Summary
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.
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Acknowledgments
We thank Dr. Isabelle Riviere for reviewing the manuscript. Our work is supported by the US National Cancer Institute (grants CA95152, CA59350), The Annual Terry Fox Run for Cancer Research (New York, NY) organized by the Canada Club of New York, William H. Goodwin and Alice Goodwin and the Commonwealth Cancer Foundation for Research and the Experimental Therapeutics Center of MSKCC, and the Bocina Cancer Research Fund.
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Lee, J., Sadelain, M., Brentjens, R. (2009). Retroviral Transduction of Murine Primary T Lymphocytes. In: Baum, C. (eds) Genetic Modification of Hematopoietic Stem Cells. Methods In Molecular Biology™, vol 506. Humana Press. https://doi.org/10.1007/978-1-59745-409-4_7
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DOI: https://doi.org/10.1007/978-1-59745-409-4_7
Publisher Name: Humana Press
Print ISBN: 978-1-58829-980-2
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