Retroviral transduction of murine primary T lymphocytes

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CD28 Antigens / immunology
  • CD3 Complex / immunology
  • Flow Cytometry
  • Mice
  • Retroviridae / genetics*
  • T-Lymphocytes / cytology
  • T-Lymphocytes / immunology
  • T-Lymphocytes / metabolism*
  • Transduction, Genetic*

Substances

  • CD28 Antigens
  • CD3 Complex