title: Improvement of gene transduction efficiency in T lymphocytes using retroviral vectors.
abstract: Successful gene transfer into T lymphocytes would provide a useful therapeutic modality for the treatment of various diseases and a valuable way to study T cell functions. Currently, most protocols involving gene transfer into T lymphocytes utilize amphotropic retroviral vectors. However, transduction efficiency using these vectors is relatively low because of the high proportion of resting cells, the concentration-dependent growth manner of T lymphocytes, and the low titer of retroviral vectors. In this article we define conditions that provide high levels of transduction by using IL-2 prestimulation and LipofectAMINE for both mouse and human T lymphocytes. We compared the effects of IL-2 prestimulation on transduction efficiencies at different time points and achieved maximum transfer levels at 72 hr after the incubation. By combining the best prestimulation time and cationic lipids-LipofectAMINE at a dose of 0.8 microM, the transduction efficiencies were increased to 45-75% (62.3 +/- 4.3%) in human T lymphocytes and to 21-33% (27 +/- 1.42%) in murine T lymphocytes as determine by FDG staining and X-Gal visualization, compared with 5% with conventional methods. These results indicate that transduction efficiencies in T lymphocytes can be significantly improved by a prolonged preincubation with IL-2 and by the addition of LipofectAMINE.
authors.affiliation: Division of Hematology/Oncology, Vincent Lombardi Cancer Center, Georgetown University School of Medicine, Washington, DC 20007, USA.
authors.forename: A G
authors.initials: AG
authors.lastname: Wu
authors.forename: X
authors.initials: X
authors.lastname: Liu
authors.forename: A
authors.initials: A
authors.lastname: Mazumder
authors.forename: J A
authors.initials: JA
authors.lastname: Bellanti
authors.forename: K R
authors.initials: KR
authors.lastname: Meehan
mesh_terms: D000818:Animals; D002411:Cation Exchange Resins; D005822:Genetic Vectors; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D008055:Lipids; D008081:Liposomes; D051379:Mice; D008811:Mice, Inbred DBA; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism*; D014161:Transduction, Genetic
chemical_list: D002411:Cation Exchange Resins; D007376:Interleukin-2; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes