Measuring Recombination Proficiency in Mouse Embryonic Stem Cells
A method is presented to measure homologous recombination in mouse embryonic stem cells by both gene targeting and short-tract gene conversion of a double-strand break (DSB). A fluorescence-based reporter is first gene targeted to the Hprt locus in a quantifiable way. A homing endonuclease expression vector is then introduced to generate a DSB, the repair of which is also quantifiable.
Key wordsRecombination Double-strand break (DSB) Hprt Mouse embryonic stem (ES) cells Green fluorescent protein (GFP) Flow cytometry Gene targeting Gene conversion I-SceI Homing endonuclease
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