Transgenic MiceSasha GardnerWill ParsonDana SunalpApplications• Test expressionof normal andaltered genes• Study animalmodels ofhuman diseases• Determinepromoter andenhanceractivityThree Methods• Embryonic StemCell• Pronucleus• RetrovirusEmbryonic Stem CellFirst Method Developed• Expose Stem Cells to DNA vectors inculture• Select for transformed cells• Inject cells into ICM of 3-5 day oldblastocyst• Transfer embryo to pseudopregnant mouse• Test offspring• Mate heterozygotes to create homozygoustransgenic strain• Results in “Mosaic Expression”• 10-20% instance of successfulincorporation of transgenePronuclear Injection• Inject plasmid directly into pronucleus of fertilizedegg prior to nuclear fusion• Implant fertilized egg into pseudopregnant female• Screen offspring for expression of transgene usingSouthern blotting technique• Mate heterozygous progency to create homozygoustransgene strain• Low percentage of progency express desired DNA• DNA may not incorporate until after first celldivisionRetrovirus Insertion• Fertilized egg is infected by retrovirusincorporated with target RNA in vitro• Retroviral enzymes insert DNA into hostcell’s genome• Egg is implanted in pseudopregnant female• Progency is screened for expression• Limited to DNA smaller than 10k bases• Highest instance of DNA incorporation• Cheapest and easiestFluorescent MiceExample of a TransgeneGFP: Green Fluorescent Protein• Tissues that express the targetgene fluoresce• Strength of fluorescenceincreases with expressionindicating higher levels oftransgene incorporation• Allows for detection ofexpression in
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