期刊
JOURNAL OF IMMUNOLOGICAL METHODS
卷 282, 期 1-2, 页码 93-102出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jim.2003.07.015
关键词
T lymphocytes; rodent; cellular differentiation; molecular biology; gene therapy
资金
- NIAID NIH HHS [AI42092] Funding Source: Medline
- NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES [R01AI042092, R29AI042092] Funding Source: NIH RePORTER
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(TM), 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. (C) 2003 Elsevier B.V. All rights reserved.
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