Aim: To construct three adenoviral vectors harboring shRNA targeting CDK1 gene, and identify its inhibitory effect on the gene expression of CDK1 in hepatoma carcinoma HepG2 cells.
Methods: Three shRNA sequences targeting CDK1 mRNA were designed. The shRNA sequences were annealed and linked with linearized pSIREN-RetroQ-ZsGreen. The recombinants were identified by PCR and DNA sequencing. CDK1-shRNA plasmid was then transfected into the cultured HepG2 cell line with lipofectamine 2000. RT-PCR and Western blot were used to detect the mRNA and protein expression of CDK1, respectively.
Results: The small hair-pin RNA sequences were successfully inserted into pSIREN-RetroQ-ZsGreen vector, and the sequences were identified by DNA sequencing. Further, Realtime PCR and Western blot results validated that the three small hair-pin RNAs effectively knockdowned the expression of endogenous CDK1 in HepG2 cells.
Conclusion: CDK1-shRNA can be effectively transfected into HepG2 cells, and induce post-transcriptional gene silencing of CDK1, which enables further functional study on CDK1.