General strategy for gene targeting in mice Step 1 Gene targeting in ES cells r Inserted DNA- Vector neo/ HSV-tk 1. ES cell culture Homologous DNA DNA Embryonic stem (ES) Blastocyst 2. Construction of targeting vector cells are cultivated The vector contains pieces of DNA that are homolo from mouse pre- gous to the target gene, as well as inserted DNA which plantation embryos changes the target gene and allows for positive- blastocysts negat ES cells Transfection Ne selection 3. ES cell transfection The cellular machinery for homologous recombination allows the targeting vector enables the target vector to find and recombine with the target gene Vector ned/ HSV-tk Rare cell carrying Target gene Targ Positive-negative selection 4. Proliferation Homologous recombination of targeted Es cell Selection for presence of neo/ and absence of HSV-tk enriches targeted Pure population of ES cells ES cells carrying targeted gene Targeted gene