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Updated: Dec 12, 2025
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Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency
Published on: January 7, 2010
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A versatile bulk electrotransfection protocol for murine embryonic fibroblasts and iPS cells
Shahin Eghbalsaied1,2, Iqbal Hyder1, Wilfried A Kues3
1Department of Biotechnology, Friedrich-Loeffler-Institut (FLI), Höltystr. 10, 31535, Neustadt, Germany.
Scientific Reports
|August 10, 2020
Summary
We developed an efficient electroporation protocol for enhanced gene transfer into primary cells. This method achieves high transfection and gene knockout rates in murine embryonic fibroblasts and stem cells using CRISPR/Cas9 technology.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetic Engineering
Background:
- Electroporation is a key method for gene transfer into cells.
- Improving electroporation efficiency in primary cells remains a challenge.
Purpose of the Study:
- To develop a novel electroporation protocol for enhanced DNA delivery into primary cells.
- To optimize gene editing efficiency using CRISPR/Cas9 in murine cells.
Main Methods:
- A square-wave pulsing protocol using OptiMEM-GlutaMAX was employed.
- Transfection efficiency was assessed using reporter genes and CRISPR/Cas9 plasmids in MEF and iPS cells.
- Gene knockout and deletion rates were quantified using a Venus reporter system.
Main Results:
- Achieved >95% electrotransfection efficiency for reporter plasmids in MEF and iPS cells.
- The protocol efficiently delivered plasmids ranging from 6.2 to 13.5 kb.
- High targeted gene knockout rates (up to 98%) and deletion rates (up to 67%) were achieved using CRISPR/Cas9.
Conclusions:
- The developed protocol offers a straightforward, cost-effective, and efficient method for plasmid electrotransfection in murine primary cells.
- This technique significantly enhances CRISPR/Cas9-mediated gene editing in primary cells.
- The protocol holds promise for advancing targeted genetic engineering applications.
