A transient three-plasmid expression system for the production of high titer retroviral vectors

Abstract

We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants.

Sea urchin insulator protects lentiviral vector from silencing by maintaining active chromatin structure

“…49,50 Briefly, 293 T cells were inoculated onto a six-well plate, Sea urchin insulator in lentiviral vector S Hino et al and 24 h later were transfected with three plasmids using Fugene reagent (Roche diagnostics). The composition of the three plasmids is as follows: 3 mg of transfer vector, 3 mg of pCMVD8/9, which encodes HIV-1 viral proteins, and 2 mg of pcDNA-VSV-G (generous gift from Hiroyuki Miyoshi, RIKEN, Tsukuba, Japan), which encodes vesicular stomatitis virus envelope glycoprotein.…”

Modulation of retinoic acid receptor function alters the growth inhibitory response of oral SCC cells to retinoids

“…293T cells were transfected with the retrovirus packaging vectors, pHIT60 and pHIT456 (Soneoka et al, 1995) as well as the MSCVpac retroviral vector containing the mouse mutant RAR-b2(R269Q) (Hawley et al, 1994;Pear et al, 1993;Tairis et al, 1995;Wu et al, 1997b). After transfection for 24 h, the medium was removed and 10 ml of fresh medium added.…”

MuLV-based vectors pseudotyped with truncated HIV glycoproteins mediate specific gene transfer in CD4+ peripheral blood lymphocytes

“…All the pHIT plasmids were obtained from AJ Kingsman (Oxford University, Oxford, UK). 36 Plasmid pSVIIIEnv713 was created by cloning the KpnI-BamHI (corresponding to nucleotide positions 5893 and 8017 of HIV HXBc2 proviral DNA, respectively; +1 = site of initiation of transcription) segment of HXBH10⌬CT, 30 a proviral DNA containing a stop codon in env (corresponding to a TCA to TAA substitution at HIV-HxBc2 nucleotide position 7902), and encoding an Env truncated of its cytoplasmic domain (obtained from Heinrich G Gö ttlinger, DFCI, Boston, MA, USA) in pSVIIIEnv, the vector used for expression of Env under the control of the HIV LTR promoter. The Env-negative proviral construct harboring a mutation in the matrix protein HXBH10Env-(KpnIfs)⌬16-18 was generated by cloning the SalI-BamHI (HXBH10 nucleotide positions 5372 and 8058, respectively; +1 = site of initiation of transcription) fragment of HXBH10Env-(KpnIfs) into HXBH10⌬16-18; both plasmids have been described elsewhere.…”

“…Infectivity assay using ␤-galactosidase activity The infectivity assay used to determine if viruses harboring the different Env glycoproteins retained infectious capacity was similar to the one described by Kimpton and Emerman, 33 with several modifications, in order to comply with the MuLV lacZ transduction strategy elaborated by Soneoka et al 36 Briefly, COS cells were lipofected with various combinations of GagPol encoding DNA and Env expression vectors as described in the previous section. Furthermore, 15 g of the pHIT111 lacZ vector was added to every transfection when pHIT60 was present, in order to obtain lacZ transduction through MuLV viruses.…”