Construction of the expression vector for short-hairpin RNA-mediated Akt gene silencing in Lovo cells / 南方医科大学学报

ABSTRACT

<p><b>OBJEVTIVE</b>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.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>