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

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• Transfecting B-cell lines and primary peripheral blood mononuclear cells (PBMCs) is challenging, hindering genome editing and gene expression studies.
• Existing methods are often inefficient, costly, or require specialized equipment, limiting their widespread application.

Purpose of the Study:

• To develop a novel, efficient, and reproducible electrotransfection protocol for various B-cell types.
• To optimize conditions for high transfection and cell viability using cost-effective reagents and standard equipment.

Main Methods:

• Electrotransfection of lymphoblastoid cells, B-cell lymphoma, leukemia cell lines, and primary B cells from PBMCs.
• Optimization of salt concentration in the electrotransfection medium and plasmid amount.
• Validation through the generation of a TP53-deficient cell line (TP53^-/- RPMI8866).

Main Results:

• Achieved routine transfection rates of approximately 79% and viability rates of 58%.
• The protocol is effective for both small and large plasmids.
• Successfully generated a TP53^-/- RPMI8866 lymphoblastoid cell line.

Conclusions:

• The developed electrotransfection protocol offers an efficient, reproducible, and cost-effective solution for B-cell manipulation.
• This method facilitates genome editing, gene silencing, and ectopic gene expression in B cells.
• The validated protocol and generated cell line will aid future research in hematological and blood cancers.