
disease models in mice rather than using dogs or non-human primates.

In the long term, a reduction in the number of animals used, for example to study human
diseases, is possible due to a greater specificity of the transgenic models developed. On the other
hand, the success of the method has led to using its potential for investigating a wider range of
diseases and conditions. The actual use of some species may be increased, in addition to the
numbers of animals which are sacrificed as donors during the creation process. The potential of
the technology has also made it possible to consider employing cattle, swine, sheep and goats as
processing units to manufacture proteins or as organ donors.

The complex interactive processes of living mammals are not reproducible in vitro. However,
transgenic animals provide a means of evaluating genetic modifications in terms of anatomical
and physiological changes in a complex system. Transgenic models are more precise in
comparison to traditional animal models, for example the oncomouse with its increased
susceptibility to tumor development enables results for carcinogenicity studies to be obtained
within a shorter time-frame, thus reducing the course of tumor development in experimentally
affected animals. However, models are not strict equivalents, so as with any other system care
must be taken in drawing conclusions from the data.

A representative, but non-inclusive, list of purposes for which transgenic animals have been used
indicates the wide ranging application of this biotechnology:
in medical research, transgenic animals are used to identify the functions of specific factors in
complex homeostatic systems through over- or under-expression of a modified gene (the inserted
transgene);

in toxicology: as responsive test animals (detection of toxicants);

in mammalian developmental genetics;

in molecular biology, the analysis of the regulation of gene expression makes use of the
evaluation of a specific genetic change at the level of the whole animal;

in the pharmaceutical industry, targeted production of pharmaceutical proteins, drug production

and product efficacy testing;

in biotechnology: as producers of specific proteins;

genetically engineered hormones to increase milk yield, meat production; genetic engineering of
livestock and in aquaculture affecting modification of animal physiology and/or anatomy;
cloning procedures to reproduce specific blood lines; and

developing animals specially created for use in xenografting.

Important general considerations include the extent to which experience acquired in the
laboratory with regard to husbandry should influence industry standards for keeping animals
created specifically as living machines for the production of proteins, antibodies, etc. What
words are appropriate to describe and evaluate the condition of animals now used as production
units? The successful cloning of Dolly underlines the fact that innovative developments in
animal science are part of the mainstream of biotechnology. In addition, the use of xenografts, at
least at the public health level makes animal and human welfare inseparable.