Summary
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.
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References
Mazzarelli P, Rabitti C, Parrella P et al. Differential modulation of Ku70/80 DNA-binding activity in a patient with multiple basal cell carcinomas. J Invest Dermatol, 2003,3:628–633
Seripa D, Persichetti P, Marangi G F et al. Homologous and non-homologous recombination differentially affect DNA damage repair in mice. EMBO J, 2000,7:1703–1710
Van Dyck E, Stasiak A Z, Stasiak A et al. Binding of double-strand breaks in DNA by human Rad52 protein. Nature, 1999,6729:728–731
Leung R K, Whittaker P A. RNA interference: from gene silencing to gene-specific therapeutics. Pharmacol Ther, 2005,2:222–239
Chi J T, Chang H Y, Wang N N et al. Genomewide view of gene silencing by small interfering RNAs. Proc Natl Acad Sci USA, 2003,11:6343–6346
Cui W, Ning J, Naik U P et al. OptiRNAi, an RNAi design tool. Comput Methods Programs Biomed, 2004,1:67–73
Taxman D J, Livingstone L R, Zhang J et al. Criteria for effective design, construction, and gene knockdown by shRNA vectors. BMC Biotechnol, 2006,6:7
Luo K Q, Chang D C. The gene-silencing efficiency of siRNA is strongly dependent on the local structure of mRNA at the targeted region. Biochem Biophys Res Commun, 2004,2:622
Kretschmer-Kazemi Far R, Sczakiel G. The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides. Nucleic Acids Res, 2003,15:4417–4424
Saetrom P, Snove O Jr. A comparison of siRNA efficacy predictors. Biochem Biophys Res Commun, 2004,1:247–253
Bohula E A, Salisbury A J, Sohail M. The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript. J Biol Chem, 2003,18:15991–15997
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Ren, J., Lin, J., Dong, X. et al. Screening of efficient siRNA target sites directed against gatekeeper genes for DNA repair. J. Huazhong Univ. Sc. Technol. 26, 640–643 (2006). https://doi.org/10.1007/s11596-006-0602-8
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DOI: https://doi.org/10.1007/s11596-006-0602-8