Gene transfection and expression in resting and activated murine CD4 T cell subsets

Abstract

It has been difficult to assess the role of specific genes in activation and differentiation of peripheral T cell subsets such as naive, effector and memory T cells due to the impairments in T cell development and immune pathologies often observed in genetically manipulated mouse models, and the lack of reliable methods for introducing genes into primary mouse T cells. In this study, we demonstrate transient transfection of genes into resting and activated mouse CD4 T cell subsets using "Nucleofection", a modified electroporation technique. Using this approach, cDNA encoding green fluorescent protein (GFP) is efficiently taken up and expressed by purified polyclonal and antigen-specific mouse naive, effector and memory CD4 T cells isolated from BALB/c or TCR-transgenic mice. The resultant transfected resting T cells are fully amenable to TCR-mediated activation. We also demonstrate that expression of endogenous gene can be turned on in resting T cells by transfection of a transcriptional transactivator. Our results demonstrate for the first time, the expression of exogenously transfected genes and the modulation of endogenous gene expression in primary mouse T cell subsets. This technology will enable a variety of mechanistic questions on T cell activation, function and signaling to be addressed in T cells that differ in activation history and functional capacities.