Establishment Of A Packaging Cell Line For Lentivirus Vector

(1 School of Pharmaceutical Engneering,Shenyang Pharmaceutical University,Shenyang, 110016 China; 2 Institute of Biotechnology, Academy of Military Medical Sciences Beijing, China)Lentivirus provides effective means for expression of exogenous genes in mammalian cells, which had proved efficient and achieved stable long-term expression of the transgenes, so it was developed as an important transgenic vector for transgenic animals and gene therapy.To generate a safe, replication-defective lentiviral vector, a genetically split gene expression was designed. The representative one is the third generation lentiviral vector based on HIV-1, which including four plasmids: (1) the packaging vector consist of gag-pol gene;(2) the plasmid encoding Rev protein;(3) the plasmid encoding the vesicular stomatitis virus G-protein (VSVG);(4) the transfer vector containing the transgene, lentiviral vectors had been generated by transient transfection of four plasmids into 293T cells.To facilitate vectors production for research on transgenic animals and gene therapy, We describe here the generation of a safe, stable tetracycline-regulated packaging cell line based on the third generation lentivirus vectors. There had been some difficulty in generating stable packaging cell line, possibly because of the toxicity of VSVG protein. Using of a single inducible expression vector pTRE2rtTAG, the cytotoxic of the envelope protein was circumvented.To establish stable cell line, 293FT cells were co-transfected with pLP1-zeo,pLP2, pTRE2rtTAG and Zeocin-resistant cell clones were expanded and analysed for PCR (polymerase chain reaction) for the presence of gag/pol,rev,VSVG and rtTA DNA. Six cell clones positive for PCR were further analyzed by reverse transcriptase-PCR(RT-PCR), among of them three were positive for RT-PCR. Then Northern blot hybridization was performed. The VSV-G mRNA in two clone was nondetectable in the absence of doxycycline,a tetracycline derivative, and induction of VSVG mRNA was observed when the doxycycline was present, then they were...