Lecture 17 Reading Lecture 17: p. 251-256, 260-261 & 264-266 Lecture 18: p. 258-264, 508-524 Transgenics Definition Overview Goals Production p.251-256, 260-261, 264-266 315
Definition A transgenic animal is an animal that has had a foreign gene or segment of DNA stably introduced into its genome and is transmitted to offspring in a Mendelian fashion. 316
Overview Transgenic animal production involves many areas of science Embryology (embryo/cell work) Physiology (hormonal sync) Genetics & Biochemistry (recombinant DNA work) New Science 317
Timeline of Transgenic Animal History 2001 2007 2015????? Prion knockout cattle 318
Goals of Transgenic Research Information/basic research Development Mechanisms of growth and disease Specialized non-agricultural purposes Pharmaceutical production Xenotransplantation Improved animals or products Disease resistance Milk modification 319
Transgenic Approach Change specific characteristics of animal Addition or modification of proteins and enzymes Incorporate change at the genetic level DNA Protein Change 320
Steps for Producing a Transgenic Animal Identify gene Clone appropriate sequences Introduce DNA Screen for transgene incorporation Test for expression Establish lines for study/use 321
DNA Construct Design 322 Gene Identified and sequenced Genomic or cdna Regulatory regions Promoter Tissue specificity 3 as well as cis and trans elements Secretory signal Routes protein in cell Clone all desired sequences into a vector
Introduction of DNA Pronuclear microinjection Embryonic stem cell transfer Nuclear transplantation Retroviral vectors Sperm-mediated gene transfer Blastomere/embryo aggregation Teratocarcinoma cell transfer Electrofusion Particle bombardment/jet injection 323
Pronuclear Microinjection First established and most used method Developed in mice Genetics and reproduction known Fast reproduction Low cost Start with one-cell zygotes and a DIC microscope 324
Pronuclear Microinjection Embryo development known Superovulation Stimulate follicles so ovary produces more eggs PMSG (mouse), FSH (goats) Synchronize eggs to be released at same time in all animals HCG (mimics luteinizing hormone (LH)) Ruptures follicles, releases eggs Fertilize and collect one cell zygotes 325
326 Microinjection Apparatus
327 Pronuclear Microinjection
Pronuclear Microinjection Surviving embryos get transferred to synchronized recipient female Done surgically into oviduct Pregnancy established Live births Screen offspring for presence of transgene Identification Transmission Expression 328 mrna Protein Phenotype
Pronuclear Microinjection: Transgene Integration Integration of DNA construct Transgene integrates at a random location in the chromosome Active or inactive chromatin Multiple copies Tandem arrays Integration after the one cell stage can occur Mosaic animal Transgene DNA not in every cell 329
Factors Affecting Microinjection and DNA Integration DNA construct itself Linear vs circular Size Ends of DNA Blunt, cohesive, non-cohesive DNA concentration Purity of DNA 330
Factors Affecting Expression of Transgenes Can be transgenic and not express DNA construct Design Multiple copies does not equate to more expression Site of integration Active or inactive chromatin Insulators Interrupt endogenous 331
Pronuclear Microinjection: Typical Results # # # # # Embryos Injected Embryos Transferred Recipients Pregnant Born Transgenic 140 88 7 15 1 332
Efficiency Microinjection is an inefficient procedure About 1% of injected embryos result in a transgenic animal Slight species variation Inefficiencies result from: Poor embryo survival Low rate of integration of transgene into genome 333
Retroviral Vectors Developed from gene therapy potential Very efficient integration Random Single site Drawbacks Size limitation Procedure complex Virus High frequency of mosaics Lentivirus 334
Definition Embryonic Stem Cells (ES cells) Embryonic stem cells are undifferentiated, pluripotent cells derived from early embryos From ICM of blastocysts Used to study: Embryogenesis Cell lineage studies Stage- & cell-specific gene expression 335
ES Cells Used to make transgenic animals Blastocyst injection ES cells injected into a blastocyst Cells incorporate into ICM Host develops into a chimera Advantages: Gene targeting enabled Knockouts Modify endogenous Disadvantages: Mice only 336
Nuclear Transfer: The fusion of a donor cell to an unfertilized egg or early embryo that has been enucleated (nucleus removed). Species: sheep, cattle, goats, pig, mice, rabbit, mule, monkey, cat, deer 337 Transgenics: sheep, cattle, pigs Homologous recombination: sheep, pigs, cattle
338 NT Procedure
Nuclear Transfer Advantages: Can modify existing genes All animals born will be transgenic (germ line) Disadvantages: Does not work for all cell types Abnormalities in offspring Inefficient 0.04% adult, 1% embryo, 1.7%fetal donor cells result in live offspring 339