Common methods for gene transfer into cells-Gene Therapy
After connecting the target gene sequence with the vector, it needs to be introduced into cells for reproduction and amplification. After screening, recombinant DNA molecular clones can be obtained. Different vectors reproduce in different host cells, and the method of introducing into cells is also different.
1、 Conversion
The genetic changes in cells caused by the entry of exogenous DNA are called transformation. As early as 1943, Avery et al. discovered the transformation phenomenon of toxic pneumococcal offspring produced by co culturing DNA of toxic pneumococcal bacteria with non-toxic pneumococcal bacteria. However, the efficiency of DNA entering cells is very low. In molecular biology and genetic engineering work, some methods can be used to treat cells. After treatment, cells are easily receptive to external DNA, called competent cells, and then interact with external sources
DNA contact can improve conversion efficiency. For example, Escherichia coli becomes a competent bacterium after being treated with cold CaCl2. When a recombinant plasmid is added and suddenly transferred from 4 ℃ to 42 ℃ for a short period of time, the plasmid DNA can enter the bacteria; Short duration application of high voltage pulses to bacteria can significantly improve conversion efficiency, which is called electroporation conversion method
2、 Infection
Bacteriophages enter host bacteria, and viruses enter host cells for reproduction, which is called infection. Using artificially modified bacteriophage live virus as a vector, after recombining its DNA with the target sequence, the recombinant DNA can be packaged into a viable bacteriophage or virus in vitro using the shell protein of the bacteriophage or virus, which can enter the host bacteria or cells through infection, allowing the target sequence to replicate and reproduce. The efficiency of infection is high, but the process of packaging DNA into bacteriophages or viruses is more complicated.
3、 Transfection
Recombinant bacteriophage DNA can also enter the host bacteria in the same way as plasmid DNA, where the host bacteria are first treated with CaCl2, electroporation, etc. to become competent bacteria and then receive DNA. The bacteriophage DNA entering the competent bacteria can replicate and reproduce in the same way, which is called transfection. The M13 bacteriophage DNA is commonly transfected into Escherichia coli. Recombinant DNA entering host cells is also commonly transfected. The most classic is the calcium phosphate method established in 1973, which utilizes the basic phenomenon that when DNA appears in the form of calcium phosphate DNA co precipitates, the efficiency of DNA uptake by cultured cells is significantly improved.
Treating cultured mammalian cells with electroporation can also improve their ability to uptake DNA, but the intensity of the applied electric field, the length of the electric pulse, and other conditions used are very different from those used to treat bacteria. Liposomes formed by encapsulating DNA with artificial lipid membranes can be introduced into cells by fusing with the cell membrane. The method is simple and effective, but the disadvantage is the cytotoxicity and serum incompatibility. In recent years, a more effective transfection reagent - cationic polymer - has been discovered, which overcomes the disadvantages of liposome transfection reagents such as high cytotoxicity and easy clearance by serum in vivo. It has high transfection efficiency and simple operation, and is increasingly being valued.
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