Abstract:
Objective To study the inhibitory effects of small interfering RNA ( siRNA) on expression of three gatekeeper genes (Rad52, Ku70 and Ku80) and screen the effective target sequences of siRNA. Methods According to the encoding sequences of gatekeeper genes, two pairs of oligonucleotide sequences targeted to every gatekeeper gene were designed and synthesized. The annealed oligonucleotide fragments were subcloned into psiRNA-h Hneo expression vector. After being identified by rest riction analysis and DNA sequencing, the recombinant plasmids (named as psiRNA1~6) were t ransfected into Hep G2 cells. Inhibitory effects of siRNAs on the expression of three gatekeeper genes were examined by RT-PCR at RNA level and Western Blot at protein level, respectively. Results The expression of gatekeeper genes in transfected cells was down-regulated significantly by siRNAs. The plasmid2derived siRNAs by psiRNA1, psiRNA4 and psiRNA5 were more effective than their counterparts. Conclusion siRNA may provide us with practical tools for further study on the three gatekeeper genes.