Construction of lentiviral VP22-EGFP and efficacy of VP22 transfection in rat neural stem cells

Objective To investigate the efficacy of VP22 on transfection in rat neural stem cells (NSC). Method The encoding VP22 fragment was got from the pUL49ep cut by BamH I,and cloned it into pHIV-EGFP to yield pHIV-VP22-EGFP and confirmed by polymerase chain reaction (PCR) and DNA sequencing. The three-plasmid system containing constructor of pHIV-EGFP plasmid, helper plasmid and envelope plasmid were co-transfected into 293T cells. The viral particles were collected, and used to infect rat neural stem cells which were cultured from pregnant SD rat. The expression of EGFP in NSC was observed by fluorescence microscopy and detected by fluorescence-activated cell sorting(FACS) ,the VP22-EGFP gene in NSC genome was assessed by PCR. For NSC proliferation, MTT assays were tested before and after lentivirus-EGFP and lentiviras-VP22-EGFP transfection. Results The cloning site of the encoding VP22 fragment in the pHIV-EGFP plasmid was between CMV and EGFP gene which was confirmed by PCR and DNA sequencing. The expression of EGFP in NSC transfected by lentivirus EGFP and lentiviros-VP22-EGFP were both observed by fluorescence microscopy. And its cell numbers which showed EGFP were (23.1±1.8)% and (34.9±2.9)% (P<0.01). The cell number of inserting of VP22 augment the transfection efficiency of lentivirus to NSC in vitro from 23.1% to 34. 9%. PCR revealed integration of VP22 gene in NSC cell genome, MTT assay revealed that VP22 had no effects on NSC proliferation. Conclusions The transfection efficiency of lentivirus could be enhanced by VP22, protein. Therefore, lentiviral vectors may provide a potent biological system for delivering genes encoding therapeutic proteins fused to VP22. It could be a useful tool for gene therapy of engineering neural stem cell. Key words: Lentivims vector; VP22shuttle protein; Efficacy of transfection; Neural stem cells