Efficient transduction of murine primary T cells requires a combination of high viral titer, preferred tropism, and proper timing of transduction.
Retroviral vectors have been used exclusively for genetic modification of primary T cells. Most T cell infection protocols have been developed for human T cells, whereas systematic investigations of the optimal conditions for transduction of murine primary T cells are limited. In this study, ecotropic and 10A1-pseudotyped retroviral vectors were compared for their efficiency in infecting murine primary T cell cells, as well as T cell lines. Various factors that affect transduction efficiency were also explored, including virus titer, times of exposure, timing of infection, low-speed centrifugation, and use of fibronectin fragment. Our results showed that up to 80% of murine primary T cells could be infected after a single exposure. Successful infection required a combination of high virus titer (>10(7) CFU/ml), proper timing of infection (within 24 h after mitogen stimulation), and preferred tropism (ecotropic vectors). These optimization results may help to establish a standard protocol for infection of murine primary T cells and provide some insight into the obstacles to retroviral infection of T cells.
Zhang
Tong
T
Gene Therapy Group, Department of Microbiology and Immunology, University of Arizona, Tucson, AZ 85721, USA.
Tsang
Tom C
TC
Harris
David T
DT
D000818:Animals; D005434:Flow Cytometry; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D011092:Polyethylene Glycols / Q000737:chemistry; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism*; D013997:Time Factors; D014161:Transduction, Genetic; D018523:Tropism; D014407:Tumor Cells, Cultured
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols