摘要:目的构建成纤维细胞生长因子3型受体(fibroblast 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.
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