[Construction and identification of adenoviral vectors expressing shRNA targeting CDK1 gene]

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.

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.

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.

CDK1-shRNA can be effectively transfected into HepG2 cells, and induce post-transcriptional gene silencing of CDK1, which enables further functional study on CDK1.