Consistent transient transfection of DNA into non-transformed human and murine T-lymphocytes.
The ability to analyze transcriptional regulation in non-transformed T-cells has been hampered by the inability to reproducibly transiently transfect these cells with DNA constructs. We have previously demonstrated that normal human whole mononuclear and CD4 T-cells can be consistently transiently transfected with plasmid DNA. Human cells were most receptive to plasmid DNA uptake between 19.5 and 20 h after prestimulation with a submitogenic dose of the polyclonal T-cell activator, PHA. Here we report an alteration and optimization of this protocol for non-transformed murine splenic T-cells, using concanavalin A instead of PHA as the preactivation stimulus. When coupled with the high sensitivity of luciferase reporter gene constructs, this protocol facilitates the analysis of a variety of T-cell-specific promoters in non-transformed T-cells. In addition, we directly demonstrate that murine T-cells are specifically transiently transfected among a population of whole mononuclear cells by using an expression vector for green fluorescent protein.
Cron
R Q
RQ
Department of Pediatrics, University of Washington, Seattle 98195-6320, USA. rqcron@u.washington.educ
Schubert
L A
LA
Lewis
D B
DB
Hughes
C C
CC
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D003208:Concanavalin A / Q000008:administration & dosage; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics*; D018274:Electroporation; D017930:Genes, Reporter; D006801:Humans; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D011401:Promoter Regions, Genetic; D015203:Reproducibility of Results; D013154:Spleen / Q000166:cytology / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D003208:Concanavalin A; D004247:DNA; D008156:Luciferases