提高逆转录病毒载体滴度和表达论文.pdfVIP

NIH Public Access Author Manuscript Biotechniques . Author manuscript; available in PMC 2006 February 3. N Published in final edited form as: I H Biotechniques . 1989 October ; 7(9): 980–990. - P A A Improved Retroviral Vectors for Gene Transfer and Expression u t h o A. Dusty Miller and Guy J. Rosman r M The Fred Hutchinson Cancer Research Center a n u Abstract s c r We describe a set of murine retrovirus-based vectors that include unique cloning sites for insertion i p t of cDNAs such that the cDNA can be driven by either the retroviral long terminal repeat, the immediate early promoter of human cytomegalovirus, or the simian virus 40 early promoter. The vectors carry the neomycin phosphotransferase gene expressed from an alternate promoter as a selectable marker. These vectors have been constructed to prevent viral protein synthesis from the remaining viral sequences, to yield high-titer virus stocks after introduction into retrovirus packaging cells, and to eliminate homologous overlap with viral DNAs present in retrovirus packaging cells in order to prevent helper virus production. Methods for generating high-titer virus are described. N I H - P A INTRODUCTION A u Retroviral vectors provide a highly efficient method for gene transfer into eukaryotic cells t h (12). This vector system can be divided into two components; the retroviral vector itself, which o r generally does not encode viral proteins, and the retrovirus packaging cell line, which provides