Construction and Identification of Lentiviral Vector-Mediated Short Hairpin RNA of AKT2 Gene

Objective: To construct a lentiviral vector-mediated RNA interference(RNAi)of AKT2 gene.Methods:In accordance with RNAi sequence design principles in the public Web site,RNAi target sequences were designed,then the target sequences of Oligo DNA were synthesized and annealed to double-stranded DNA,which were connected with PLVTHM-GFP vector digested by MluI and ClaI.Short hairpin RNA lentiviral vectors were constructed.293T cells and U87 cells were transfected by shRNA lentiviral vector,and virus titer was determined.Transfection efficiency U87 cell was determined by flow cytometry.The expression of the AKT2 gene in U87 cells was identified by PCR and Western blot.Results: The lentiviral vector of AKT2-shRNA-oligonucleotide chain was inserted correctly;Infection efficiency of 293T cells observed under fluorescence microscope was more than 90%,the virus titer was 3.59 × 107 TU/ml;Transfection efficiency was 86.93%.AKT2 interference rate was 68%.shRNA lentiviral vectors inhibited the expression of AKT2.Conclusion: A lentiviral vector of AKT2-gene RNAi was constructed successfully by the genetic engineering technology,and it provides a condition forfurther research in vitro and vivo.