High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation

Mol Ther. 2006 Jan;13(1):151-9. doi: 10.1016/j.ymthe.2005.07.688. Epub 2005 Sep 2.


The use of nonviral gene transfer methods in primary lymphocytes has been hampered by low gene transfer efficiency and high transfection-related toxicity. In this report, high gene transfection efficiency with low transfection-related toxicity was achieved by electroporation using in vitro-transcribed mRNA. Using these methods, >90% transgene expression with >80% viable cells was observed in stimulated primary human and murine T lymphocytes transfected with GFP or mCD62L. Electroporation of unstimulated human PBMCs or murine splenocytes with GFP RNA yielded 95 and 56% GFP+ cells, respectively. Electroporation of mRNA for NY-ESO-1, MART-1, and p53 antigen-specific TCRs into human T lymphocytes redirected these lymphocytes to recognize melanoma cell lines in an MHC-restricted manner. The onset of gene expression was rapid (within 30 min) and durable (up to 7 days postelectroporation) using both GFP and TCR-mediated recognition of target cells. There was no adverse effect observed on the T lymphocytes subjected to RNA electroporation evaluated by cell growth rate, annexin-V staining of apoptotic cells, BrdU incorporation, tumor antigen-specific recognition or antigen-specific TCR affinity. The results of this study indicate that mRNA electroporation provides a powerful tool to introduce genes into both human and murine primary T lymphocytes.

MeSH terms

  • Animals
  • Antigens, Neoplasm / metabolism
  • Apoptosis
  • Cell Proliferation
  • Cell Survival
  • Cells, Cultured
  • Electroporation
  • Green Fluorescent Proteins / biosynthesis
  • Green Fluorescent Proteins / genetics
  • Humans
  • L-Selectin / genetics
  • MART-1 Antigen
  • Membrane Proteins / metabolism
  • Mice
  • Neoplasm Proteins / metabolism
  • RNA, Messenger / biosynthesis*
  • RNA, Messenger / genetics
  • Receptors, Antigen, T-Cell / biosynthesis
  • Receptors, Antigen, T-Cell / genetics
  • Spleen / cytology
  • T-Lymphocytes / cytology
  • T-Lymphocytes / metabolism*
  • Transfection*
  • Tumor Suppressor Protein p53 / metabolism


  • Antigens, Neoplasm
  • CTAG1B protein, human
  • MART-1 Antigen
  • MLANA protein, human
  • Membrane Proteins
  • Mlana protein, mouse
  • Neoplasm Proteins
  • RNA, Messenger
  • Receptors, Antigen, T-Cell
  • Tumor Suppressor Protein p53
  • L-Selectin
  • Green Fluorescent Proteins