title: Continuous high-titer HIV-1 vector production.
abstract: Human immunodeficiency virus type 1 (HIV-1)-based vectors are currently made by transient transfection, or using packaging cell lines in which expression of HIV-1 Gag and Pol proteins is induced. Continuous vector production by cells in which HIV-1 Gag-Pol is stably expressed would allow rapid and reproducible generation of large vector batches. However, attempts to make stable HIV-1 packaging cells by transfection of plasmids encoding HIV-1 Gag-Pol have resulted in cells which secrete only low levels of p24 antigen (20-80 ng/ml), possibly because of the cytotoxicity of HIV-1 protease. Infection of cells with HIV-1 can result in stable virus production; cell clones that produce up to 1,000 ng/ml secreted p24 antigen have been described. Here we report that expression of HIV-1 Gag-Pol by a murine leukemia virus (MLV) vector allows constitutive, long-term, high-level (up to 850 ng/ml p24) expression of HIV-1 Gag. Stable packaging cells were constructed using codon-optimized HIV-1 Gag-Pol and envelope proteins of gammaretroviruses; these producer cells could make up to 10(7) 293T infectious units (i.u.)/ml (20 293T i.u./cell/day) for at least three months in culture.
authors.affiliation: Department of Immunology and Molecular Pathology, Windeyer Institute, University College London, 46 Cleveland St., London W1T 4JF, UK.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Ikeda
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takeuchi
authors.forename: Francisco
authors.initials: F
authors.lastname: Martin
authors.forename: Francois-Loic
authors.initials: FL
authors.lastname: Cosset
authors.forename: Kyriacos
authors.initials: K
authors.lastname: Mitrophanous
authors.forename: Mary
authors.initials: M
authors.lastname: Collins
mesh_terms: D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D005822:Genetic Vectors / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D015497:HIV-1 / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D011786:Quality Control; D014162:Transfection / Q000379:methods*