THE PCL VECTOR SYSTEM - RAPID PRODUCTION OF HELPER-FREE, HIGH-TITER, RECOMBINANT RETROVIRUSES

We describe the construction and characterization of retroviral vector s and packaging plasmids that produce helper-free retrovirus with tite rs of 1 x 10(6) to 5 x 10(6) within 48 h. These vectors contain the im mediate early region of the human cytomegalovirus enhancer-promoter fu sed to the Moloney murine leukemia virus long terminal repeat at the T ATA box in the 5' U3 region, yielding the pCL promoter. By selecting v ectors designed to express genes from one of four promoters (dihydrofo late reductase, Rous sarcoma virus, long terminal repeat, or cytomegal ovirus), the pCL system permits the investigator to control the level of gene expression in target cells over a 100-fold range, while mainta ining uniformly high titers of virus from transiently transfected prod ucer cells. The pCL packaging plasmids lack a packaging signal (Delta Psi) and include an added safety modification that renders them self-i nactivating through the deletion of the 3' U3 enhancer. Ecotropic, amp hotropic (4070A), and amphotropic-mink cell focus-forming hybrid (10A1 ) envelope constructions have been prepared and tested, permitting fle xible selection of vector pseudotype in accordance with experimental n eeds. Vector supernatants are free of helper virus and are of sufficie ntly high titer within 2 days of transient transfection in 293 cells t o permit infection of more than 50% of randomly cycling target cells i n culture. We demonstrated the efficacy of these vectors by using them to transfer three potent cell cycle control genes (the p16(INK4A), p5 3, and Rbl genes) into human glioblastoma cells.