Transgenesis in Mouse--转基因鼠

1 Transgenesis in Mouse Chi-Kuang Leo Wang September 22nd, 2008 2 Genetic Manipulation • Transgenesis • Gene Targeting • ENU • Embryonic Stem Cells & Embryology • Congenic, Assisted reproduction, ICSI & Cloning 3 Science reporters, BBC News 4 Transgenic Research BSA injection: 1966, Lin SV40: 1974, Jaenisch & Mintz Viral infection: 1976, Jaenisch HSVtk: 1980, Anderson, Capecchi Pronuclei injection: mRNA 1980, Brinster DNA 1980, Gordon YAC 1992, Schedl BAC 1997, Antoch, Nielsen, Yang 2 5 Transgenic and Gene Targeting Related Paper Numbers 0 1000 2000 3000 4000 5000 6000 7000 8000 19 81 19 82 19 83 19 84 19 85 19 86 19 87 19 88 19 89 19 90 19 91 19 92 19 93 19 94 19 95 19 96 19 97 19 98 19 99 20 00 20 01 20 02 20 03 20 04 20 05 Transgenic Targeting 6 Transgenic Technology • Microinjection • Retroviral infection • Transformation • Sperm-mediated gene transfer • Transposon “Overexpression” “Random integration; Multiple copy” 7 基因型鑑定 14天採樣檢驗 19天懷孕期 輸卵管胚移植手術 胚原核顯微注射 取單細胞受精卵 賀爾蒙刺激排卵 PolII EGFPEGFP pA 設計建構-轉殖基因 Transgenic Production 8 Overexpression • Reporter • Wild type • Inhibition • Competition • Replacement • Rescue 3 9 Heterochromatin HeterochromatinActive chromatin (Euchromatin) LCR: Locus Control Region E: Enhancer P: Promoter S: Silencer 10 Reporter genes • Secretory- hgh • Enzyme activity- CAT, luc • Positive selection- neo, hygro, puro • Positive & negative selection- HyTK, tk*, mhprt* • Visual- lacZ, FPs (GFP, EGFP, hrGFP etc.) • Combination-βgeo 11 Accurate Transgene Expression ‹ Pattern Temporal Spatial ‹ Level ‹ Completeness 12 Transgene expression (overexpression) Endogenous gene (expressing) Endogenous gene (inactive) • Functional (wild type) • Mutated (active) • Dominant negative (inactive) • Anti-sense / dsRNAi • Expression analysis (reporter) • Gene dosage • Miss expression (ectopic) • Rescue / complementation (wild type) • Replacement (mutated or different gene) 4 13 Limitations in conventional transgenesis • Construct capacity • Expression accuracy • Expression level • Position effects, ectopic expression • Copy number, repeats • Transgene stability; expression variation, rearrangement 14 Now and Future • Accurate control- site, format, level • Tools- animal model, drug testing, research material, research tools • Biofactory- DNA/ RNA/ protein products, cell/ organ donor, cell/ organ recipient • Gene delivery/ gene therapy 15 Giraldo & Montoliu, Tg Res 10, 2001 16 Giraldo & Montoliu, Tg Res 10, 2001 5.5 kb 0.28 kb 250 kb 5 17 Recombinant DNA Vectors • Plasmids 20+ kb • Lambda vectors 30 kb • Cosmid 40 kb • PAC (P1 artificial chromosome) 80-90 kb • BAC (bacterial artificial chromosome) 100-300 kb • YAC (yeast artificial chromosome) 100-1000+ kb 18 Giraldo & Montoliu, Tg Res 10, 2001 19 YAC (1993) • Tyrosinase 250 kb • Myf-5 680 kb • Imprinting (Igf2 & H19) 130 kb • Immunoglobulin loci 1.3 Mb • Alzheimer disease 400 kb • Down’s syndrome 2 Mb • Human b-globin locus 250 kb 20 BAC (1997) • Mouse apolipoprotein B 207 kb • Mouse clock 140 kb • Goat α–lactalbimin 160 kb • Mouse α 9acetilcholine receptor 140 kb • Human β–globin locus 100 kb • Mouse OCTN2 204 kb • Human Spinal Muscular Atrophy 115 kb 6 21 YAC / BAC Transgenesis • Study gene structure-function relationships • Human disease model • Complement for mouse