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Expression of HTLV-1 Genes in T-Cells Using RNA Electroporation

  • Mariangela Manicone
  • Francesca Rende
  • Ilaria Cavallari
  • Andrea K. Thoma-Kress
  • Vincenzo CiminaleEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1582)

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) infects about 20 million people world-wide. Around 5% of the infected individuals develop adult T-cell leukemia (ATL) or a neurological disease termed tropical spastic paraparesis (TSP) after a clinical latency of years to decades. Through the use of two promoters and alternative splicing HTLV-1 expresses at least 12 different proteins. HTLV-1 establishes a life-long persistent infection by inducing the clonal expansion of infected cells, a property largely ascribed to the viral genes Tax and HBZ. However, the fact that ATL arises in a minority of infected individuals after a long clinical latency suggests the existence of factors counterbalancing the oncogenic potential of HTLV-1 in the context of natural infection.

To study the role of the different HTLV-1 gene products in the HTLV-1 life cycle, we optimized a transfection protocol for primary T-cells using an approach based on the electroporation of in vitro-transcribed RNA. Results showed that the RNA transfection technique combines a high transfection efficiency with low toxicity, not only in Jurkat T-cells but also in primary T-cells. These findings suggest that RNA electroporation is preferable for experiments aimed at investigating the role of HTLV-1 gene products in the context of primary T-cells, which represent the main target of HTLV-1 in vivo.

Key words

HTLV-1 ATL In vitro transcription RNA electroporation Peripheral blood mononuclear cells (PBMCs) 

Notes

Acknowledgment

The authors are grateful to Prof. Ugur Sahin (Research Center for Immunotherapy (FZI), Mainz, Germany; TRON—Translational Oncology at the University Medical Center of Johannes Gutenberg University, Mainz, Germany; Biopharmaceutical New Technologies (BioNTech) Corporation, Mainz, Germany) for providing the plasmid pST1-eGFPmut-2hBgUTR-A120. We are grateful to Armin Ensser and Benjamin Vogel (Institute of Clinical and Molecular Virology, Erlangen, Germany) for helpful discussions.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Mariangela Manicone
    • 1
  • Francesca Rende
    • 1
  • Ilaria Cavallari
    • 3
  • Andrea K. Thoma-Kress
    • 2
  • Vincenzo Ciminale
    • 1
    • 3
    Email author
  1. 1.Department of Surgery, Oncology and GastroenterologyUniversity of PadovaPadovaItaly
  2. 2.Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-NürnbergErlangenGermany
  3. 3.Istituto Oncologico Veneto, IRCCSPadovaItaly

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