Electroporation---an Efficient Biotransformation Method


  • Abstract: Before introducing electroporation, an important concept-gene library is introduced. A gene library is a recombinant population obtained by fragmenting and completely cloning a certain genome, and constructing a highly abundant gene library greatly increases the effectiveness and targeting of subsequent screening. This technology is widely used in many fields because it is easy to perform in vitro screening and obtain a target fragment and facilitate further research. In library construction, electroporation technology is of paramount importance due to its extremely high conversion efficiency. With the continuous maturity of electroporation method, it is used in the transmission of nucleic acid vaccines, transfection of foreign genes, killing of tumor cells by non-thermal effects, introduction of therapeutic proteins and growth factor genes, tissue regeneration, and gene knockout. It shows unique advantages and has a wide range of applications. The success of library screening depends to some extent on the size and diversity of the storage capacity, and the efficiency and stability of electroporation is an important limiting factor. In addition, electroporation has an important use, which is for cell transfection. Cell transfection methods mainly include viral vector transfection and non-viral vector transfection. The viral vector method has not been widely used due to problems such as safety and immune rejection; the non-viral vector method mainly includes liposome and electroporation. A liposome is a vesicular vector composed of a lipid bilayer, which can be wrapped with a foreign gene and then endocytosed with a cell membrane to introduce a foreign gene. The electroporation rule is a transient change in the structure of the cell membrane caused by high-intensity electrical pulses, resulting in an increase in transient reversible membrane permeability and the appearance of hydrophilic pores on the membrane. Therefore, non-permeable exogenous macromolecular substances such as genes, drugs, and the like are promoted into cells. This paper mainly introduces the principle of electroporation, the factors affecting the transformation efficiency and its application, especially electroporation for gene transfer.

    Keywords: electroporation, conversion efficiency, application

    Principles and advantages of electroporation

    Electroporation is the introduction of a foreign gene into an animal's target tissue or organ by an electric field. Because this method can effectively introduce foreign genes, it can be applied to a variety of tissues and organs, and is highly efficient. The principle of the living electroporation method is very simple. At the moment of the DC electric field, the surface of the cell membrane produces hydrophobic or hydrophilic microchannels of 105 to 115 μm. This channel can be maintained for several milliseconds to several seconds and then recovered by itself. During this time, macromolecules such as DNA can enter the cell through this tiny channel. The main advantages are as follows:

    The selection of target organs is wide. In theory, any tissue and organ can be used as a target organ for living electroporation.

    There is no limit to the size of the imported foreign gene fragment. From the expression vector of a few KB or more than a few kilobytes, to the YAC and BAC genomes of 100 to 200 KB, there have been reports of successful introduction and expression.

    The operation is simple and fast. Electroporation takes only a few seconds and the DNA fragments do not require special purification procedures.

    Factors affecting conversion efficiency

    As an efficient transformation method, electroporation is widely used in many fields such as transfection of foreign genes. However, in practice, the conversion efficiency of electroporation is affected by many factors such as operator and electro transformation conditions, such as cell culture temperature, growth state, exogenous gene concentration, washing buffer composition, instrument, field strength, and the like. Other factors, such as the size of the plasmid, the structure of the plasmid, the purity of the water and wash buffer, and the centrifugation conditions, can affect the efficiency of electro transformation.

    Application of Electroporation in Quantum Dot Labeled Cells

    Quantum dots are an emerging fluorescent marker. There are many reports on the method of labeling cells with quantum dots, in which the endocytosis is mostly used, but these methods need to connect biological reagents with membrane-permeating function on quantum dots, which may lead to quantum dot aggregation and fluorescence. The strength is reduced or even disappeared, so that it cannot be uniformly dispersed in the cytoplasm. Another common method is to inject quantum dots into cells by microinjection technique. The advantage of this method is that the transfer efficiency is high. The disadvantage is that the number of cells that can be transferred each time is very limited, and the operation is cumbersome, time-consuming and laborious.

    Cell electroporation is a technique that uses a pulsed electric field to change the permeability of a cell membrane to achieve DNA fusion and cell fusion. Under the optimal conditions, electroporation can minimize cell damage and allow cells to penetrate effectively. Therefore, endonucleases and antibodies can be introduced into human cells, and have remarkable characteristics such as high transfer efficiency and no damage to cells. It has been widely used in transgenic and tumor treatment. Quantum dots have the same volume as macromolecular proteins, and can smoothly transfer through the cell membrane to achieve efficient transfer, thus providing an efficient and simple method for quantum dots to enter human cells.

    Application of electroporation in gene therapy

    In the treatment of skin tumors and brain tumors, electroporation is effective in introducing a drug gene into a treatment site. After electroporation introduced human monocyte chemo attractant protein gene into rat brain tumor, the protein was expressed locally in the tumor, and aggregation of macrophages and leukocytes was observed at the site of gene introduction.

    Some scientists have studied the role of electroporation in the treatment of tumors from another angle. The steep pulse of high electric field intensity is used to induce irreversible electrical breakdown of tumor cells and eventually die. This method is very suitable for the treatment of local tissue tumors.

    Electroporation can also be involved in the treatment of cancer. After the gene is introduced into the body by in vivo electroporation, the introduced tissue can become a temporary secretory organ that secretes antibodies and other substances to resist the erosion or compensation of cancer cells due to the erosion of cancer cells. The body's multiple physiological functions are lost.

    In addition to the local treatment of tumors by electroporation, it can also treat diseases of the body's endocrine system and immune system. The gene encoding the secreted protein can be introduced into the body by living electroporation. The recombinant therapeutic protein can be distributed to various tissues and cells of the body along the blood circulation for treatment. For example, after the EPO gene is introduced into the living electroporated skeletal muscle, the increase in the expression product of the EPO gene can be detected by the hematocrit value.

    References

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