Cell transfection refers to a technology that introduces foreign genes into cells. According to the protein expression process of mammalian cells, cell transfection is required after cell culture is completed, and different transfection methods are selected according to different experimental purposes. At present, commonly used cell transfection methods are mainly divided into three categories: physically mediated (electroporation, gene gun, microinjection), chemically mediated (lipofectamine transfection, calcium phosphate precipitation, cationic lipid transfection, cationic polymer transfection), biologically mediated (virus-mediated transfection, protoplast transfection).
An ideal cell transfection method should have the advantages of high transfection efficiency and low cytotoxicity. Currently, commonly used transfection methods in laboratories include liposome transfection, cationic polymer transfection and viral transfection. Different transfection methods have their own advantages and disadvantages. The principles of some commonly used transfection methods are listed below.
Calcium Phosphate Transfection
The calcium phosphate transfection technique involves the precipitation of DNA and calcium phosphate. Precipitation was facilitated by mixing HEPES-buffered saline solution containing sodium phosphate with calcium chloride solution and DNA. Although this technique is cost-effective and can be used for transient or stable transfection of a variety of cells, relatively small changes in pH can affect transformation efficiency. Furthermore, reagents must be maintained homogeneous to achieve reproducible analytical results. However this transfection method will not work in RPMI or other media with high phosphate concentrations.
Liposome-Mediated Transfection
Liposome-mediated transfection technology involves the use of cationic lipids or nonlipid polymers capable of forming liposomes. Liposome-based transfection reagents are chemicals that form positively charged lipid aggregates that smoothly fuse with the host cell's phospholipid bilayer, allowing entry of foreign genetic material with minimal resistance. Liposome-mediated transfection technology is suitable for transfecting a variety of DNA, small RNA, and CRISPR/Cas9 components into a variety of cell lines. Lipofectamine transfection reagents are expensive, but their high transfection efficiency makes them worth it in the long run.
Electroporation Transfection
The technique involves exposing cell membranes to high-intensity electrical pulses, which cause certain areas of the cell to temporarily lose stability. During this transient destabilization, the cell membrane becomes highly permeable and allows entry of a variety of foreign molecules, including DNA. Electroporation is a simple, nonchemical technique that produces high transformation efficiencies in a variety of cell types. Although this technique does not alter target cell morphology and function, this method can lead to cell death if transfection is not performed under optimal conditions.
DEAE-dextran Transfection
DEAE-dextran, a polycationic derivative of the carbohydrate polymer dextran, was one of the first chemical reagents used to transfer nucleic acids into cultured mammalian cells. In DEAE dextran-mediated transfection, cationic DEAE dextran molecules bind tightly to the negatively charged nucleic acid backbone. The net positive charge of the resulting nucleic acid-DEAE-dextran complex enables it to adhere to the cell membrane and enter the cytoplasm via DMSO- or glycerol-induced endocytosis or osmotic shock. DEAE-dextran transfection is simple and inexpensive. However, this method has potential cytotoxicity and low transfection efficiency.
Viral Transfection
The method involves using viral vectors to deliver nucleic acids into cells. Viral delivery systems, such as lentiviral, adenoviral, and retroviral vectors, can transfect nucleic acids even in complex cells. Viral transfection offers advantages in many aspects, including high in vivo transfection efficiency and sustained gene expression due to integration of the viral vector into the host genome, making this system the first choice for gene delivery in clinical trials. However, there are many drawbacks to using viral delivery, including biosafety requirements, cytotoxicity, and differences in infectivity between viral vector formulations.