mutation • Bioreactors / biofactories • Genome / library screening in vivo 22 Obstacles in YAC / BAC Transgenesis • Construct engineering / modification capabilities • DNA amplification / purification • Construct release, linearization or uncut • Construct purification / handling • Injection; skill • Genotyping; integrity 23 Future-1 HACs - Next generation gene therapy vectors “Synthetic, nonimmunogenic gene-transfer vector” • Retroviruses Proliferating cells only • Adenoviruses Do not stably integrate Inflammatory response • DNA-liposome complexes Inefficient & transient • Origin of replication DNA synthesis • Telomeres Stabilize chromosome ends • Centromere/Kinetochore sequence Mitotic stability 24 Future-2 HACs - Next generation gene therapy vectors • Ethical issue • Stability • Removable / controllable / changeable • Integrated form / free form • Inheritable? 7 25 Giraldo & Montoliu, Tg Res 10, 2001 26 Nat Rev Gen, 2, 743-755, 2001 27 Nat Rev Gen, 2, 743-755, 2001 28 RNA interference (RNAi) - Knockdown Nat Chem Biol, 2, 2006 8 29 Nat Chem Biol, 2, 2006 30 Gene overexpression v.s. gene repression Conventional BAC/LCR Knockdown • DNA + + + • RNA + +++ --- • Protein + +++ --- 31 RNAi – advantages; faster, easier, cheaper • Tolerate polymorphism – Interstrains, interspecies • Homologous sequence targeting • Variable repression efficiency - Dosage analysis • Less construction effort • Small: Easy in cloning • Activity higher than antisense oligonucleotides • Effective at low concentration • pre in vitro test • Often potent with dramatic phenotype • Powerful tool for experiments at pre-implantation stages 32 Mol Biotech, 34, 2006 9 33 J Allergy Clin Immunol, 2008 34 Human Gene Therapy 19, 2008 35 Human Gene Therapy 19, 2008 36 Molecular Cell, 17, 1-10, 2005 RNAi – Off-target effects 10 37Nat Methods, 3, 2006 38 Nat Methods, 3, 2006 39 Neuron, 53, 2007 40 RNAi - Methodology Int. # Copy # Inheritable • ES Single Single + Preselectable • P. injection Single Mult. + • Viral infection Mult. Single + • Hydrodynamic - Single - • Electroporation - Single - • Sonoporation - Single - • Lipo-transfection - Single - • Magnetofection - Single - 11 41 Methods Mol Biol 437, 2008 42 Nat Chem Biol, 2, 2006 43 Methods Mol Biol 437, 2008 44 Nature Clin Pract 3, 2007 12 45Nature Biotech 25, 2007 46 Nat Chem Biol, 3, 2007 47 J Allergy Clin Immunol, 2008 48 Methods Mol Biol 437, 2008 13 49 Nat Rev Genetics, 8, 2007 50 Nat Rev Genetics, 8, 2007 51 Pharm Res 25, 2008 52 Pharm Res 25, 2008 14 53 BioTechniques 41, 2006 54 BioTechniques 41, 2006 55 Inducible siRNA • tet H1: – van de Wetering et al. EMBO reports 4, 2003 – Wiznerowics & Trono J. Virology 77, 2003 – Szulc et al. Nature Methods 3, 2006 • tet U6: – Ohkawa & Taira Hum. Gene Ther. 11, 2000 – Chen et al. Cancer Research 63, 2003 – Matsukura et al. Nucleic Acids Res. 31, 2003 • tet Cre U6 EGFP: – Chang et al. Am. J. Pathology 165, 2004 • tet polII shRNA-mirs: – Dickins et al. Nature Genetics 37, 2005 – Stegmeier et al. PNAS 102, 2005 • tet polII multiple shRNAs: – Xia et al. BioTechniques 41, 2006 56 EMBO reports 4, 2003 15 57 Nat Methods, 3, 2006 58 Nat Methods, 3, 2006 59 Trends in Genetics 20, 2004 60 PNAS 37, 2005 16 61 Trends in Biotechnology 23, 2005 62 Nature 441, 2006 Fatality due to oversaturation of miRNA/shRNA pathways