High-level transfection of primary rabbit T lymphocytes

J Immunol Methods. 2008 Jul 20;336(1):85-9. doi: 10.1016/j.jim.2008.03.006. Epub 2008 Apr 7.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • CD4 Antigens / genetics
  • CD4 Antigens / immunology
  • Cell Survival / immunology
  • Electroporation / methods
  • Female
  • Flow Cytometry / veterinary
  • Formazans / chemistry
  • Green Fluorescent Proteins / genetics
  • Models, Animal
  • Rabbits / physiology*
  • T-Lymphocytes / immunology*
  • Tetrazolium Salts / chemistry
  • Transfection / methods*
  • nef Gene Products, Human Immunodeficiency Virus / immunology

Substances

  • CD4 Antigens
  • Formazans
  • Tetrazolium Salts
  • nef Gene Products, Human Immunodeficiency Virus
  • nef protein, Human immunodeficiency virus 1
  • Green Fluorescent Proteins
  • MTT formazan