Evaluation of techniques for the insertion of nucleic acids in PBMCs

• Grant number: 22/00862-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2022
• End date: December 31, 2022
• Field of knowledge: Biological Sciences - Immunology - Cellular Immunology
• Principal Investigator: Gerson Dierley Keppeke
• Grantee: Kethellen Graziele Gomes da Silva
• Host Institution: Escola Paulista de Medicina (EPM). Universidade Federal de São Paulo (UNIFESP). Campus São Paulo. São Paulo , SP, Brazil
• Associated research grant: 17/20745-1 - Autoantibodies depletion on autoreactive plasmocytes by genome editing with the CRISPR/Cas9 system, AP.JP

Abstract

The insertion of nucleic acids such as DNA plasmids or RNA fragments, aiming for genomic editing, into peripheral blood mononuclear cells (PBMC), which include plasma cells, is a difficult task, mainly due to low efficiency. Several methods are commercially available, so the objective of this work will be to test the different methods of inserting nucleic acids into plasma cells, to find the most efficient and with lower toxicity. Total plasma cells isolated with magnetic microbeads from the PBMC of three healthy donors will be cultured and subjected to transfection by lipid particles containing a circular DNA plasmid. The same plasmid DNA will also be inserted using electroporation. In a third group, the desired gene will be inserted into plasma cells using viral carrier particles, more precisely lentiviruses. The plasmid or lentivirus must contain at least one gene capable of producing a green fluorescent protein, such as GFP. The observation of transfected or transduced plasma cells under a fluorescence microscope will allow the proportional estimation of the efficiency of the method of inserting genetic material. The toxicity of the procedure will be evaluated by the incorporation of Propidium Iodide, a red marker that indicates the destabilization of the plasma membrane, that is, cells in the process of death. In this work, we hope to identify the best method for inserting Cas9 genes and guide RNAs into plasma cells to perform genomic editing techniques in these cells.(AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

GOMES, K.; AIRES, P. P.; KEPPEKE, G. D.. Evaluation of commercially available chemical reagents and electroporation for insertion of nucleic acids into hard-to-transfect cells. Genetics and Molecular Research, v. 22, n. 2, p. 10-pg., 2023-06-02. (17/20745-1, 22/00862-1)