[Construction of a Flp "exchange cassette" contained vector and gene targeting in mouse ES cell]

Using the HPRT genomic DNA fragment and synthesized oligonucliotides, pSP-HPRT-F-Neo-F3 was designed and constructed as a replacement gene targeting vector by usual molecular cloning techniques. Structure of pSP-HPRT-F-Neo-F3 was identified by restrictive digestion analysis and partly sequencing. Then linearized pSP-HPRT-F-Neo-F3 DNA was electroporated into ES cells, and transfected cells were screened by being cultured in medium containing 200 micrograms/mL G418 and 2 micrograms/mL 6-GT. Twenty-four double drug resistant clones were picked up and analyzed, among them, two clones were proved to have taken place the required recombination by PCR and southern blotting analysis.