恒河猴P21基因在COS-7细胞中沉默位点的验证

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

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541-561、542-562、215-239、624-648 bp。四个靶位点在mRNA水平的沉默效率分别为(91.82±3.21)%、(82.47±2.48)%、(81.31±2.69)%和(87.35±4.59)%。相应的蛋白表达量为(11.97±0.70)%、(20.22±0.65)%、(23.21±0.63)%和(14.42±0.86)%。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。

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.