恒河猴 p53基因沉默靶点在细胞水平的验证

Rhesus monkey P53 gene silencing at the cellular level

目的：为建立恒河猴 p53基因沉默模型，首先在细胞水平筛选和测定恒河猴 p53基因有效沉默靶点。方法测定非洲绿猴肾来源成纤维细胞 COS-7中 p53基因的表达水平；对恒河猴 p53基因做生物信息学分析，优选靶序列设计 shRNA，克隆入慢病毒载体 FUGW-TDT，转染 COS-7细胞，以 real-time PCR 检测 p53 mRNA 抑制水平，以 Western blot 检测 p53蛋白水平表达变化。结果在 COS-7细胞检测到 p53基因的表达；生物信息学筛选到3个靶片段，分别位于 p53 mRNA 的238～258 bp，681～701 bp，169～189bp，均在开放阅读框内； p53三个靶位点的沉默效率在 mRNA 水平分别为（87.17±4.03）％，（72.62±14.11）％，（76.22±0.98）％；在蛋白水平的沉默效率分别为（84.44±2.18）％，（71.0±1.18）％，（74.17±0.95）％。结论在细胞水平筛选得到3个有效的 p53基因沉默靶点，可用于后续的恒河猴基因沉默研究。

Objective In order to establish a rhesus monkey model of p53 gene silencing, firstly we screened and determined the effective silencing targets of p53 gene at the cellular level in rhesus monkey.Methods The expression of p53 gene was detected in COS-7 cells （ derived from the kidney of the African Green Monkey, Cercopithecus aethiops）.Three small hairpin RNA （ shRNA） sequences targeting rhesus monkey p53 gene were designed, analysed by bioinformatics, and inserted into lentivirus-based gene silencing constructs FUGW-TDT.The plasmids of p53-RNAi and control vector were transfected into the COS-7 cells, respectively.The suppression of p53 mRNA was detected by real-time PCR, and the changes of p53 protein expression were detected by Western blot assay.Results p53 gene expression was detected in COS-7 cells.Bioinformatics analysis showed that three gene-silencing sequences were screened which lied in the open reading frame （ ORF） region and targeted 238 -258bp, 681 -701bp, 169 -189bp of the rhesus monkey p53 mRNA.At 48 hrs after transfection of the three silencing constructs, p53 mRNA was suppressed by（87.17 ＆#177;4.03）%, （ 72.62 ＆#177;4.11）% and（76.22 ＆#177;0.98 ）%, and p53 protein was suppressed by （ 84.44 ＆#177;2.18 ）%, （ 71.04 ＆#177;1.18）% and （ 74.17 ＆#177;0.95 ）%, respectively. Conclusions We obtained three effective target sequences showing high efficiency in p53silencing, which can be＆amp;nbsp;used in further studies on gene silencing in rhesus monkey.