Retroviral transduction of murine primary T lymphocytes.
In comparison to human T cells, efficient retroviral gene transfer and subsequent expansion of murine primary T cells is more difficult to achieve. Herein, we describe an optimized gene transfer protocol utilizing an ecotropic viral vector to transduce primary murine T cells activated with magnetic beads coated with agonistic anti-CD3 and CD28 antibodies. Activated T cells are subsequently centrifuged (spinoculated) on RetroNectin-coated tissue culture plates in the context of retroviral supernatant. Variables found to be critical to high gene transfer and subsequent efficient T cell expansion included CD3/CD28 magnetic bead to cell ratio, time from T cell activation to initial spinoculation, frequency of T cell spin-oculation, interleukin-2 concentration in the medium, and the initial purity of the T cell preparation.
Lee
James
J
Department of Medicine and Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Sadelain
Michel
M
Brentjens
Renier
R
D000818:Animals; D018106:CD28 Antigens / Q000276:immunology; D017252:CD3 Complex / Q000276:immunology; D005434:Flow Cytometry; D051379:Mice; D012190:Retroviridae / Q000235:genetics*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D014161:Transduction, Genetic
D018106:CD28 Antigens; D017252:CD3 Complex