Screening of efficient siRNA target sites directed against gatekeeper genes for DNA repair.
To investigate the RNA interference (RNAi) effect induced by vector-derived small interfering RNA (siRNA) targeting the three gatekeeper genes (Rad52, Ku70, Ku80) and screen the more effective target sites from candidates for further research, by using siRNA design tools online, we selected 2 candidate sequences directed to every gatekeeper gene. According to the sequences, six vector-derived siRNAs (denoted psiRNA1-6) and one mocking psiRNA7 were constructed. Among them, psiRNA1 and psiRNA2 targeted Rad52, psiRNA3 and psiRNA4 to Ku70, psiRNA5 and psiRNA6 to Ku80. The mocking psiRNA7 was used as control. After sequence identification, the seven plasmids were transfected into HepG2 cell line. siRNA-induced silencing of gatekeeper genes was determined by using RT-PCR at RNA level and Western Blot at protein level. The results showed that the six plasmids specifically targeting the coding region of gatekeeper genes were successfully designed and constructed. To some extent, the six plasmids could reduce the expression of target gene. Comparatively, the plasmid-derived siRNA psiRNA1, psiRNA4 and psiRNA5 were more effective than their counterparts. The results suggest that the gene silencing efficiency of siRNA is different, depending on their targeted region, and siRNA may provide us with practical tools for further study on the three gatekeeper genes, i.e. Rad52, Ku70, Ku80.
Ren
Jinghua
J
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Lin
Jusheng
J
Dong
Xuyang
X
Xu
Dong
D
Chen
Qiong
Q
Liu
Yao
Y
Chang
Ying
Y
Yao
Jinjian
J
Han
Siyuan
S
D034961:Antigens, Nuclear / Q000235:genetics*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics*; D005822:Genetic Vectors; D056945:Hep G2 Cells; D006801:Humans; D000072200:Ku Autoantigen; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D051721:Rad52 DNA Repair and Recombination Protein / Q000235:genetics*; D014162:Transfection
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C497194:RAD52 protein, human; D034741:RNA, Small Interfering; D051721:Rad52 DNA Repair and Recombination Protein; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen