Electroporation, also called electropermeabilization (EP), is a physical method of introducing genetic material, proteins, drugs, or other desired molecules into cells for various purposes with the application of electric pulses to create temporary pores in the cell membrane, delivering the molecules to the cells.
Neumann et al. first introduced the concept of electroporation in 1982. Over the past few decades, electroporation has made significant advancements. Initially, it was limited to simple in vitro experiments. It can now be used in gene transfer, gene therapy, drug delivery, and various in vivo therapeutic approaches.
Electroporation, also known as electropermeabilization, is a technique used in molecular biology and biotechnology to introduce substances such as DNA, RNA, or other molecules into cells by creating temporary pores in the cell membrane through the application of an electric field. This method is widely used in various applications, including genetic engineering, drug delivery, and cell-based therapies.
Principle of Electroporation
The working principle of electroporation involves applying electric pulses to cells, which creates temporary pores in the cell membrane. These pores allow the entry of molecules, such as DNA, proteins, drugs, and other substances, into the cells.
The application of electric pulses to cells causes disruption of the cell membrane. The pulses change the electrical potential across the membrane, leading to the formation of temporary pores in the phospholipid bilayer that forms the cell membrane. These pores allow molecules to pass through the membrane.
Once the electric pulse is turned off, the pores start to close and the cell returns to its normal state. Some of the molecules taken up by the cell are trapped inside as the membrane reseals. If the electric field allows the membrane defects to reseal, it is reversible electroporation, while if the defects persist, it is irreversible electroporation, leading to cell death.
Once the molecules enter the cells through the temporary pores, they can interact with the cellular mechanism, influencing cellular processes and functions.
Steps of Electroporation
The specific steps and materials involved in electroporation may vary based on the cell type, and electroporation equipment. The steps below provide a general overview of the electroporation process.
- Cells are first harvested and suspended in an electrolyte buffer solution. This buffer helps conduct the electric current and maintains the integrity of the cells during the electroporation process.
- The cell suspension is placed in an electroporation chamber or cuvette, which is typically made of a conductive material like metal or plastic. The cuvette has two parallel electrodes that are positioned on opposite sides of the cell suspension.
Application of Electric Field:
- An electric field is applied across the cell suspension by connecting the electrodes to an electrical pulse generator. The electric field disrupts the cell membrane, creating temporary pores or holes in the lipid bilayer.
- When the electric field is applied, it causes a rearrangement of the lipid molecules in the cell membrane. This rearrangement leads to the formation of transient nanopores in the membrane.
- During the brief period when these pores are open, exogenous molecules (e.g., DNA, RNA, proteins, or drugs) present in the surrounding medium can diffuse into the cell through the pores. This allows for the delivery of foreign genetic material or other substances into the cell's cytoplasm.
- Once the electric pulse is terminated, the pores begin to reseal. The resealing process occurs relatively quickly, and the cell membrane returns to its normal state.
- After electroporation, the treated cells are typically transferred to a growth medium, allowing them to recover and resume normal cellular functions. Some cells may not survive the process, but those that do can express the introduced genetic material or respond to the delivered substances.
The parameters of the electric field, such as voltage, pulse duration, and pulse number, are crucial factors in determining the efficiency and safety of electroporation. Optimizing these parameters is essential for achieving successful delivery of molecules into the target cells while minimizing cell damage.
Electroporation is a versatile and powerful technique widely used in biological research and various biotechnological applications, including gene therapy, CRISPR-Cas9 genome editing, and the development of genetically modified organisms.
Applications of Electroporation
Some major applications of electroporation are:
- Electroporation is widely used in the transformation of bacterial cells to make them competent and capable of taking up foreign DNA for various purposes like recombinant protein production, cloning, and other biotechnological applications.
- Electroporation is also used in transfection. Similar to bacterial transformation, electroporation allows the manipulation of eukaryotic cells by engineering their genetic material.
- Electroporation is widely used in genetic engineering to introduce foreign DNA into cells. For example, it can be used for the transformation of plant cells, allowing the introduction of foreign DNA into plants for crop improvement.
- Electroporation can be used for delivering drugs and vaccines. It allows the delivery of large substances that cannot easily cross the skin. DNA vaccines against various diseases, such as plague, and SARS-CoV-2, have also been developed using electroporation as a delivery method.
- Electroporation is also used in gene therapy to deliver modified or additional genetic material.
- Irreversible electroporation is used in the treatment of cancer. This method uses high-voltage electric pulses to cause permanent damage and cell death in tumors.
Yeast Transformation Electroporation
Advantages of Electroporation
- Electroporation is a versatile method that can be used in various cell types, including bacteria, yeast, plant cells, and mammalian cells.
- Electroporation can be used to deliver genetic material into cells that are difficult to transfect.
- Electroporation provides consistent and reproducible results.
- Electroporation does not require the use of vectors for gene transfer. It is a safe and non-toxic method.
Disadvantages of Electroporation
- High-voltage electric pulses used in electroporation can cause cell damage or cell death if the parameters are not optimized.
- Electroporation requires specialized equipment, including an electroporator, cuvettes, and appropriate voltage settings.
- Electroporation may lead to variable transfection efficiency. Some cells may take up the introduced molecules more efficiently than others.
- Very large DNA fragments or complex structures may not be efficiently delivered into the cells.
- Cells are delicate following electroporation and must be treated carefully during recovery to avoid cell death.