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Methods Mol Biol. 2009;506:83-96. doi: 10.1007/978-1-59745-409-4_7.

Retroviral transduction of murine primary T lymphocytes.

Author information

1
Department of Medicine and Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

Abstract

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.

PMID:
19110621
PMCID:
PMC5003426
DOI:
10.1007/978-1-59745-409-4_7
[Indexed for MEDLINE]
Free PMC Article

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