Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro
September 12th, 2011
Gene transfection by electroporation is improved approximately two times when orientation of electric field is changed during pulse application, while cell viability is not affected. The increase in gene transfection is caused by the increase of the membrane area which is made competent for DNA entry into the cell.
Gene electrode transfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses. In successful gene electrode transfer, several steps are involved formation of a complex between DNA and cell membrane translocation of DNA across the ized membrane transfer of DNA from cytoplasm into the nucleus and gene expression for gene electro transfer. Different electric pulse protocols are used in order to achieve maximum gene transfection.
One of them is changing the electric field orientation during the pulse delivery. In this video, we demonstrate the difference in gene electrode transfer efficiency when all pulses are delivered in the same direction and when pulses are delivered. By changing electric field orientation experiments were performed on Chinese hamster ovary cells, CHOK one For the experiment.
Follow these steps, grow cells in a culture flask in the incubator for 24 hours. On the day of the experiment, prepare cell suspension by trypsin with 0.25%trips in EDTA solution and count the cells centrifuge cells for five minutes at 1000 rotations per minute at four degrees Celsius and resuspend cell pellets in ISO OSM Miller sodium phosphate buffer to a cell density of 5 million cells per milliliter. And move the sample to the appropriate dish cells are exposed to electric field in pipe tip with integrated electrodes connected to a high voltage prototype generator.
These electrodes allow application of a relatively homogeneous electric field in different directions. The tip and the generator were developed at the laboratory of Biocybernetics faculty of electrical engineering. University of Luana add plasmid, E-G-F-P-N one to a cell suspension concentration of 10 micrograms per milliliter.
Mix cells with plasmid DNA thoroughly and incubate the mixture for two to three minutes at room temperature before applying electric pulses. Aspire 100 microliters of cell suspension into the pipee tip with integrated electrodes. In this experiment, a train of eight rectangular pulses is applied using high voltage prototype generator to different electric field protocols are used in the first protocol.
All pulses are delivered in the same direction, whereas in the second protocol, pulses are delivered by changing electric field orientation. The second protocol can only be used with appropriate pulse generator, which allows application of electric pulses in different directions. Immediately after the pulse application, transfer the cells from pipe tip into six well plates and add fetal bovine serum.
25%of sample volume incubate cells for five minutes at 37 degrees CELs. To allow cell membrane resealing add two milliliter of HAMF 12 to each sample in six well and incubate cells for 24 hours at 37 degrees Celsius in a humidified 5%CO2 atmosphere in the incubator. Efficiency of gene electro transfer is determined as the percentage of cells expressing GFP 24 hours after pulse application.
The cells are observed using a fluorescence microscope in the acquisition software. Set the excitation wavelength to 488 nanometers and choose an appropriate band pass filter for green fluorescent image to detect the cells that are expressing GFP. Acquire at least five images, face contrast and green fluorescence at 20 times.
Objective magnification count cells in face contrast image and cells that are expressing GFP in green fluorescence image determine the percentage of gene electro transfer efficiency by dividing the number of cells that are expressing GFP with a number of all cells in each corresponding image. The percentage of cells expressing GFP when all pulses are delivered in the same direction and when pulses are delivered by changing electric field orientation is presented by changing the electric field orientation. The percentage of cells expressing GFP increases significantly.
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Overview
This study investigates gene transfection via electroporation, highlighting the impact of electric field orientation on transfection efficiency. The findings indicate that altering the electric field direction during pulse application can double gene transfection rates without compromising cell viability.
Key Study Components
Area of Science
- Gene therapy
- Cell biology
- Electroporation techniques
Background
- Gene electrode transfer is a method for delivering genes into cells.
- It involves applying short, intense electric pulses to facilitate DNA entry.
- Successful transfection requires DNA to cross the cell membrane and enter the nucleus.
- Different electric pulse protocols can enhance transfection efficiency.
Purpose of Study
- To evaluate the effect of electric field orientation on gene transfection efficiency.
- To determine if changing the orientation affects cell viability.
- To demonstrate the process using Chinese hamster ovary (CHO) cells.
