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. CONCLUSION:FGFR2 conditional knockout depending on Cre-loxP can be successfully used in ES cells.

译文

目的:探讨成纤维细胞生长因子受体2(FGFR2)在不同分化阶段的功能。根据Cre-loxP,开发了具有条件敲除FGFR2的工程鼠胚胎干(ES)细胞。
方法:将Cre-loxP系统用于条件靶向载体。表达Cre重组酶的感受态AM-1细菌用于确认Cre介导的FGFR2外显子7和8的缺失。将靶向载体电穿孔到ES细胞中,并用G418和更昔洛韦筛选转染的ES细胞。最后,通过Southern印迹和PCR鉴定具有正确靶向事件的ES克隆。
结果:成功构建了有条件敲除鼠FGFR2的靶向载体,并通过PCR和酶切分析鉴定。通过用G418和更昔洛韦选择性培养收集了86个ES克隆。发现86个ES克隆中有四个在基因组DNA中包含靶向基因序列,该DNA由Southern Blot用5'端侧翼探针证实。四个ES克隆中的两个具有正确的靶向事件,包括在基因组DNA中插入了靶向基因序列,并使用3'端侧翼探针通过Southern Blot检测。最后,通过Southern印迹和PCR在两个ES克隆之一中鉴定出loxP(loxP3)在基因组DNA的外显子8和9之间的插入。
结论:依赖于Cre-loxP的FGFR2条件敲除可成功用于ES细胞。

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