Genetic manipulation of Animals How and Why? Chris Tuggle Department of Animal Science

1. Genetic manipulation of Animals • How and Why? • Chris Tuggle • Department of Animal Science • Iowa State University

2. Overview • A. Why transfer genes into animals? B. Methods of gene transfer • C. Effects of gene transfer into animals

3. Why transfer genes into animals? Basic Research • Model Biological processes • Study gene regulation/function in context of animal. • Identify and isolate new genes of interest • Mark cell lineages for developmental studies Several advantages of Transgenesis over other methods to study Biology

4. Why transfer genes into animals? Applied Research • Produce therapeutic proteins • Cure disease; correct of genetic defects. • Generate models for human disease. • Production of xenotransplant donors. • Production of superior animals.

5. Why transfer genes into animals?i.e., Why not do this research in other ways? 1. Process under study not applicable to culture systems. • Cell type to be studied cannot be cultured. • Inability to replicate in vivo environment in tissue culture (cell<->cell; tissue interactions).

6. Why transfer genes into animals? • 2. Developmental studies require a living mammal • to study certain aspects of: • Implantation events/physiology • Maternal:fetal interactions • Birth events/physiology

7. Why transfer genes into animals? 3. Animal tissue expression required for protein effectiveness or activity • Mammal-specific modifications to protein for activity. 4. Protein production in transgenic animal cost-effective 5. Scientific goal requires use of animal • Human gene therapy, xenotransplantation • Animal genetic improvement

8. Sidelight: The Mouse as the model research mammal • Advantages for the Use of Mice in Genetic Manipulation Research • Many genetic manipulations possible (add genes, delete genes) • Fast reproductive cycle for a mammal -egg to egg about 3.5 months • Many genetic strains and mutants described • Best characterized animal which is closest to humans; of laboratory animals, most directly useful for biomedical genetics research

9. Sidelight: The Mouse as the model research mammal • Disadvantages for the Use of Mice in Genetic Research • Large genome (can be difficult to screen for certain manipulations) • Long gestation time (relative to invertebrates; flies, worms) • Inaccessibility of the fetus for observation/manipulation; especially relative to vertebrate species like chicken and zebrafish • Expense (complicated growth media [uterus]) • Ethical concerns require good justification

10. Categories of Genetic Manipulation • Genetic modification is performed early enough in developmental life of the animal that all cells of resulting transgenic animal carry the modification. Germ-line Genetic Modification • Thus germ cells (sperm or egg) are modified and will transmit the transgene to the next generation as any other chromosomal gene • Examples: • Expressing foreign proteins in milk of transgenic mice • Modifying pigs to serve as xenotransplantation donors • Creating livestock with superior production traits

11. Categories of Genetic Manipulation Somatic Cell Genetic Modification • Modification is performed relatively late in the life of the animal (often in the young adult) so that one or only a few cell types are genetically changed. • Examples: • Thus as germ cells are not modified, the transgene which may exist elsewhere in the animal cannot be passed to the next generation. • Human ex vivo gene therapy • Injection of DNA into mammary tissue to identify optimal transgene construct

12. Detect color Flanking DNA Promoter-Reporter Types/Uses of Transgenes Constructs 1. Reporter Gene Fusions • Used to dissect gene expression mechanisms -to find DNA elements controlling expression • Reporter is an easy-to-analyze protein; usually an enzyme that produces a colored or fluorescent product.

13. Gene Regulation Without Enhancer With Enhancer

14. Types/Uses of Transgenes Constructs 2. Knockout/Knock-in Constructs • For targeted disruption or specific modification of a gene using homologous recombination. • To study the function of the gene. • KO: to determine general function • KI: to precisely modify genetics. • Selectable marker gene is inserted into a gene in cultured cells, then these cells are used to create a mouse with the disruption in every cell.

15. Positive-Negative Selection- Technology to Enrich for True Homologous Recombinants Negative: TK+ activity can be selected against Generalized contruct for HR Positive- select for Neomycin resistance Neor cassette X Exon disrupted X Thymidine kinase gene Genomic sequences available for homologous recombination Gene function disrupted at this allele Neor cassette Exon disrupted Exon disrupted

16. Types/Uses of Transgenes Constructs 3. Directed RNA/Protein Expression • For specific production of a specific gene product such as a pharmaceutical protein which is missing in a patient with inborn error of metabolism. • Many levels of control may have to understood for accurate direction of expression: • Tissue specificity • Time or stage specificity • Subcellular localization signals • Excretion signals

17. Methods of Gene Transfer 1. Pronuclear microinjection of fertilized embryos • Many papers describing successful use • Production of multi-subunit enzyme activity by multiple gene transfer Prunkard (1996) 2. Recombinant retroviral infection • May be used more in the future with high resolution ultrasound injection in utero 3. Injection of blastocysts with manipulated embryonic stem cells for “knockout/knock-in mice” production • Homologous recombination, many papers 4. Transfer of in vitro modified nuclei into enucleated embryos • Again homologous recombination • Few papers in field- mouse, pigs (sheep?) • KO work published only

19. Methods of Gene Transfer 1. Pronuclear microinjection of fertilized embryos • Many papers describing successful use • Production of multi-subunit enzyme activity by multiple gene transfer Prunkard (1996) 2. Recombinant retroviral infection • May be used more in the future with high resolution ultrasound injection in utero 3. Injection of blastocysts with manipulated embryonic stem cells for “knock-out/knock-in mice” production • Homologous recombination, many papers 4. Transfer of in vitro modified nuclei into enucleated embryos • Again homologous recombination • Few papers in field- mouse, pigs (sheep?) • KO work published only

