Construction of lentivial RNA interference vectors of rat PER2 gene and determination of its interference efficiency.

Objective To construct the lentiviral RNA interference ( RNAi) vectors of rat PER2 gene and evaluate the effects of silencing PER2 gene expression by siRNA. Methods The target sequence of siRNA- PER2 in rats was designed, and the complementary DNA containing both sense and antisense oligonucleotides was synthesized. After phosphorylation and annealing, the double-stranded DNA was cloned to pGLV-U6-EGFP to construct pLentivirus-per2-rat. Then the effects of RNAi in reducing gene expression were further confirmed by real-time PCR in transfected rat A7r5 aortic vessel smooth muscle cells expressing PER2. Results The rat lentivirus vectors expressing PER2-specific shRNA were synthesized, and the specificity of the designed target sequence was primarily identified by electrophoresis and gene sequence analysis. Rat A7r5 aortic vessel smooth muscle cells were successfully transfected with pLentivirus-per2-rat, and the transfection rate was 70%. The real-time PCR analysis confirmed that the level of PER2 mRNA was decreased significantly as compared with that in the negative control group, and suppressed by approximately 84% in cells transfected with pLentivirus-per2-rat. Conclusion The successful construction of rat lentivirus vectors expressing PER2-specific shRNA may lay a foundation for further study on the clock gene's function. [ABSTRACT FROM AUTHOR]