Inhibition of Chk1 and Chk2 gene expression in U251 cell line using Lentivirus-mediated shRNA

Objective To construct eukaryotic expression vector of RNA interference specific for Chk1 and Chk2 gene,and to screen the stably transfected cell clones.Methods Genomic sequences of Chk1 and Chk2 gene were retrieved from Genbank and cDNA were designed and synthesized,and one scrambled shRNA sequences served as negative control.The cDNA were synthesized and inserted into plasmid pLKO.1.Recombinant vectors were transformed into competent E.coli.The positive clones were selected and recombinant plasmids were extracted.The plasmids were digested with EcoR 1 and Nco I,then loaded in agarose gel electrophoresis.The three shRNA vectors were transfected into U251,the stably transfected cell clones were obtained after being screened with puromycin.Western blotting and Reverse transcriptase-polymerase chain reaction (RT-PCR) were performed to examine the inhibitory effect at the RNA level and protein level. Results The stably transfected pLKO.1-TRC-Chk1-shRNA and pLKO.1-TRC-Chk2-shRNA cell clones were significantly down-regulated by siRNA as validated by Western blotting and RT-PCR. Conclusion RNA interfering(RNAi) mediated by the shRNA expressing vectors can significantly down-regulate the expression of Chkl and Chk2 in glioma cell line U251.The stably transfected cell clones were obtained for further study.