慢病毒感染方法构建稳定干涉PDRG1的HeLa细胞系

PDRG1(p53 and DNA-damage regulated 1)是实验室通过文库筛选发现的参与NF-kB信号通路调控的一个新基因。已知PDRG1在多种肿瘤中高表达,并参与p53信号通路;同时该基因受UV以及基因毒性等刺激调控。但其在NF-κB信号通路中作用尚未见报道,为了深入研究PDRG1对NF-κB信号通路的调控机理,构建了稳定干涉PDRG1的HeLa细胞系。选择了基于慢病毒载体pLKO.1的感染体系,该慢病毒体系可以同时感染分裂期与非分裂期的细胞,将目的基因干涉序列稳定整合至靶细胞基因组中;再经过抗生素压力筛选后在短时间内获得稳定干涉目的基因表达的细胞株。设计合成PDRG1干涉序列并克隆至pLKO.1载体,转染病毒包装细胞293TN后收集慢病毒上清感染HeLa细胞系,进一步经过嘌呤霉素压力筛选成功获得稳定表达PDRG1干涉序列的细胞株。该稳定细胞株的建立为PDRG1的功能研究提供了必要的实验工具。

Construction of HeLa-PDRG1-knock down stable cell line by lentivirus infection

PDRG1 (p53 and DNA-damage regulated1) is one of the proteins that play a role in the regulation of NF-kB signalling pathway and was found in our lab by library screening. There is evidence that the mRNA of PDRG1 was up-regulated by UV and genotoxicity while down-regulated by p53. PDRG1 was also over-expressed in relevant tumors, but its function in the NF-?B signalling pathway remains elusive. In order to make a further study of the function of PDRG1 in NF-?B signaling pathway, a stable cell line that can stably interfere the expression of PDRG1 is constructed with the Hela cell by the integration of the PDRG1 inference sequence into the target cell through the lentivirus vector pLKO.1 and then the antibiotic screening to get the Anti-Puro Positive cells. By combining the lentivirus infection and antibiotic screening, the efficiency of selection is markedly increased. This stable cell line will serve as a necessary element in the further study of PDRG1.