Genetic Manipulation of Animals How and Why? Chris Tuggle AnS 451/551 lecture December 2004 Department of Animal Science - PowerPoint PPT Presentation

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Genetic Manipulation of Animals How and Why? Chris Tuggle AnS 451/551 lecture December 2004 Department of Animal Science PowerPoint Presentation
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Genetic Manipulation of Animals How and Why? Chris Tuggle AnS 451/551 lecture December 2004 Department of Animal Science

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  1. Genetic Manipulation of Animals • How and Why? • Chris Tuggle • AnS 451/551 lecture • December 2004 • Department of Animal Science • Iowa State University

  2. Overview • A. Why transfer genes into animals? B. Methods of gene transfer • C. Examples 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 or studies requiring specific cell populations 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. 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: • Mutating genes to study structure/function in transgenic mice • Modifying pigs to serve as xenotransplantation donors • Creating livestock with superior production traits

  9. 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

  10. 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

  11. 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

  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. • 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. • To study the function of the gene. • KO: to determine general function • KI: to precisely modify genetics • To mark specific cells

  15. 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

  16. 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

  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 “knock-out/knock-in mice” production • Homologous recombination, many papers

  18. 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

  19. Generation of Specific Mutation In Living Mice What is a “Targeting Vector”? How does one screen for a targeted cell?

  20. 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

  21. Knock-in: adding or changing genetic information, not just disrupting it Positive- select for Neomycin resistance Simple Knock-in: New activity added Reporter (GFP, B-gal)-IRES-neo Exon Exon X TK X Genomic sequences available for homologous recombination Gene function can be disrupted or not Reporter-neo Exon Exon

  22. loxP loxP loxP loxP neo neo Simplest Point mutations- requires Cre or flp recombination to remove selectable marker(s) Specific sequence required for CRE recombinase to function Point mutation Exon 3 TK Exon 2 X X Genomic sequences available for homologous recombination Exon 2 Exon 3

  23. loxP loxP loxP neo Simplest Point mutations- requires Cre or flp recombination to remove selectable marker(s) Positive- select for Neomycin resistance Point mutation Exon 2 Exon 3 Complete removal at ES cell level, transfect CRE into floxed-neo cells to create single point mutant (not regulate-able) Exon 2 Exon 3 Point mutation

  24. loxP loxP loxP loxP neo CRE Conditional Knock-out- Select time or Place for recombination to remove exon Positive- select for Neomycin resistance Exon 2 Exon 3 Selective removal: create mice with floxed-neo allele, breed heterozygous mouse with mouse expressing CRE in specific cell type- eliminates everything between loxP sites Exon

  25. loxP loxP loxP loxP neo CRE Conditional Knock-out- Select time or Place for recombination to remove exon Positive- select for Neomycin resistance Exon 2 Exon 3 Selective removal: create mice with floxed-neo allele, breed heterozygous mouse with mouse expressing CRE in specific cell type- eliminates everything between loxP sites Exon

  26. loxP loxP loxP loxP neo CRE Conditional Knock-out- Select time or Place for recombination to remove exon Positive- select for Neomycin resistance Exon 2 Exon 3 Selective removal: create mice with floxed-neo allele, breed heterozygous mouse with mouse expressing CRE in specific cell type- eliminates everything between loxP sites Promoter expressing CRE in specific: Cells Stage Tissue Exon

  27. Methods of Gene Transfer (Cont’d) 4. Sperm-mediated DNA transfer (? Controversial) 5. Injection of naked DNA or modified DNA into tissue • Skin (Hengge, 1995), muscle, mammary gland or into bloodstream (DNA coupled to ligand or liposomes for cell uptake; Zhu, 1993). • Muscle injection of skeletal alpha-actin promoter driving GHRH in pigs • > detected expression and increased growth Draghia-Akli et al., 1997)

  28. Methods of Gene Transfer (Cont’d) 4. Sperm-mediated DNA transfer (? Controversial) 5. Injection of naked DNA or modified DNA into tissue • Skin (Hengge, 1995), muscle, mammary gland or into bloodstream (DNA coupled to ligand or liposomes for cell uptake; Zhu, 1993). • Muscle injection of skeletal alpha-actin promoter driving GHRH in pigs • > detected expression and increased growth Draghia-Akli et al., 1997)

  29. Methods of Gene Transfer (Cont’d) 4. Sperm-mediated DNA transfer (? Controversial) 5. Injection of naked DNA or modified DNA into tissue • Skin (Hengge, 1995), muscle, mammary gland or into bloodstream (DNA coupled to ligand or liposomes for cell uptake; Zhu, 1993). • Muscle injection of skeletal alpha-actin promoter driving GHRH in pigs • > detected expression and increased growth Draghia-Akli et al., 1997)

  30. Methods of Gene Transfer (Cont’d) 6. Transfer of (manipulated) nuclei into enucleated embryos • By using unmanipulated adult cells: • Dolly (not transgenic) Wilmut et al., 1997 • Cloned mice (not transgenic) Wakayama et al., 1998. • By using manipulated fetal fibroblast cells: • Transgenic sheep Schnieke et al., 1997, work on other species reported as well.

  31. Steps in Nuclear Transfer and Cloning

  32. Precise Genetic Modification: NT of Gene Targeted Nuclei Embryonic Stem cells Transfer embryo into recipient female Gene Targeting by Homologous Recombination Cloned animal born

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

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

  35. Examples 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

  36. 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.

  37. 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.

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

  39. 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. • NRAMP1- genetic proof of positionally cloned genes with variant explaining disease

  40. Vital dye “mutations or non-mutations” • Fuse GFP, EGFP, etc. to protein to see where protein is within living cells, or to tag specific cell types for sorting (GFP) and analysis 3. Mutagenesis - Study Gene Function • Combination Recessive/Dominant mutations • Null mutations in one gene, replacement (by knock-in) of another gene product which may be dominant • Examples: gene family member replacement-- to show redundant and non-redundant functions

  41. 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 addition studies “Gene Pharming” • Blood clotting factors • Nutrition additives • Other metabolism enzymes

  42. 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. • Pigs and sheep with deletion of enzyme responsible for hyperacute rejection (alpha 1, 3 galactosyl transferase) • 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)). • Direct injection of plasmids- growth effect of expressed GHRH. • 2001-present • 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 • Many new results with NT- cattle, sheep, mice, goat, rabbit, cat, mule, horse, pig, rat

  43. Uses for “Gene Trap” technology in Functional Genomics • Gene Trap- an insertional mutagenesis technique where the mutagen is an 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

  44. 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

  45. Gene trap transgenic embryos