[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.
Zheng
J M
JM
Department of Medical Genetics, Second Military Medical University, Shanghai 200433, China.
Li
J
J
Fu
J L
JL
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002999:Clone Cells; D004247:DNA / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016133:Polymerase Chain Reaction; D014162:Transfection
D004274:DNA, Recombinant; D004247:DNA; D007041:Hypoxanthine Phosphoribosyltransferase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase