Evaluation of commercially available chemical reagents and electroporation for insertion of nucleic acids into hard-to-transfect cells

Autor(es)

Gomes, K. ; Aires, P. P. ; Keppeke, G. D.

• Tipo de documento: Artigo Científico
• Fonte: Genetics and Molecular Research; v. 22, n. 2, p. 10-pg., 2023-06-02.

Resumo

Insertion of nucleic acids into cells unlocks the possibility of modulating gene expression; however, some cells such as primary cells and those that grow in suspension are hard-to-transfect. New therapies for cancer and possible autoimmune diseases, such as those that involve chimeric antigen receptor T cells, rely on the insertion of plasmids into lymphocytes, which fit into the hard-to-transfect category. In such cases virus-based transduction is usually applied, but the carrier vector tends to be incorporated into the cell's own DNA in a stable manner, with unpredictable consequences. Thus, highly efficient nonviral gene/plasmid delivery is a sought-after technology. We evaluated several commercially available chemical transfection methods as well as electroporation in difficult-to-transfect cells, including a human lymphocyte cell-line (Jurkat) and fresh peripheral blood mononuclear cells (PBMCs), both grown in suspension. The cell-toxicity of the methods was also evaluated. Twenty-four hours after transfection of the plasmid pCMV-GFP, the proportion of GFP positive (GFP+) cells was evaluated by cytometry. The cationic polymer TurboFect yielded similar to 7.8% of GFP+ Jurkat cells on average, while the other reagents (Lipofectamine 3000, FuGENE HD and X-tremeGENE HP) presented <3% of GFP+ cells. In PBMCs, none of the chemical reagents yielded >3% transfected cells. Electroporation was more efficient, with similar to 45% of GFP+ in Jurkat and similar to 15.7% GFP+ in PBMCs. However, it proved to be highly toxic, with similar to 80% of the cells considered non-viable 24h after the procedure, while TurboFect showed little-to-no toxicity. In conclusion, it was found that despite its high toxicity electroporation was the only method with applicable transfection efficiency in PBMCs, while in Jurkat the reagent TurboFect can be applied with acceptable results. The strategy for insertion of nucleic acids needs to be fine-tuned for each target cell type and experimental condition. (AU)

• Processo FAPESP: 17/20745-1 - Depleção de autoanticorpos por edição gênica com o sistema CRISPR/Cas9 em plasmócitos autorreativos
• Beneficiário: Gerson Dierley Keppeke
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores
• Processo FAPESP: 22/00862-1 - Avaliação de técnicas para inserção de ácidos nucleicos em plasmócitos
• Beneficiário: Kethellen Graziele Gomes da Silva
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica