Abstract:

To construct a lentiviral vector of RNA interference (RNAi) of PAK4 gene and to study the interference  efficiency of  PAK4 shRNA in SACC-83 cells in vivo.Methods  The complementary oligo DNA was designed according to the effective sequence of siRNA targeting PAK4 gene and then synthesized and cloned into the pGCSIL-GFP vector.The obtained lentiviral vector containing PAK4i shRNA was named as shPAK4i-LV,and it was confirmed by PCR and sequencing.The HEK-293T cells were cotransfected with lentiviral vector shPAK4i-LV，pHelper1.0 and pHelper2.0.All virus stocks were produced by LipofectamineTM 2000 transfection.The titer of virus was tested according to the expression level of GFP.Results  PCR and DNA sequencing demonstrated that the lentiviral vector shPAK4i-LV of PAK4 shRNA was constructed successfully.The titer of concentrated virus was 2E+9TU•mL^-1.The SACC-83 cell line was infected with shPAK4i-LV and the expression of PAK4 gene was inhibited about 70% which was confirmed by real-time PCR.Conclusion The shPAK4i-LV is constructed successfully and SACC-83-shPAK4i transient transfection cell model is established.It provides the basis for research on PAK4 signal transduction pathway in tumor.

Key words: RNA interference;PAK4 gene;plasmid construction;lentivirus;carcinoma,adenoid cystic