20. Methods of Gene Transfer 1. Pronuclear microinjection of fertilized embryos • Many papers describing successful use • Production of multi-subunit enzyme activity by multiple gene transfer Prunkard (1996) 2. Recombinant retroviral infection • May be used more in the future with high resolution ultrasound injection in utero 3. Injection of blastocysts with manipulated embryonic stem cells for “knock-out/knock-in mice” production • Homologous recombination, many papers 4. Transfer of in vitro modified nuclei into enucleated embryos • Again homologous recombination • Few papers in field- mouse, pigs (sheep?) • KO work published only

21. Generation of Specific Mutation In Living Mice How does one screen for a targeted cell?

22. Methods of Gene Transfer 1. Pronuclear microinjection of fertilized embryos • Many papers describing successful use • Production of multi-subunit enzyme activity by multiple gene transfer Prunkard (1996) 2. Recombinant retroviral infection • May be used more in the future with high resolution ultrasound injection in utero 3. Injection of blastocysts with manipulated embryonic stem cells for “knock-out/knock-in mice” production • Homologous recombination, many papers 4. Transfer of in vitro modified nuclei into enucleated embryos • Again homologous recombination • Few papers in field- mouse, pigs (sheep?) • KO work published only

23. Steps in Nuclear Transfer and Cloning

24. Precise Genetic Modification: NT of Gene Targeted Nuclei Embryonic Stem cells Fetal Fibroblasts Gene Targeting by Homologous Recombination

25. Effects of Gene Transfer into Animal/Humans 1. Disease Models/Curing Disease 2. Studying Gene Regulation in the Whole Animal 3. Mutagenesis - Study Gene Function 4. Gene Product Synthesis/Breed Improvement 5. Human Gene Therapy

26. Effects of Gene Transfer into Animal/Humans • Dominant disease models • Oncogenesis - breast cancer mouse model (mice with strong promoter expressing oncogene are cancer-prone) • Spongiform encephalopathy (mad cow disease) is caused by “prions”. Transgenice mice expressing hamster “prion” gene have scrapie lesions and transgenic mice are more easily infected by scrapie agent. 1. Disease Models/Curing Disease • Human immune system model for AIDS research • Replacement of parts of mouse immune system with that unique to humans. • Curing inborn errors of metabolism • Usually recessive mutations, so only need to ADD gene information- many examples.

27. 2. Studying Gene Regulation in the Whole Animal • Main method is to use reporter genes to identify regulatory regions that express reporter in specific cell types. • Tissue-specific--mammary gland, liver, muscle. • Region-specific--developmentally regulated genes. • Stage-specific-example; globin gene family.

28. In vivo testing of DNA regulatory sequences Wild Type Mutant Flanking Sequence Flanking Sequence

29. Dominant mutations • Excess human Growth Hormone - giant mouse 3. Mutagenesis - Study Gene Function • Interfering with formation of multiprotein structures and assemblies • Recessive mutations • Null mutations (targeted mutations using ES cells) many examples now: • Examples: • transgenic mice with knockout in tumor suppressor gene (p53) are tumor-prone. • NRAMP genetic proof

30. 4. Gene Product Synthesis/Breed Improvement • History of Transgenic Farm Animals • 1985-1992 • Modification of animal for production trait improvement • GH or IGF overexpressing transgenes • 1988-present • Modification of animal to produce pharmaceuticals- “Gene Pharming” • Blood clotting factors • Nutrition additives • Other metabolism enzymes

31. 4. Gene Product Synthesis/Breed Improvement • 1985-present • Modification of animal to serve as donor for human transplantation: • “Xenotransplantation”; major problem that needs to be overcome is hyperacute rejection • Transgenic organ transfer not performed in humans yet. • 1997-2000 • Use of nuclear transfer to clone animals; adult, fetal cells as nuclei donor. Use of genetically manipulated fibroblast cells and subsequent nuclear transfer to generate transgenic sheep, cattle, mice (gene addition and locus-specific studies(sheep and mice)). • 2001-2 • Cell culture approaches to modify developing chicken cells. (2001) • Use of nuclear transfer to produce targeted modifications in pigs (2002)- first knock-out in pigs

32. Uses for “Gene Trap” technology in Functional Genomics • Gene Trap- a insertional mutagenesis technique where the mutatgen is assayable marker gene (lacZ) • Used to identify and study genes involved in development because different blue stain patterns can be easily screened. • Used extensively now for “functional genomics” • a) generate mutations in many new genes • b) see expression pattern in single copy; also • c) if breed to homozygosity, observe • phenotype of insertional mutation at this gene

33. Use transgenesis and “Gene Trap” construct to find new genes Gene Trap: - promoterless lacZ gene - no activity unless integrates into gene - blue stain shows where new gene is normally expressed LacZ Gene B-like Expression possible No Expression Gene A Gene B Gene C

34. Gene trap transgenic embryos

35. Conclusions • Many useful methods to modify the genome of mice to understand and model biology • Recent developments in cloning and nuclear transfer holds promise in gene transfer into large animals for genetic improvement • New uses for transgenic mice in the Human Genome Project • All such manipulations should be ethically justified

36. DNA Microinjection of Fertilized Oocyte