Chapter 7

1. Chapter 7 Animal Biotechnology

2. Benefits of Genetically Engineered Animals • Used to develop new medical treatments • Improve our food supply • Enhance our understanding of biology of all animals, including humans

3. Animal Models • Animal systems are a model for the human system • Polio vaccine was developed using animals as test systems • Cataract surgical procedures were developed with animals • Dialysis was tested first in animals before being applied to human conditions

4. Regulation of animal research • Animal Welfare Act • Sets specific regulations regarding, housing, feeding, cleanliness and medical care of animals • Researchers must first develop a plan describing • Appropriateness of species to be used • Minimum number of animals needed for test • Oversight committee reviews and approves plan • Government agencies monitor welfare of the test animals

5. Phase Testing • Testing a new product for safety in humans involves vigorously following scientific methodology developed for animal systems • Involves collecting data from a statistically significant number of trials (experiments) in lab cell tissue cultures, in live animals and in human subjects. • 3-stages of testing Human trials Animal model Tissue culture if successful if successful

6. Testing • If test results using cell cultures indicates toxicity of product, then product will never be tested on live animals. • Testing on live animals requires evaluation of more than one species, since different species may respond differently.

7. Phase Testing • Animal models can provide the following information on a new product • Absorption of chemical by body • Body metabolism of chemical • Time require for chemical or product to be excreted • If significant problems are encountered with product in live animals, then product is never tested in humans.

8. Side-effects of new drugs discovered in animal models • Example • Propecia • Used to encourage hair growth • Animal studies indicated that serious birth defects occurred in male offspring when pregnant animals were given large doses of drug • As a result of animal tests, warnings were put on containers of Propecia to avoid birth defects in humans using drug.

9. How do you select appropriate animal as a model for the human system? • Look for genetic homology between animal and human systems. • In addition, identify animal that • Has short time between generations • Can produce lots of offspring in each generation • Can be easily maintained and manipulated in the laboratory

10. Lung and cardiovascular Immune system HIV and AIDS research Dog Mice Monkey and chimpanzee Matching animal systems as models for the human system SystemBest animal model for human

11. Zebrafish as a model organism • Popular hardy aquarium fish • Size of a paperclip • Can live in small spaces • Spawn continuously • 3 months between generations • 200 progeny/week/female • Complete organ development within 120 hours of birth • Because the embryos inside a female are easily visible to naked eye, they are ideal animal systems for evaluating the effect of a new drug on development

12. Homology Testing Oxford Grid human Dots represent similar genes Boxes with more than one dot represent conserved sequences

13. Easy to follow drug effect on embryo development under microscope, since egg can mature outside female.

14. Zebrafish • Lots of genetic similarity to humans • Egg lends itself to genetic transfer • no need to implant an egg inside a donor mother for gestation. • Embryos are transparent, making it possible to study cell division under microscope from first hour of creation. • transplant gene into embryo • Because the genetics of zebrafish and humans are similar, they are ideal animal systems for determining whether a new drug induces genetic mutations

15. Exchanging genes between individuals stopped Select for recombinant before somatic cells stop dividing Somatic cell of human Cloned in tissue culture Reconstructed embryo Chromosome 5 Homologous Recombination (rare event) Look for effect of gene disruption or insertion on organ development Targeted gene disruption or insertion

16. Homologous Recombination flawed gene Person 1 chromosome good gene Person 2 chromosome gccatt ccgtc cggtaa ggcag Mix chromosomes and promote DNA replication by mitosis. gccatt ccgtc cggtaa ggcag Exchange section of DNA on one chromosome with a section of DNA containing good gene on another chromosome. Offspring now has a copy of good gene from Person 2 in allele donated from Person 1

17. Embryo Reconstruction Cells generated from original somatic cell in which homologous recombination occurred

18. Nuclear Transfer Step 1: Remove the nucleus from an egg egg Suction to hold egg Perforate egg with needle and withdraw intact nucleus

19. Reconstructed embryo Genetically modified somatic cells Step 2: insert nucleus from transformed cell Nucleus from somatic cell Egg divides to produce differentiated cells An new clone, a genetic copy of the donor, forms when the egg starts to divide Functional tissue or organ

20. Cloning Creating Dolly: A breakthrough in cloning

21. Embryo twinning (conventional approach) • splitting embryos in half to produce artificially created twins • commonly practiced in cattle industry today • limitation is that organisms being copied is unknown • you may or may not end up with an animal that has the desired characteristics and you have to wait until the animals is full-grown to find out. • Dolly was created from an adult cell-not an embryo • Dolly was an exact copy of an adult with known characteristics. • How is this done?

