RPL11基因稳定敲低细胞系的构建及验证

Construction and Verification of RPL11 Gene Stable Knockdown Cell Lines

针对RPL11基因设计三条不同的shRNA,并分别克隆至带绿色荧光标签的pRNAT-U6.1载体,以用于构建RPL11稳定敲减的细胞系。将重组质粒转染至HeLa细胞中并加入G418进行筛选,随后通过有限稀释法将阳性细胞亚克隆纯化。将扩大培养的亚克隆细胞进行Western blot和qPCR实验验证获得RPL11基因稳定敲低的细胞株。利用CCK-8细胞活力检测试剂盒和Puromycin标记法测定RPL11基因稳定敲低细胞系的生长活力和新生蛋白合成能力,结果表明,RPL11基因稳定敲低后并不影响细胞的活力和新生蛋白合成能力,该细胞系为后续研究RPL11的非核糖体功能奠定了基础。

In order to construct stable knockdown cell lines of RPL11,three different shRNA target sequences of RPL11 gene were designed and ligated to PrNAT-U6.1 with green fluorescent label to obtain recombinant plasmids.The recombinant plasmids were transfected into HeLa cells,and G418 was added for pressure screening.The positive cells were subcloned and purified by limited dilution method.Western blot and qPCR were used to verify the expanded culture of monoclone cells,and RPL11 gene stable knockdown cell line was obtained.CCK-8 cell viability test kit and puromycin labeling method were used to determine the growth activity and new protein synthesis ability of RPL11 stable knockdown cell lines.The results showed that RPL11 stable knockdown did not affect the cell viability and protein synthesis level,which laid a foundation for the follow-up study of RPL11 non ribosomal function.