Easy and robust electrotransfection protocol for efficient ectopic gene expression and genome editing in human B cells

Reynand Jay Canoy (1, 2) , Franck M. André (1, 3, 4) , Anna Shmakova (1, 5, 4) , Joëlle Wiels (1, 5, 4) , Marc Lipinski (1, 5, 4) , Yegor Vassetzky (1, 6, 5, 4) , Diego Germini (1, 4, 5)

1 METSY - Aspects métaboliques et systémiques de l'oncogénèse pour de nouvelles approches thérapeutiques
2 UP System - University of the Philippines
3 UMR 8203 - Vectorologie et thérapeutiques anti-cancéreuses [Villejuif]
4 IGR - Institut Gustave Roussy
5 UMR8126 - Signalisation, noyaux et innovations en cancérologie
6 Koltzov Institute of Developmental Biology

B-cell lines and primary PBMCs are notoriously hard to transfect, thus making genome editing, ectopic gene expression, or gene silencing experiments particularly tedious. Here we propose a novel efficient and reproducible protocol for electrotransfection of lymphoblastoid, B-cell lymphoma, leukemia cell lines, and B cells from PBMCs. The proposed protocol requires neither costly equipment nor expensive reagents; it can be used with small or large plasmids. Transfection and viability rates of about 79% and 58%, respectively, have been routinely achieved by optimizing the salt concentration in the electrotransfection medium and the amount of plasmid used. A validation of the protocol was obtained via the generation of a TP53 −/− RPMI8866 lymphoblastoid cell line which should prove useful in future hematological and blood cancer studies.

Domaines: Biotechnologies

Reynand Jay Canoy, Franck M. André, Anna Shmakova, Joëlle Wiels, Marc Lipinski, et al.. Easy and robust electrotransfection protocol for efficient ectopic gene expression and genome editing in human B cells. Gene Therapy, In press, ⟨10.1038/s41434-020-00194-x⟩. ⟨hal-02988662⟩