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Chronic Lymphocytic Leukemia

Efficient gene transfer in CLL by mRNA electroporation

Leukemia volume 22, pages 323–329 (2008)Cite this article

Abstract

Chronic lymphocytic leukemia (CLL) consists of at least two major prognostic subgroups, characterized by different cellular and molecular markers. This observation sparked studies on the function and clinical importance of these markers. In order to address their function adequately, an efficient and reliable method for gene transfer is needed. In this study, we compared efficiency and utility of different gene transfer techniques in CLL. Lenti-, retro- and adenoviral transduction did not yield appreciable numbers of marker gene enhanced green fluorescent protein (EGFP) positive CLL cells, despite various prestimulation protocols. Efficient transgene expression was observed after nucleofection of CLL cells with plasmid DNA, at the expense of low survival rates. After optimization, electroporation of in vitro transcribed mRNA yielded up to 90% EGFP+CLL cells without affecting survival. Transgene expression remained detectable for at least 2 weeks after electroporation. Furthermore, we could demonstrate overexpression of ZAP70 and of a ZAP70-EGFP fusion protein after electroporation with ZAP70 or ZAP70-EGFP mRNA. We conclude that mRNA electroporation is a novel and straightforward method for highly efficient gene transfer in CLL. The application of this technique should facilitate functional studies on CLL cells, as well as clinical research.

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Acknowledgements

This work was supported by Grant No G.0026.06 from the Research Foundation—Flanders (FWO-Vlaanderen), Grant No 01J02006 from the Bijzonder Onderzoeksfonds from Ghent University and Grant 2007 from Centrum voor Gezwelziekten, Ghent University Hospital and the Interuniversity Attraction Poles Programme—Belgian State—Belgian Science Policy. BV is a Senior Clinical Investigator and FVB is a Research Assistant of the Research Foundation—Flanders (FWO-Vlaanderen). We thank Dr Fritz Offner and Dr Ann Janssens for providing blood samples, Dr Nathalie Cools for critical reading of the manuscript, Christian De Boever for help with artwork and Drs Reuven Agami, Chiara Bonini, Laure Coulombel, Dimitar Efremov, Eli Gilboa, Harry Heimberg, Lawrence Samelson, Hergen Spits, Kris Thielemans, Mireille Van Gele and Juan-Carlos Zùñiga-Pflücker for providing cell lines and research reagents. The personnel of the Aids Reference Laboratory kindly provided technical assistance with sequencing.

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Authors and Affiliations

  1. Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium

    F Van Bockstaele, V Pede, E Naessens, S Van Coppernolle, B Verhasselt & J Philippé

  2. Laboratory of Experimental Hematology, Antwerp University Hospital, University of Antwerp, Edegem, Belgium

    V Van Tendeloo

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  1. F Van Bockstaele
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Correspondence to J Philippé.

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The work was done in Ghent University Hospital, Ghent, Belgium and Antwerp University Hospital, Edegem, Belgium.

Note added in proof

While this manuscript was in review, a paper appeared in Leukemia (Seiffert M, Stilgenbauer S, Döhner H and Lichter P. Efficient nucleofection of primary human B cells and B-CLL cells induces apoptosis which depends on the microenvironment and on the structure of transfected nucleic acids. Leukemia 2007; 21:1977–83.), that confirms increased cell mortality after nucleofection with plasmid DNA. Furthermore, these authors also reported transfection efficiencies of 80–90% after nucleofection of EGFP mRNA.

Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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Van Bockstaele, F., Pede, V., Naessens, E. et al. Efficient gene transfer in CLL by mRNA electroporation. Leukemia 22, 323–329 (2008). https://doi.org/10.1038/sj.leu.2405007

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