Construction and identification of a lentiviral vector mediating RNA interference of tyrosine kinase B gene / 中华行为医学与脑科学杂志

ABSTRACT

Objective To construct a tyrosine kinase B(TrkB) targeted RNA interference (RNAi) lentiviral vector.Methods Four oligonucleotides targeting rat TrkB gene were synthesized and cloned into lentiviral vector pXZRNAi 1.0 to construct recombinant lentiviral vectors pXZRNAi-shTrkB-1,2,3,4.Neural stem cells prepared from rat hippocampus were infected with these high-titer viruses.Real-time PCR was employed to detect the TrkB mRNA expression and western blot was used to assess the gene silencing efficacy of these recombinants.Results Enzyme digestion and DNA sequencing results demonstrated that these shRNAs were correctly inserted into lentiviral vectors and the four recombinants were constructed successfully with the titer of 8.6 × 105cfu/ml.The infection efficiency of the letivirus on neural stem cells reached 80％.Compared with the uninfection group,the expression levels of TrkB mRNA in neural stem cells decreased significantly after transfected with pXZRNAi-shTrkB-3 and 4((66.7 ± 5.5) ％ and(76.8 ± 4.9) ％ respectively,P ＜ 0.05) ; and the protein expression levels were also significantly decreased ((68.5 ± 4.3)％ and (78.2 ± 5.1)％ respectively,P ＜ 0.05).Conclusion The lentiviral vectors for TrkB have been successfully constructed with high yield of lentivirus,which provides versatile method for assessing gene function in neural stem cells.