What is Electroporation?
is a technique used in microbiology that allows plasmid DNA or other chemicals to be transferred into the cell. An electroporation machine applies an electrical field to enhance permeability and allow media to enter the cell membrane.
One way to use electroporation in molecular biology is gene transfer. An electroporation machine is proven to be a highly efficient way to target cells for gene editing and gene delivery. However, there are plenty of other reasons to use electroporation.
So how does electroporation work? Read on to find out.
The first recorded incident of electroporation transfection (the process of artificially introducing DNA or RNA into cells) using high-voltage electrical currents was performed in 1982 by Wong and Neumann. After successfully transfecting cells using fibroblasts – the technique was refined for use with all cell types.
Cell Types Frequently Used in Electroporation
- Plasmid DNA
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How Does Electroporation Work?
In vitro electroporation is achieved by transmitting high voltage through suspended cells in disposable electroporation cuvettes. Transient and reversible breakdown of the cell membrane can be achieved by applying an external electrical field.
In this transient state, cells can be loaded with different cell solutions or molecules. This can be achieved with simple diffusion or electrophoretic processes, which will allow media to pass through the permeable cell membrane.
Although electroporation was initially developed for gene transfer, the phenomena is now used to transfer large molecules. These molecules include antibodies, tracers, and drugs. Electroporation is also used in vivo, in vitro, and, more specifically, on cancer cells.
An understanding of cell biology is vital to ensure cell viability and prevent cell death. Anywhere from 50-70% of cells subjected to high voltage fields
die. Cell death is most likely caused by irreparable damage to the structure of the cell membrane, which causes the cell membranes to rupture.
Basic Electroporation Protocol
This basic in vitro electroporation protocol can be used for electroporation transfection of multiple cell types, including mammalian cells. Cell suspension is achieved using an electroporation buffer, where cells are placed into an electroporation cuvette.
The cells are then exposed to high-voltage electric shock with a specific level of strength and length of time. The cells are given a period of recovery before being placed in a non-selecting cell growth medium.
Materials for Mammalian Cell Transfection
- Complete medium with and without selective agents
- Electroporation buffer – 32° temperature
- Linear or supercoiled, purified DNA preparation
- Electroporation cuvettes and a power source
- Additional reagents and equipment for stable transformation for selective medium and harvesting the transfected cells
Alternate Protocol: Plant Protoplasts
Using this protocol, plant cells are stripped of their cell walls, allowing DNA to be introduced. This alternate protocol results in protoplasts.
Similar to mammalian cells, electroporation of plant protoplasts can be performed using various electrical parameters. Some cell lines electroporate more efficiently at room temperature, like mammalian cells.
After Wong and Neumann performed electroporation transfection of fibroblasts, electroporation was used to transfer DNA into embryonic stem cells. Targeting vectors that allow DNA to recombine with homologous regions in the genome were later designed.
This could not have been accomplished without electroporation. Electroporation introduces DNA into cells in a naked form that allows for participation in homologous recombination.
In vivo electroporation was originally used for delivering chemotherapy to cancer cells and tumors. The first successful in vivo transfection with electroporation was reported in the 1990s.
Today, electroporation is the most efficient way to transfect plasmid DNA.
Electroporation is used in the process of creating knockout mice, gene and cell-based therapies, and treatment of solid malignant tumors. Knockout mice are genetically modified mice that have had an existing gene replaced with an artificial piece of DNA.
Knockout mice facilitate the studying of genes which have been sequenced, but whose function is unknown. Genetic modification can be studied in a proactive fashion to help predict human responses to certain genes. The act of knocking out gene activity can unveil indicators about how the gene normally functions.
Since mice and humans share many of the same genes, studying knockout mice allows us to better understand how a shared gene may cause diseases in humans. Electroporation machines are crucial in the creation of knockout mice.
Researchers use one of two methods to deliver DNA into the chromosomes within the nuclei of embryonic stem cells. These methods are homologous recombination and gene trapping.
Performed by using electroporation to introduce a piece of artificial DNA that shares an identical gene sequence to the gene. The nucleus of the cell recognizes the identical DNA sequence and knocks out the function of the existing gene.
Involves manipulation of an embryonic stem cell using a random process. In gene trapping, artificial DNA containing a tracker gene known as a “reporter gene” is delivered into a random gene.
Ways to Optimize Electroporation
- Gap Size: The field strength is determined using the voltage divided by gap size. By using the appropriate gap size, the voltage can be reduced while maintaining the same field strength.
- Cell Diameter: Typically, larger cell diameters require lower voltages and smaller cells require higher voltages to successfully permeate the cell membrane.
- Temperature: Cell temperature affects the efficiency of electroporation. For example, most mammalian cells can be successfully electroporated at room temperature.
- Pulse Length: Any increase in voltage should be accompanied by an incremental decrease in pulse duration to optimize parameters. Increased cell death can occur when higher voltages are used for longer durations.Your Trusted Partner for Electroporation ProjectsIf you are looking to achieve high-caliber electroporation, RBC Medical can help. We have over 25 years of experience developing electroporation machines or other medical devices, and our success has led to 93% of clients requesting repeat business with us.Partner with an organization that brings clarity to medical device projects, from initial ideas to marketable products. For more more information, please contact us today.