Genetic Engineering of Embryonic Stem Cells Using the Cre/lox System

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Biology | Life Sciences


Michael Reagan, Biology


While lox site recombination efficiency has been studied in E. coli, similar studies have not completed using ES cells. In the present study we assess Cre-induced recombination efficiency between a pair of loxP sites or between mutant lox FAS and lox 2272 sites in mouse ES cells by creating DNA substrates with these lox sites flanking an antibiotic resistance gene (conferring hygromycin resistance). Subsequent electroporation of these DNAs into ES cells and the quantification of antibiotic resistant colonies will indicate whether or not homologous and heterologous pairs of loxP sites can be induced to recombine in the presence of Cre recombinase. We also will survey the level of Cre at which its toxic effect(s) can be seen on cell survival and growth after gene transfer.