Notes Life Sciences (Raffles Institution Year 4 Biology)

Cloning Cloning is creating an organism that is an exact genetic copy of another. Every bit of DNA is same between the two. Types of Cloning 1. Artificial Embryo Twinning 2. Somatic Cellular Nuclear Transfer a. Therapeutic Cloning b. Reproductive Cloning 3. Vegetative Propagation (descendants of single plant) 4. DNA/Molecular Cloning Recombinant DNA Technology Artificial Embryo Twinning 1. Zygote divides into two-celled embryo, and the two cells separate. 2. Each cell continues dividing on its own, developing into separate individuals. 1. Manually separates early embryo into individual cells. 2. Plated in Petri dishes. 3. Resulting embryos placed in surrogate mother, where they are carried to term and delivered. 4. All the embryos came from the same zygote, and hence they are genetically identical. Somatic Cellular Nuclear Transfer 1. 2. 3. 4. Nucleus of somatic cell (diploids) removed Inserted into unfertilisted and enucleated egg cell (nucleus removed) Egg with donated nucleus divides, to form an embryo Embryo placed inside surrogate and develops

Reproductive Cloning The blastocyst is placed into a surrogate mother. Therapeutic Cloning The embryonic stem cells are separated for tissue culture. Dolly the Sheep Created by Somatic Cellular Nuclear Transfer

Done by Goh Zuo Min

Not truly an identical clone of donor animal because only chromosomal or nuclear DNA is the same as the donor Some genetic materials come from mitochondria in the cytoplasm of the enucleated egg DNA/Molecular Cloning Recombinant DNA Technology 1. Identify and isolate a gene associated with a phenotype (location, DNA sequence) 2. Replicate to form many copies of the gene, using restriction enzymes to remove sections of DNA at a target sequence 3. Gene inserted into plasmids, which are structures that form from bacterial DNA, using DNA ligase to seal the DNA fragments 4. Plasmids are inserted into bacterial cells and then cultured Cloning an Organism vs. Gene Cloning an organism is making an exact genetic copy of that organism. Cloning a gene is isolating an exact copy of a single gene from the entire genome of an organism. Used to study the function of the individual gene in the laboratory. Risks of Cloning 1. High failure rate a. Success rate 0.1 3.0% b. Enucleated egg and transferred nucleus may not be compatible c. Egg with newly transferred nucleus may not begin to divide or develop properly d. Implantation of the embryo into the surrogate mother might fail e. The pregnancy itself might fail 2. Problems during later development a. Large Offspring Syndrome (LOS) b. Cloned animals that survive tend to be much bigger at birth than their natural counterparts, and have abnormally large organs c. Lead to breathing, blood flow and other problems d. LOS doesnt always occur; scientists cannot reliably predict its occurrence e. Some clones without LOS have developed kidney or brain malformations and impaired immune systems 3. Abnormal gene expression patterns a. Clones may not express the right genes at the right time Done by Goh Zuo Min

b. Re-programming the transferred nucleus to behave like in an early embryonic cell, and for subsequent differentiation, is difficult 4. Telomeric differences a. As cells divide, their chromosomes get shorter. DNA sequences at both ends of a chromosome, called telomeres, shrink in length every time the DNA is copied. b. The older the animal, the shorter its telomeres. c. Transferred nucleus is already old shortened telomeres may affect its development or lifespan] Frozen Ark 1. Scientists take small tissue samples from animals, so life is not endangered 2. Tissue may be frozen for safe-keeping 3. DNA extracted from tissue sample straight after it was obtained or after freezing 4. DNA can be used for research, which may one day lead to resurrection of extinct species 5. Some DNA samples are sent to other labs as an insurance against damage or loss 6. Unused DNA can be frozen, potentially for thousands of years Ethical Considerations 1. What is the moral status of the organisms created by cloning? 2. Is it permissible to create a developing human entity only to destroy it? 3. Is it right to seek human eggs for scientific research? 4. What are the ethical issues relating to the person whose cells are being cloned? 5. Will therapeutic cloning facilitate reproductive cloning, the birth of a cloned baby?

Done by Goh Zuo Min

Stem Cells The body is made up of over 200 different types of cells. All these cell types come from a pool of cells, named the embryonic stem cells (ESCs), totipotent stem cells, in the early embryo from the inner cell mass in the blastocyst. Stem cell is a single cell that can replicate itself and has the potential to differentiate into many cell types. Properties of Stem Cells 1. Unspecialised 2. Capable of dividing themselves for long periods to produce more stem cells 3. Can differentiate into specialized cell types under appropriate conditions Unspecialised stem cells Long term self-proliferation (under appropriate conditions) Differentiate into many cell types Types of Stem Cells 1. 2. 3. 4. 5. Early Embryonic Stem Cells (Totipotent) Blastocyst Embryonic Stem Cells (Pluripotent) Fetal Stem Cells (Pluripotent) Umbilical Cord Stem Cells (Multipotent) Adult Stem Cells (Unipotent)

Stem Cell Potency Totipotent means that it can differentiate into all cell types. Pluripotent means that it can differentiate into cells derived from any of the three germ layers. Multipotent means that it can differentiate into a number of cells, but only those of a closely related family of cells. Unipotent means that it can produce only one cell type, their own. Uses of Stem Cells 1. Human Development (how cells differentiate and function) 2. Drug Development (test for effects of drugs) Done by Goh Zuo Min

3. Stem Cell Therapy (replace damaged tissue, e.g. heart failure, diabetes, Parkinsons) Stem Cell Therapy 1. Define the Problem 2. Finding the Right Type of Stem Cell 3. Match Stem Cells with Recipient a. Immune system attacks foreign material including stem cells and tissues b. Transplanted stem cells must match recipient closely c. Tissue typing test performed using blood samples from both individuals d. However, immune responses are typically muted in the brain compared to other areas of the body e. Fetal tissues should not trigger an intense immune response in recipient 4. Put Stem Cells in Right Place 5. Make the Transplanted Stem Cells Perform Control 1. Receive treatment 2. Do not receive treatment 3. Receive surgical procedure, and pre- and post- surgical care with no fetal tissue implant (placebo effect)

Done by Goh Zuo Min

Model Organisms

Apotiosis Importance

microRNA Role Process of RNA interference in modulating the expression of DNA Revolutionize personalized disease therapy

Bacterial Transformation GFP gene carried on a plasmid

Done by Goh Zuo Min