小鼠FGFR3可控性RNAi载体的构建和抑制效率验证

目的 构建小鼠成纤维细胞生长因子受体3(fibroblast growth factor receptor 3,FGFR3)可控性RNAi载体及RNAi载体,并在体外验证后者效率.方法 以含有pLoxPneo基因的pBSK/U6载体为骨架,首先构建针对FGFR3的可控性RNAi载体pBSU6/FGFR3i,然后经Cre重组酶去除neo基因后得到RNAi载体FGFR3-RNAi.将FGFR3-RNAi与FGFR3表达载体分别共转染RAW264.7和HEK293T细胞株,经半定量RT-PCR和Western blot分别检测FGFR3 mRNA和蛋白表达情况.结果 成功构建针对FGFR3的可控性敲低载体和RNAi载体;FGFR3载体在细胞水平可明显降低FGFR3 mRNA的丰度及蛋白表达.结论 为进一步获得FGFR3可控性敲低的小鼠模型奠定基础.

Construction and validation of murine FGFR3 RNAi vector

Objective To establish and validate a conditional RNAi vector and RNAi vector of murine FGFR3. Methods The conditional RNAi vector based on pBSK/U6 with pLoxPneo targeting to FGFR3 gene were constructed. The FGFR3-RNAi vector without neo gene and FGFR3 expressing vector were co-transfected into RAW264.7 and HEK293T cell line. The expressions of FGFR3 mRNA and protein were evaluated by semi-quantitative RT-PCR and Western blot, respectively. Results The FGFR3 conditional RNAi and RNAi vectors were constructed successfully. And the FGFR3 RNAi vector could inhibit not only mRNA but also protein expression of FGFR3. Conclusion A FGFR3 knockdown vector has been constructed, which could contribute to a conditional knockdown mouse modle for the further research of FGFR3 biological functions.