Objevtive: To construct a eukaryotic expression vector of short-hairpin RNA (shRNA) targeting human Akt gene and assess the effect of Akt gene silencing on the growth of colon cancer Lovo cells.
Methods: Two shRNAs targeting human Akt gene were cloned into pENTRTM/U6 plasmid to obtain the entry clones, and the positive clones were verified by sequencing. After recombination of the pENTRTM/U6 entry constructs and Plenti6/Block-iT DEST vector, the positive clones were confirmed by sequencing. Lovo cells were transfected by the entry vector and DEST Vector, and RT-PCR and Western blotting were performed to detect the interference of Akt gene expressions.
Results: The pENTRTM/U6 entry clones carrying Akt shRNA and pLenti6/DEST-pENTRTM/U6-Akt shRNA were successfully constructed. Both of the vectors were transfected into Lovo cells and resulted in obvious knockdown of the mRNA and protein expressions of Akt.
Conclusion: The Akt siRNA expression vector constructed can significantly inhibit Akt gene expression in Lovo cells, which facilitates further studies of Akt function and tumor gene therapy.