transfection definition and cell transfection method
transfection is the process of introducing DNA, RNA or protein into eukaryotic cells , and is used to study and regulate gene expression . Therefore, transfection technology, as an analysis tool, helps characterize genetic functions, protein synthesis, cell growth, and development. Transfection analysis can not only promote the progress of cell research, but also enhance drug development strategies. Similar strategies such as viral transfection or viral transduction are to use lentivirus microparticles to insert exogenous substances into eukaryotic cells. Bacterial transformation is the horizontal gene transfer process by which bacteria ingest exogenous genetic materials. For more content, please visit Merck Life Sciences official website: www.sigmaaldrich.cn
Transfection type
Currently there are a variety of transfection methods, including physical, chemical and biotechnological methods. These techniques generally involve incorporating nucleic acid into cells using transient or stable transfection methods.
transient transfection technology involves introducing DNA into cells, but in this method, DNA does not integrate with cell chromosomes. This technology has high transfection efficiency and can analyze gene transcription after 1-4 days. For large-scale transient gene expression (TGE) of mammalian cell cultures, transfection vectors such as polyethyleneimine (PEI) and calcium phosphate (CaPi) can be used. In addition, a large-scale TGE method using Chinese hamster ovary (CHO) cells in the absence of serum has been developed.
stable transfection technology involves integrating transfected DNA into cell chromosomes or forming appendages. Selectable markers can then be used to identify stably transfected cells, including dihydrofolate reductase (DHFR), hygromycin B phosphotransferase (HPH), and adenosine deaminase (ADA), among others. Commonly used transfection techniques in
part include calcium phosphate precipitation, lipid transfection, electroporation and virus delivery. Additionally, these methods can be used for co-transfection. These techniques involve the simultaneous delivery of two different nucleic acids into the same cell and are often used to achieve stable transfection. Transfection methods have developed many new methods, such as Biolistic delivery systems that use high-speed microparticles to deliver cellular nucleic acids, and in vivo transfection protocols that facilitate systemic delivery of siRNA molecules.
calcium phosphate transfection
calcium phosphate transfection technology involves the precipitation of DNA and calcium phosphate. Precipitation was promoted by mixing HEPES buffered saline solution containing sodium phosphate with calcium chloride solution and DNA. Glycerol shock is often used to enhance DNA uptake in certain cells. Although this technique is cost-effective and can be used for transient or stable transfection of multiple cells, relatively small changes in pH value (±0.1) will affect the conversion efficiency. Furthermore, the reagent must be kept homogenized to achieve reproducible analytical results. However, this transfection method does not work in RPMI or other culture media with high phosphate concentrations.
liposome mediated transfection (lipid transfection) technology involves the use of cationic lipids or non-lipid polymers capable of forming liposomes. For example, the lipid transfection agent may contain DOTMA (N-[1-(2,3,-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and X-tremeGENE™ transfection agents suitable for transfecting a variety of DNA, small RNA and CRISPR/Cas9 components into a variety of cell lines. With corresponding modifications, lipid transfection can also be used in cost-effective high-throughput systems, but this transfection technique is usually cell type-specific.
This technique involves exposing cell membrane to high-intensity electrical pulses, which causes temporary loss of stability in certain areas of the cell. During this instantaneous instability, the cell membrane becomes highly permeable and allows various exogenous molecules to enter, including DNA4. Electroporation is a simple non-chemical technique that can produce high conversion efficiency in various cell types. Although this technique does not change the morphology and function of target cells, this method can lead to cell death if transfected under optimal conditions.
This method involves the delivery of nucleic acid into cells using viral vector . Viral delivery systems such as lentivirus, adenovirus and tumor retroviral vectors can be transfected with nucleic acids even in complex cells. Although the virus delivery method is very effective, it can be quite labor-intensive. In addition, most viruses require control and careful monitoring of biosafety levels. It is also important to consider several limiting factors before viral transfection, such as the lytic nature of the viral vector, cell line packaging, and host cell specificity.
With the development of transfection experimental protocols and the increasing simplification of transfection assay methods, it is necessary to select suitable transfection reagents in order to achieve the optimal transfection efficiency.
When considering suitable transfection reagents, it is important to identify the assayed cell type and culture conditions. Rare cell cultures, neurons and primary cells are often more difficult to transfect, so there is a need for reagents that can promote transfection, especially for such difficult-to-transfect cells.
In addition, reagent levels and cytotoxicity parameters should be considered before selecting a suitable transfection agent. For the desired cell type, the ideal agent should have low cytotoxicity and high transfection efficiency.
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