A simple, rapid method for evaluation of transfection efficiency based on fluorescent dye

Area of Science:

• Biotechnology
• Cell Biology
• Molecular Biology

Background:

• Transient gene expression (TGE) and electroporation are crucial for producing recombinant therapeutic proteins in mammalian cells.
• Improving transfection efficiency is key to enhancing the yield of these valuable proteins.
• Current methods for evaluating transfection efficiency can be time-consuming and indirect.

Purpose of the Study:

• To develop and validate a novel, rapid method for evaluating transfection efficiency in Chinese Hamster Ovary (CHO) cells.
• To optimize electroporation conditions for enhanced transient gene expression of recombinant Factor VII (rFVII).
• To demonstrate the applicability of the method for assessing transfection without reporter genes.

Main Methods:

• CHO cells were engineered to express recombinant FVII (rFVII).
• Cells were labeled with fluorescent dye and analyzed using confocal microscopy and flow cytometry.
• Electroporation parameters (voltage, repetitions, plasmid/carrier DNA amounts) were systematically varied.
• Transfection efficiency was quantified by detecting cells with the rFVII encoding gene.

Main Results:

• A novel method enabled direct detection and quantification of transfected cells via flow cytometry.
• The highest transfection efficiency achieved using optimized electroporation conditions was approximately 86%.
• The method successfully evaluated transfection efficiency without the need for co-expressed reporter genes.

Conclusions:

• The developed method provides a rapid and accurate assessment of transfection efficiency in mammalian cells.
• This technique is valuable for optimizing recombinant protein production processes.
• The method's adaptability with antibodies allows for evaluating transfection of other recombinant proteins.