恒河猴P21基因在COS-7细胞中沉默位点的验证

【摘要】 目的 在细胞水平筛选恒河猴 p21基因的有效沉默靶点。方法 检测 COS-7 中 p21基因的表达水平；设计shRNA并构建p21-RNA干扰慢病毒载体FUGW-TDT-p21shRNA转染 COS-7 细胞，通过real-time PCR检测沉默效率，并以 Western Blot 在蛋白水平进行验证。结果 筛选到四个有效的靶位，分别位于p21 mRNA 的541-561bp，542-562bp，215-239bp，624-648bp。四个靶位点在mRNA水平的沉默效率分别为 (91.82?.21)%， (82.47?.48)%， (81.31?.69)%和(87.35?.59)%。相应的蛋白表达量为(11.97?.70)%， (20.22?.65)%， (23.21?.63)%和(14.42?.86)%。结论 在细胞水平筛选得到四个有效的 p21基因沉默靶点，可用于恒河猴基因沉默研究。

Validation of the silencing site of rhesus monkey P21 gene in COS-7 cells

Objective To screen the effective silencing targets of P21 gene at the cellular level in rhesus monkey. Methods To detect the expression of P21 gene in COS-7 cells (derived from the kidney of African green monkey, Cercopithecus aethiops). Four small hairpin RNA (shRNA) sequences targeting rhesus monkey P21 gene were designed and inserted into lentivirus-based gene silencing constructs FUGW-TDT. The vectors were transfected into COS-7 cells respectively. The suppression of P21 mRNA was detected by real-time PCR, and the expression of P21 protein was detected by Western blot assay. Results Four gene-silencing sequences were screened that lied in 541-561 bp, 542-562 bp, 215-239 bp, and 624-648 bp of the rhesus monkey P21 mRNA. Their silencing rate was (91.82±3.21)%, (82.47±2.48)%, (81.31±2.69)% and (87.35±4.59)%, and the protein expression was (11.97±0.70)%, (20.22±0.65)%, (23.21±0.63)% and (14.42±0.86)%, respectively. Conclusions Four effective silencing target sequences are screened at cellular level, which can be used in gene silencing research of rhesus monkeys.