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The complex formed by the positively charged DEAE-dextran and the negatively charged phosphate backbone of the nucleic acid is transiently transfected by endocytosis. The result is reproducible, but the results are reproducible.
High pulse voltage destroys cell membrane potential, DNA is introduced into the pores formed on the membrane, and stable transfection is transiently transfected. All cells are widely applicable, but the cell death rate is high, and the amount of DNA and cells is large. It is necessary to optimize the electroporation experimental conditions according to different cell types.
Integration of foreign genes into chromosomes by infecting host cells. Stable transfection can be used for cells that are difficult to transfect, primary cells, cells in vivo, etc.
Specific host cells, but carrying genes can not be too large, cells need to be divided, need to consider safety factors
The DNA is precipitated with microscopic heavy metal particles, and the coated particles are projected into the cells by a ballistic device. The DNA is gradually released intracellularly, and the transient transfection can be used for: human epidermal cells, fibroblasts, lymphocyte lines. Primary cell
Micro-injection of DNA directly into target cells, stable transfection, transient transfection, transfection, and limited number of embryonic cells for engineering or transgenic animals.
In addition to the above traditional methods, in recent years, some cationic polymer gene transfection technologies have been introduced internationally, which are widely used by the host, are easy to operate, have low cytotoxicity, and have high transfection efficiency, which are favored by researchers. The polymer (Dendrimers) and polyethyleneimine (PEI) have the best transfection performance, but the structure of the dendrimer is not easy to be further modified, and the synthesis process is complicated. Polyethyleneimine is a kind of High cationic charge density organic macromolecules separated by two carbon atoms, ie each "third atom is a protonated amino nitrogen atom, allowing the polymer network to act as an effective "proton sponge" at any pH. "Proton sponge". This polycation can transfer various reporter genes into various species of cells, and its effect is better than that of lipid polyamide. After further modification, its transfection performance is better than dendrimer polymerization. And its cytotoxicity is low. A large number of experiments have proved that PEI is a very promising gene therapy vector. PEI is often used as a core component in the design of more complex gene vectors.
Principle and application of cell transfection technology
Principle and application of cell transfection technology
Conventional transfection techniques can be divided into two broad categories, one is transient transfection, and the other is stable transfection (perfect transfection). The former exogenous DNA/RNA does not integrate into the host chromosome, so it can exist in one host cell. Multiple copy numbers produce high levels of expression, but usually only last for a few days, and are used for analysis of promoters and other regulatory elements. In general, supercoiled plasmid DNA transfection efficiency is higher, 24-72 after transfection. Within hours (depending on a variety of different constructs) analysis results, often using some reporting systems such as fluorescent protein, β-galactosidase, etc. to help detect. The latter also known as stable transfection, foreign DNA can be integrated into the host In the chromosome, it may also exist as an episome. Although the linear DNA is lower than the supercoiled DNA, the integration rate is high. The probability of integration of the foreign DNA into the chromosome is very small, about 1/104 of the transfected cells can Integration, usually through the use of a number of selectable markers, such as aminopropyl transferase (APH; neomycin resistance gene), hygromycin B phosphotransferase (HPH), thymidine kinase (TK) and other repeated screening, stabilized Transfected homologous cell lines.
The choice of transfection technology has a great influence on the transfection results. Many transfection methods need to optimize the ratio of DNA to transfection reagent, cell number, culture and detection time, etc. Some traditional transfection techniques, such as DEAE dextran method, Calcium phosphate method, electroporation method and liposome method have their own advantages and disadvantages. The main principles and application characteristics are as follows:
Calcium phosphate method
Calcium phosphate DNA complex adsorbs cell membrane and is stably transfected by endocytosis. Transient transfection is not suitable for primary cells. Simple operation but poor reproducibility. Some cells are not suitable.
DEAE-dextran method
Electroporation
Virus-mediated method
Retroviral
Adenovirus
Integration of foreign genes into chromosomes by infecting host cells Transiently transfecting specific host cells for use in difficult-to-transfect cells requires safety considerations
The cationic liposome positively charged liposome forms a complex with the negatively charged phosphate group of the nucleic acid. It is stably transfected by endocytosis and transiently transfected with all cells. It has wide applicability, high transfection efficiency and good repeatability. Serum should be removed during transfection. The transfection effect varies greatly with cell type.
Biolistic particle transfer method
Microinjection
Comparison of various transfection methods:
The linear PEI (Line PEI, LPEI) and its derivatives used as gene transfection vectors were earlier than the branched PEI (Branched PEI, BPEI). Past studies have concluded that LPEI/DNA transfection complexes are not considered in specific conditions. The cytotoxicity of the substance is low, which is conducive to cell localization, so the transfection efficiency should be higher than that of BPEI. However, recent studies have shown that the high degree of branching of BPEI is beneficial to the formation of small transfection complexes, thereby improving transfection efficiency. At the same time, the cytotoxicity is also increased. The ultra-high-branched, flexible PEI derivative contains additional secondary and tertiary amine groups. It is found in the dyeing experiment that the PEI has low toxicity but high transfection efficiency. .
GenEscort is a series of high-branched degradable PEI derivatives synthesized by cross-linking various branched and ultra-high-branched small-molecule PEI with various cross-linking agents containing degradable bonds under physiological conditions. The branched structure of the polymer makes it highly positively charged, so it is easy to efficiently coat various DNA, RNA molecules and plasmids to form small nanoparticles, thereby improving transfection efficiency, when the formed complex enters the cell, The chemical bond degradable under physiological conditions is hydrolyzed intracellularly, and the crosslinked polymer is decomposed into a non-cytotoxic small molecule PEI. The transfection reagent of the structure can achieve high transfection efficiency and low in vitro application. Cytotoxicity, its degradability is also of great significance for in vivo applications.