22. Cells collected from donor animal and put in a culture medium that keeps them alive but prevents their replication and stops gene expression. Egg of an animal has it’s nucleus (DNA) removed (enucleation) Nucleus of cultured somatic cells from donor animal are then inserted into a recipient animal’s egg next to its cytoplasm. Apply low-level electric charge and fuses with egg cytoplasm to produce a 1-cell cloned embryo. New cell containing egg behaves as if it were an embryonic cell rather than an adult cell. Cell division occurs just as it would in an ordinary fertilized egg. Transfer embryo to surrogate mother for gestation. Newborn will be genetically identical to donor

23. Successfully cloned species • Sheep • goat • pig • cow • endangered cow (gaur) • house cat

24. Limits of cloning • Viable cell is required • Success rate is still low • Dolly was successful only after 277 failed attempts • only 29 implanted embryos lived longer than 6 days • Many clones are born with defects • kidney problems • diabetes • crippling disabilities • old before their time-telomere length • Dolly was diagnosed with arthritis -premature aging?

25. Cloning as a means of producing replacement body parts? • Idea is to reduce chance of cloned tissue from being rejected by original “parent”. • It would take years for clone to produce the organs to be used for transplant

26. Benefits of Cloning • Reduce variability of responses of a population being used to test new drugs, etc. • avoids confounding factor of different genetic predispositions • Preservation of endangered species • cloning pandas using common black bear as surrogate host. • Reduce time to produce new breeds of farm animals from 6-9 years 3 years

27. Early experiments on transgenic animals • A new gene was added to a cell grown in a tissue culture and the effects on that one cell were observed. • With the introduction of cloning, a gene could be added to many cells, and all the cells could be screened to see which one(s) contained the gene. • Each cell that contained the gene could then be used to grow a complete animal using cloning technology

28. Transgenic techniques • Retrovirus-mediated transgenesis • infect mouse embryo with retroviruses before the embryos are implanted into an animal for gestation. • Retrovirus acts as a vector for the new DNA • size of new DNA is limited • viruses genetic material can interfere with embryo development • not very efficient cell nucleus embryo retrovirus

29. Pronuclear injection • Introduction of foreign DNA at earliest possible stage of development of the zygote (fertilized egg) • Just before the egg and sperm cells join, DNA is injected into the nucleus of either cell. • Since the DNA is injected with a syringe, no vector is required and no vector genetic material is introduced that could complicate outcome

30. blastocyte blastocyte Foreign DNA Embryonic stem cell method • Embryonic stem cells are collected from inner cell mass of blastocytes • Cells are mixed with foreign DNA • some cells take up the foreign DNA and incorporate it into cell’s own DNA in the nucleus and are “transformed” • Transformed cells are injected into the inner cell mass of the host blastocyte for differentiation and development Transformed cell

31. Transgenics to make milk healthier for humans • Lactoferrin-protein that binds iron needed by human babies for development • introduce gene for this protein into cells of cow that are responsible for milk production • Human immune genes introduced into cows as a factory for human antibody production.

32. Transgenics as a means of deleting genes and their functions • Deleting a gene is a way of determining what its function is in the cell • Active gene is replaced with a gene that has no functional information • When the gene is “knocked out” by the useless DNA, the trait controlled by the active gene is eliminated from the animal .

33. Knockout Mice Knockout mice begin as embryonic stem cells with specifically modified DNA that has been prepared by recombinant techniques. The modification results in a nonsense mutation in the normal gene of the animal.

36. Homologous recombination within target gene Chromosome with normal gene normal gene Plasmid with useless DNA Useless DNA gccatt ccgtc cggtaa ggcag Recombination between vector and chromosome gccatt ccgtc cggtaa ggcag insert section of DNA of gene on vector into a section of DNA containing good gene on chromosome of stem cells. Chromosome is modified with a useless form of the gene. Look for a trait that has changed

38. Random insertion of useless gene at a location other than the target gene Chromosome with normal gene normal gene Vector with useless DNA Useless DNA gccatt ccgtc cggtaa ggcag Recombination between vector and chromosome gccatt ccgtc cggtaa ggcag Insert section of DNA of useless gene on vector into a section of chromosome that does not disrupt target gene. Chromosome is modified with a useless form of the gene at some other site than target gene

40. chimera Transformed stem cell Blastocyte Knockout mouse with nonfunctional gene in all its differentiated somatic cells Not all cells had the trait changed Need to crossbreed for 2 generations to get all cells to lose trait.

41. Antigen (Ag) Antibody (Ab) Producing human antibodies in animals • Antibodies are proteins whose structure gives it the ability to bind very specifically to other proteins Region of antigen protein that is specifically recognized and bound by antibody

42. Antibodies could be designed that target and inactivate cancer cells in our bodies. • Myelomas: antibody-secreting tumors • Monoclonal Abs (mAb) are produced from myeloma cells that produce an Ab that reacts with only one region of an antigenic protein

43. Making cells that produce monoclonal antibodies The specific antibody is released into the culture medium and recovered Once a cell line is identified that produces an antibody against a specific antigen, it can be replicated and the cells frozen until needed to make the specific antibody

44. Review • Approaches to change genomes of animals • Nuclear transfer of genetically modified somatic cell into an egg. Rapid growth of organs for transplant into donor animal. • Nuclear transfer of somatic cell into egg implant into surrogate to produce viable organism (Dolly) • Retrovirus mediated genetic modification in animal genome. • Nuclear transfer of embryonic stem cell into egg. Implant into surrogate to produce viable organism

45. Pronuclear injection