Gene transfection and expression in resting and activated murine CD4 T cell subsets.
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.
Lai
Wendy
W
Department of Surgery, University of Maryland School of Medicine, MSTF Building, Room 400, Baltimore, 685 W Baltimore St, Baltimore, MD 21201, USA.
Chang
Cheong-Hee
CH
Farber
Donna L
DL
D000818:Animals; D017252:CD3 Complex / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D015870:Gene Expression; D015316:Genetic Therapy; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D014162:Transfection / Q000379:methods*
D017252:CD3 Complex; D011948:Receptors, Antigen, T-Cell