Ku80基因RNAi逆转录病毒载体的构建及稳定转染人肾癌786-O细胞株的建立

Construction of recombinant retroviral vector for RNA interference of Ku80 gene and establishment of stably transfected human renal carcinoma 786-O cell line

目的构建并鉴定逆转录病毒介导的Ku80基因RNA干扰表达体系,并建立稳定、高效、长久低表达Ku80的人肾癌786-O细胞株。方法针对Ku80基因,设计并合成3对特异性RNA干扰靶序列,退火形成双链DNA,与BglⅡ和HindⅢ双酶切后的pSUPERretro-puro载体连接,构建pSUPERretro-puro-siKu80干扰表达载体,并对重组载体进行测序鉴定。筛选出基因沉默效应最强的干扰序列后使用磷酸钙法共转染293FT细胞,产生的病毒颗粒随后感染786-O细胞,经嘌呤霉素筛选得到阳性克隆。用RT-PCR检测Ku80基因mRNA表达,Western blot测定转染后786-O细胞中Ku80蛋白的表达量,以鉴定稳定细胞株。结果测序结果证实目的片断正确插入pSUPERretro-puro表达载体中,成功构建了pSUPERretro-puro-siKu80干扰表达载体,稳定转染干扰表达载体的786-O细胞Ku80基因的表达显著降低。结论成功构建了抑制Ku80基因表达的逆转录病毒载体,并获得Ku基因稳定干扰的人肾癌786-O细胞株,为下一步利用小干扰RNA技术研究肾癌的基因治疗奠定了基础。

Objective To construct and identify the expression system of RNA interference of Ku80 gene mediated by retrovirus, and establish the human renal carcinoma 786-0 cells with stable, efficient, long-term low expression of Ku80. Methods We designed and synthesized three pairs of effective RNA interference sequences to target KuS0 gene. Then the sequences were annealed to form double-stranded DNA and inserted to pSUPERretro-puro vectors digested by Bgl II and Hind l]I. The recombinant vectors were called pSUPERretro-puro-KuS0 and identified by sequencing. The in- terference sequence with the strongest silencing effect of KuS0 gene was screened and then co-trans- fected in 293FT cells through the calcium phosphate method. Generated virus particles subsequently infected 786-0 cells, the positive clone was obtained following puromycin selection. In order to iden- tify the stable cell line, the expression levelof Ku80 was detected from both mRNA level and protein level by RT-PCR and Western blot, respectively. Results Sequencing results showed that the tar- get fragments were inserted correctly into pSUPERretro-puro vectors . We successfully constructed pSUPERretro-puro-siKu80 vectors. RT-PCR and Western blot results showed that compared with the control group, 786-0 cells stably transfected with RNA interference vector significantly reduced Ku80 gene expression. Conclusions We successfully constructed retroviral vector inhibiting Ku gene expression, and established stable human renal carcinoma 786-0 cell line interfering Ku80 gene expression. This study provides the foundation for further investigation of gene therapy of kidney cancer by RNA interference.