High-level transfection of primary rabbit T lymphocytes.
Efficient gene delivery is essential for the assessment of transgene function in untransformed hematopoietic cells. Here, we explored the utility of different non-viral and viral gene delivery techniques for primary T cells from New Zealand White rabbits. We find that electroporation and nucleofection result in high-level transgene expression from both small and large GFP reporter constructs in activated rabbit T cells at moderate cytotoxicity. Both non-viral gene delivery methods were vastly superior to retroviral, lentiviral, or adenoviral transduction approaches. The effectiveness of non-viral gene delivery for functional analyses was demonstrated by downregulation of CD4 cell surface molecules through transient expression of the endocytosis-inducing Nef protein from human immunodeficiency virus in a signature motif-specific manner. This study establishes conventional electroporation as an efficient and inexpensive procedure to render primary rabbit T cells accessible to rapid functional ex vivo analyses.
Tervo
Hanna-Mari
HM
Department of Virology, University of Heidelberg, Heidelberg, Germany.
Allespach
Ina
I
Keppler
Oliver T
OT
D000818:Animals; D030801:Animals, Genetically Modified; D015704:CD4 Antigens / Q000235:genetics / Q000276:immunology; D002470:Cell Survival / Q000276:immunology; D018274:Electroporation / Q000379:methods; D005260:Female; D005434:Flow Cytometry / Q000662:veterinary; D005562:Formazans / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics; D023421:Models, Animal; D011817:Rabbits / Q000502:physiology*; D013601:T-Lymphocytes / Q000276:immunology*; D013778:Tetrazolium Salts / Q000737:chemistry; D014162:Transfection / Q000379:methods*; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000276:immunology
D015704:CD4 Antigens; D005562:Formazans; D013778:Tetrazolium Salts; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D049452:Green Fluorescent Proteins; C079782:MTT formazan