T-Helper Cells pp 217-226 | Cite as

mRNA Transfection of T-Lymphocytes by Electroporation

Part of the Methods in Molecular Biology book series (MIMB, volume 2285)


Electroporation enables the transfection of different cell types including microbial, plant, and animal cells with charged molecules, such as nuclear acids or proteins. During electroporation, an electrical field is applied to the cells leading to a transient permeabilization of the cell membrane allowing exogenous molecules to enter the cells. Here we report the electroporation of human primary CD4+ -T cells with in-vitro transcribed mRNA to facilitate gene editing (knockout) of the CC-chemokine receptor 5 (CCR5), the coreceptor of the human immunodeficiency virus 1 (HIV1) predominantly used during primary infection. Using such strategy of transient expression of a CCR5-specific Transcription-activator-like-effector nuclease (TALEN), we aim to protect helper T cells from de novo HIV infection.

Key words

Electroporation mRNA transfection CD4+-T cells isolation Gene editing CCR5-knockout Gene therapy 



Our work has been supported by the German Center for Infection Research (DZIF) within TTU HIV (04.803). We wish to thank Tanja Sonntag, the UKE Cytometry & Cell Sorting Facility and the Institute for Transfusion Medicine of the UKE for expert technical assistance and support.


  1. 1.
    Shi J, Ma Y, Zhu J et al (2018) A review on electroporation-based intracellular delivery. Molecules 23:3044. Scholar
  2. 2.
    Wong T-K, Neumann E (1982) Electric field-mediated gene transfer. Biochem Biophys Res Commun 107:584–587CrossRefGoogle Scholar
  3. 3.
    Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982) Gene transfer into electric fields. EMBO J 1:841–845CrossRefGoogle Scholar
  4. 4.
    Potter H, Weir L, Leder P (1984) Enhancer-dependent expression of human κ immunoglobulin genes introduced into mouse pre-B-lymphocytes by electroporation. Proc Natl Acad Sci U S A 81:7161–7165. Scholar
  5. 5.
    Tsong TY (1990) On electroporation of cell membranes and some. Relat Phenom 299:271–295Google Scholar
  6. 6.
    Rosazza C, Meglic SH, Zumbusch A et al (2016) Gene Electrotransfer: a mechanistic perspective. Curr Gene Ther 16:98–129CrossRefGoogle Scholar
  7. 7.
    Baum C, Forster P, Hegewisch-Becker S, Harbers K (1994) An optimized electroporation protocol applicable to a wide range of cell lines. BioTechniques 17:1058–1062PubMedGoogle Scholar
  8. 8.
    Deora AA, Fernando D, Ryan S, Rodriguez-Boulan E (2008) Efficient electroporation of DNA and protein into confluent and differentiated epithelial cells in culture. Bone 23:1–7. Scholar
  9. 9.
    Batista Napotnik T, Miklavčič D (2018) In vitro electroporation detection methods—an overview. Bioelectrochemistry 120:166–182. Scholar
  10. 10.
    Aksoy P, Aksoy BA, Czech E, Hammerbacher J (2018) Electroporation characteristics of human primary T cells bioRxiv 466243.
  11. 11.
    Zhang Z, Qiu S, Zhang X, Chen W (2018) Optimized DNA electroporation for primary human T-cell engineering. BMC Biotechnol 18:1–9. Scholar
  12. 12.
    Saulis G, Saule R (2012) Size of the pores created by an electric pulse: microsecond vs millisecond pulses. Biochim Biophys Acta Biomembr 1818:3032–3039. Scholar
  13. 13.
    Weaver JC (1993) Electroporation: a general phenomenon for manipulating cells and tissues. J Cell Biochem 51:426–435. Scholar
  14. 14.
    Zerbib D, Amalric F, Teissié J (1985) Electric field-mediated transformation: Isolation and characterization of a TK+ subclone. Biochem Biophys Res Commun 129:611–618. Scholar
  15. 15.
    Biasco L, Baricordi C, Aiuti A (2012) Retroviral integrations in gene therapy trials. Mol Ther 20:709–716. Scholar
  16. 16.
    Cesana D, Sgualdino J, Rudilosso L et al (2012) Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations. J Clin Invest 122:1667–1676. Scholar
  17. 17.
    Cesana D, Volpin M, Serina Secanechia YN, Montini E (2017) Safety and efficacy of retroviral and lentiviral vectors for gene therapy. In: Brunetti-Pierri N (ed) Safety and efficacy of gene-based therapeutics for inherited disorders. Springer International Publishing, Cham, pp 9–35CrossRefGoogle Scholar
  18. 18.
    Porteus M (2016) Genome editing: a new approach to human therapeutics. Annu Rev Pharmacol Toxicol 56:163–190. Scholar
  19. 19.
    Berdien B, Mock U, Atanackovic D, Fehse B (2014) TALEN-mediated editing of endogenous T-cell receptors facilitates efficient reprogramming of T-lymphocytes by lentiviral gene transfer. Gene Ther 21:539–548. Scholar
  20. 20.
    Mock U, Machowicz R, Hauber I et al (2015) mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV coreceptor CCR5. Nucleic Acids Res 43:5560–5571. Scholar
  21. 21.
    Chicaybam L, Sodre AL, Curzio BA, Bonamino MH (2013) An efficient low-cost method for gene transfer to T-lymphocytes. PLoS One 8:1–11. Scholar
  22. 22.
    Mock U, Hauber I, Fehse B (2016) Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases. Nat Protoc 11:598–615. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.Research Department of Cell and Gene Therapy, Department of Stem Cell TransplantationUniversity Medical Center Hamburg-EppendorfHamburgGermany
  2. 2.German Center for Infection Research (DZIF)HamburgGermany

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