Construction of engineered murine embryonic stem cells with conditional knockout of FGFR2 depending on Cre-loxP.
OBJECTIVE
To investigate the functions of Fibroblast Growth Factor Receptor-2 (FGFR2) at different stages of cell differentiation. The engineered murine embryonic stem (ES) cells with conditional knockout of FGFR2 were developed depending on Cre-loxP.


METHODS
Cre-loxP system was used in a conditional targeting vector. The competent AM-1 bacteria, which expressed Cre-recombinase, was used to confirm the Cre-mediated deletion of the floxed exons 7 and 8 of FGFR2. The targeting vector was electroporated into the ES cells, and the transfected ES cells were screened with G418 and Ganciclovir. Finally, the ES clones with correct targeting events were identified by Southern Blot and PCR.


RESULTS
The targeting vector with conditional knockout of murine FGFR2 was successfully constructed and confirmed by PCR and digestion analysis in bacteria. 86 ES clones were collected by selective culture with G418 and Ganciclovir. Four of the 86 ES clones were found containing the targeting gene sequence in genomic DNA proved by Southern Blot with a 5'-end flank probe. Two of the four ES clones had the correct targeting events that included the insertion of the targeting gene sequence in genomic DNA and were checked by Southern Blot with a 3'-end flanking probe. Finally, the insertion of loxP (loxP3) between exons 8 and 9 in genomic DNA was identified in one of the two ES clones by Southern Blot and PCR.


CONCLUSIONS
FGFR2 conditional knockout depending on Cre-loxP can be successfully used in ES cells.
Jianmin
Wang
W
Laboratory of trauma center, Daping Hospital, The Third Military Medical University, Chongqing 400042, China. jmwang@cta.cq.cn
Ruihua
Song
S
Lei
Chen
C
Liangjun
Yin
Y
Bo
Chen
C
Jing
Sun
S
Yuanbing
Gou
G
Ling
Zhao
Z
Lin
Chen
C
D000818:Animals; D001483:Base Sequence; D004622:Embryo, Mammalian / Q000166:cytology*; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics; D016678:Genome / Q000235:genetics; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D051497:Receptor, Fibroblast Growth Factor, Type 2; C045073:Cre recombinase; D019426:Integrases