Methods Used
- Application of electric pulses to CHO cells.
- Comparison of transfection efficiency with varying electric field orientations.
- Assessment of cell viability post-transfection.
- Analysis of DNA entry into cells and subsequent gene expression.
Main Results
- Gene transfection efficiency improved by approximately two times with altered electric field orientation.
- No significant impact on cell viability was observed.
- Increased membrane area contributed to enhanced DNA entry.
- Demonstrated the effectiveness of different electric pulse protocols.
Conclusions
- Changing the orientation of the electric field during pulse application significantly enhances gene transfection.
- This method can be optimized without compromising cell health.
- Further research may explore additional protocols for improved gene delivery.
Frequently Asked Questions
Executive Industry Relevance
Optimizing electric field orientation during electroporation enhances plasmid gene transfer efficiency, offering a non-viral strategy to improve transfection yields in early-stage target validation and lead identification workflows. This approach supports mechanistic de-risking by increasing DNA entry through expanded membrane competence without compromising cell viability, thereby improving predictive confidence in preclinical models. The method enables reproducible, scalable gene delivery for assay development and phenotypic screening applications in biopharma R&D.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Increases membrane area competent for DNA entry, improving transfection efficiency and supporting functional target validation in recombinant protein expression studies.
- Operational Value: Enables consistent plasmid delivery across cell populations, reducing variability in early-stage hypothesis testing and pathway clarification.
Screening & Assay Development
- Scientific Value: Provides quantitative, fluorescence-based readouts of gene transfer efficiency compatible with high-content screening platforms.
- Operational Value: Standardizes electroporation parameters using integrated electrode tips and programmable pulse generators, improving assay reproducibility across laboratories.
- Strategic Value: Supports scalable preparation of transfected cell lines for downstream target engagement and biomarker assays.
Translational & Preclinical Research
- Scientific Value: Maintains cell viability post-transfection, enabling longitudinal studies of gene expression and phenotypic effects in disease-relevant systems.
- Operational Value: Facilitates generation of stable or transiently transfected preclinical models using CHO cells, a widely used host for biologics production and pathway analysis.
Pipeline & Workflow Integration
This method fits within the discovery continuum from target validation through lead identification, where efficient gene delivery is required to assess target function and modulate pathways in cellular models.
- Discovery Biology: Supports hypothesis testing by enabling reliable overexpression or knockdown of genes of interest in mammalian cells.
- Screening: Delivers standardized, quantitative transfection outputs that allow comparison of genetic constructs or regulatory elements.
- Analytics: Generates percentage-based efficiency metrics (GFP+ cells/total cells) that enable statistical comparison between pulse protocols.
- Translational Research: Connects to preclinical continuity by producing viable transfected cells suitable for further validation in 3D models or co-culture systems.
- Enterprise Reuse: Represents a reusable electroporation capability applicable across multiple projects requiring plasmid delivery into adherent or suspension cell lines.
Operational & Enterprise Impact
- Scientific Value: Enhances predictive confidence in gene delivery outcomes by increasing transfection efficiency through biophysical optimization of pulse parameters.
- Operational Value: Improves reproducibility and standardization of gene transfer procedures through defined pulse trains and field orientation control.
- Strategic Value: Reduces biological risk in target validation by ensuring sufficient transgene expression for phenotypic assessment, minimizing false negatives.
- Portfolio Impact: Enables risk-adjusted prioritization of genetic targets based on achievable expression levels in cellular assays.
Implementation Considerations
- Requires expertise in mammalian cell culture, plasmid preparation, and fluorescence microscopy for transfection efficiency assessment.
- Dependent on access to high-voltage pulse generators capable of programmable electric field orientation changes and compatible electrode-integrated tips.
- Necessitates standardization of pulse parameters (voltage, duration, number) and cell density across experiments to ensure comparability.
- Involves optimization of DNA-to-cell ratio and incubation times to balance transfection efficiency with cellular stress.
- Limited to adherent or suspension cell lines compatible with electroporation; primary or sensitive cells may require alternative delivery methods.
