Validation of the silencing site of rhesus monkey P21 gene in COS-7 cells / 中国实验动物学报

ABSTRACT

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 , Cerco-pithecus aethiops).Four small hairpin RNA (shRNA) sequences targeting rhesus monkey P21 gene were designed and in-serted into lentivirus-based gene silencing constructs FUGW-TDT.The vectors were transfected into COS-7 cells respective-ly.The suppression of P21 mRNA was detected by real-time PCR, and the expression of P21 protein was detected by West-ern 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 cel-lular level , which can be used in gene silencing research of rhesus monkeys .