fgfr3基因敲除载体的构建及中靶ES细胞的筛选与鉴定

Construction of Targeting Vector for Knockout of Murine fgfr3 and the Screening of Targeted ES Cells

目的构建成纤维细胞生长因子3型受体(nbroblast growth factor receptor-3,FGFR3)基因敲除载体,并对中靶ES细胞进行筛选与鉴定,为最终建立FGFR3条件性敲除小鼠模型或FGFR3功能减低小鼠,研究FGFR3在小鼠骨骼等器官发育、损伤修复过程中的作用打下基础。方法在分析小鼠fgfr3基因组DNA序列结构的基础上,设计并构建了针对小鼠fgfr3外显子9、10的条件性基因敲除载体,采用电穿孔法转染ES细胞,用G418与Gancyclovir对电转的ES细胞进行正负筛选,最后对ES细胞克隆做Southern与PCR鉴定。结果对ES细胞正负筛选后共挑取180个克隆；用5′端探针进行Southern杂交鉴定后发现2株阳性克隆；经PCR检测发现这两株克隆fgfr3的8号内含子内的LoxP序列丢失。结论得到两株fgfr3基因中只含有neo基因的ES细胞克隆,fgfr3的表达是降低的,可用来建立FGFR3功能降低的小鼠。

Objective To explore the functions of FGFR3 signals in mouse organogenesis. Method We first made a fgfr3 knockout construct, in which the exon 9 and 10 of fgfr3 were floxed and could be deleted by Cre-mediated recombination of LoxP. Then, we obtained and identified the targeted ES clones. On the basis of analysis of Cre-mediated recombination of LoxP, a conditional targeting construct was made. At first, the Cre-mediated deletion of floxed exon 9 and 10 of fgfr3 was confirmed by introduction of the targeting vector into Cre-expressing AmI bacteria. The transfection of targeting vector into ES cells was then carried out by electroporation, transfected ES cells were screened with G418 and Gancyclovir, and finally, the ES clones with correct targeting event were identified by Southern blot and PCR. Results PCR, restriction enzyme digestion and sequence analysis confirmed that a targeting vector for knockout of murine fgfr3 was successfully made. 180 ES clones were collected after screening by both G418 and Gancyclovir, and two positive ES clones were found by Southern blot of 5＇-end flank probe, but their LoxP inside intron 8 were found lost by PCR. Conclutsion＇ After successful making of targeting construct for knockout of murine fgfr3, two targeted ES clones were obtained with neo insertion in intron 8 of fgfr3, which can be used to generate a mouse strain with downregulated expression of fgfr3.