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.