Gene electrotransfer

Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or DNA to be introduced into the cell (also called electrotransfer). In microbiology, the process of electroporation is often used to transform bacteria, yeast, or plant protoplasts by introducing new coding DNA. If bacteria and plasmids are mixed together, the plasmids can be transferred into the bacteria after electroporation, though depending on what is being transferred cell-penetrating peptides or CellSqueeze could also be used. Electroporation works by passing thousands of volts across a distance of one to two millimeters of suspended cells in an electroporation cuvette (1.0 – 1.5 kV, 250 – 750 V/cm). Afterwards, the cells have to be handled carefully until they have had a chance to divide, producing new cells that contain reproduced plasmids. This process is approximately ten times more effective than chemical transformation. Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, or DNA to be introduced into the cell (also called electrotransfer). In microbiology, the process of electroporation is often used to transform bacteria, yeast, or plant protoplasts by introducing new coding DNA. If bacteria and plasmids are mixed together, the plasmids can be transferred into the bacteria after electroporation, though depending on what is being transferred cell-penetrating peptides or CellSqueeze could also be used. Electroporation works by passing thousands of volts across a distance of one to two millimeters of suspended cells in an electroporation cuvette (1.0 – 1.5 kV, 250 – 750 V/cm). Afterwards, the cells have to be handled carefully until they have had a chance to divide, producing new cells that contain reproduced plasmids. This process is approximately ten times more effective than chemical transformation. Electroporation is also highly efficient for the introduction of foreign genes into tissue culture cells, especially mammalian cells. For example, it is used in the process of producing knockout mice, as well as in tumor treatment, gene therapy, and cell-based therapy. The process of introducing foreign DNA into eukaryotic cells is known as transfection. Electroporation is highly effective for transfecting cells in suspension using electroporation cuvettes. Electroporation has proven efficient for use on tissues in vivo, for in utero applications as well as in ovo transfection. Adherent cells can also be transfected using electroporation, providing researchers with an alternative to trypsinizing their cells prior to transfection. One downside to electroporation, however, is that after the process the gene expression of over 7,000 genes can be affected. This can cause problems in studies where gene expression has to be controlled to ensure accurate and precise results. Although bulk electroporation has many benefits over physical delivery methods such as microinjections and gene guns, it still has limitations including low cell viability. Miniaturization of electroporation has been studied leading to microelectroporation and nanotransfection of tissue utilizing electroporation based techniques via nanochannels to minimally invasively deliver cargo to the cells. Cell fusion is of interest not only as an essential process in cell biology, but also as a useful method in biotechnology and medicine. Artificially induced fusion can be used to investigate and treat different diseases, like diabetes, regenerate axons of the central nerve system, and produce cells with desired properties, such as in cell vaccines for cancer immunotherapy. However, the first and most known application of cell fusion is production of monoclonal antibodies in hybridoma technology, where hybrid cell lines (hybridomas) are formed by fusing specific antibody-producing B lymphocytes with a myeloma (B lymphocyte cancer) cell line. Electroporation is performed with electroporators, purpose-built appliances which create an electrostatic field in a cell solution. The cell suspension is pipetted into a glass or plastic cuvette which has two aluminium electrodes on its sides. For bacterial electroporation, typically a suspension of around 50 microliters is used. Prior to electroporation, this suspension of bacteria is mixed with the plasmid to be transformed. The mixture is pipetted into the cuvette, the voltage and capacitance are set, and the cuvette is inserted into the electroporator. The process requires direct contact between the electrodes and the suspension. Immediately after electroporation, one milliliter of liquid medium is added to the bacteria (in the cuvette or in an Eppendorf tube), and the tube is incubated at the bacteria's optimal temperature for an hour or more to allow recovery of the cells and expression of the plasmid, followed by bacterial culture on agar plates. The success of the electroporation depends greatly on the purity of the plasmid solution, especially on its salt content. Solutions with high salt concentrations might cause an electrical discharge (known as arcing), which often reduces the viability of the bacteria. For a further detailed investigation of the process, more attention should be paid to the output impedance of the porator device and the input impedance of the cells suspension (e.g. salt content). Since the cell membrane is not able to pass current (except in ion channels), it acts as an electrical capacitor. Subjecting membranes to a high-voltage electric field results in their temporary breakdown, resulting in pores that are large enough to allow macromolecules (such as DNA) to enter or leave the cell. Additionally, electroporation can be used to increase permeability of cells during in Utero injections and surgeries. Particularly, the electroporation allows for a more efficient transfection of DNA, RNA, shRNA, and all nucleic acids into the cells of mice and rats. The success of in vivo electroporation depends greatly on voltage, repetition, pulses, and duration. Developing central nervous systems are most effective for in vivo electroporation due to the visibility of ventricles for injections of nucleic acids, as well as the increased permeability of dividing cells. Electroporation of injected in utero embryos is performed through the uterus wall, often with forceps-type electrodes to limit damage to the embryo. In vivo gene electrotransfer was first described in 1991 and today there are many preclinical studies of gene electrotransfer. The method is used to deliver large variety of therapeutic genes for potential treatment of several diseases, such as: disorders in immune system, tumors, metabolic disorders, monogenetic diseases, cardiovascular diseases, analgesia….