Construction of lentiviral vector of RNA interference of PKCY gene / 中华麻醉学杂志

ABSTRACT

Objective To construct a lentiviral vector of RNA interference (RNAi) of PKCγ gene. Methods The effective sequence of siRNA targeting PKCγ gene was confirmed in our previous study. The complementary DNA containing both sense and antisense oligo DNA of the targeting sequence was designed, synthesized and cloned into the pGCSIL-GFP vector, which contained U_6 promoter and green fluorescent protein (GFP) . The resulting lentiviral vector containing PKCγshRNA was named lentivinis RNAi vector of PKCγ, and it was confirmed by realtime PCR and sequencing. 293T cells were cotransfected with lentiviral vector pGCSIL-CTP, pHelper 1.0 and pHelper 2.0. All virus stocks were produced by calcium phosphate-mediated transfection. The titer of virus was tested according to the expression level of GFP. Results PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of PKCγ producing PKCγshRNA was constructed successfully. The titer of concentrated virus was 1 ×10~9 TU/ml. Conclusion The lentivinis RNAi vector of PKCy was constructed successfully.