Comparison of Cell Transfection Methods

For mammalian cell transfection and protein expression, choosing the right transfection method and transfection reagent plays a decisive role in the success rate of transfection. This article mainly lists some methods, principles and applicability of transient transfection and stable transfection, and lists the steps and precautions of liposome cell transfection.

Cell transfection refers to a technique for introducing exogenous genes into cells. According to the mammalian cell protein expression process, cell transfection is required after cell culture is completed. Different transfection methods are selected according to different experimental purposes. At present, the commonly used cell transfection methods are mainly divided into three categories: physical mediation (electroporation, gene gun, microinjection), chemical mediation (liposome transfection, calcium phosphate coprecipitation, cationic polymer mediation), and biological mediation (virus-mediated transfection, protoplast transfection).

The ideal cell transfection method should have the advantages of high transfection efficiency and low cytotoxicity. At present, the commonly used transfection methods in the laboratory are liposome transfection, cationic polymer transfection and virus transfection. Different transfection methods have their own advantages and disadvantages. Choose a relatively suitable transfection method according to the habits of cells and different experimental purposes. The principles, advantages, disadvantages and applicability of some commonly used transfection methods are listed below.

Comparison of Cell Transfection Methods

Method	Principle	Features
Cationic liposome transfection	The positively charged liposomes bind to DNA through electrostatic interaction to form a DNA-liposome complex, which then enters the cell through endocytosis.	a) Easy to operate b) Applicable to various naked DNA and RNA fragments c) Suitable for transfection of various cells d) Certain requirements for DNA concentration e) Certain toxicity to cells
Cationic polymers	Positively charged cationic polymers form positively charged complexes with the phosphate groups of nucleic acids. The complexes come into contact with negatively charged cell membranes and enter cells through endocytosis.	Similar to liposome transfection, but with very low toxicity, simple operation, and wide applicability
Calcium phosphate method	Calcium phosphate can promote the binding of exogenous DNA to cells. Calcium phosphate-DNA complexes can attach to the cell surface and enter the cell through endocytosis.	a) Simple operation b) High DNA concentration requirement c) Limited applicability (not suitable for primary cells)
Electroporation	The high pulse voltage destroys the cell membrane, forming pores on the cell membrane surface, and DNA enters the cell through the pores.	a) Wide applicability, suitable for plasmids and large genomic fragments b) Experimental conditions should be optimized for different cells c) High cell lethality
Microinjection	Directly inject DNA into cells using micromanipulation and microinjection techniques	a) High integration rate, suitable for engineering transformation and establishment of transgenic animals b) Complex operation, and requires expensive and sophisticated equipment c) Unable to control the integration site and copy number of exogenous genes, resulting in fragment deletion and mutation
Retroviral transfection	It enters the cell through the interaction between the viral membrane protein and the receptor on the cell surface, uses the host cell enzymes to transcribe and replicate to synthesize DNA, and randomly integrates into the cell genome.	a) High transfection efficiency, suitable for transfection of difficult-to-transfect cells b) Virus-mediated transfection, stable integration of exogenous genes c) Retroviruses only infect dividing cells d) Accommodates exogenous gene length < 8kb

Liposome Transfection Steps

(1) Plate cells, add a certain amount of cell culture medium, and culture in a 37℃ carbon dioxide incubator. When the cell density grows to 80-90%, prepare for transfection.

(2) Add serum-free diluted transfection reagent to the EP tube and place at room temperature for 5 minutes

(3) Add DNA diluted in serum-free culture medium to the EP tube and place at room temperature for 5 minutes.

(4) Mix the two substances obtained in steps 2 and 3 to obtain liquid C, mix gently, and place at room temperature for 15-30 minutes.

(5) Wash the cells twice with serum-free culture medium and add an appropriate amount of serum-free culture medium to the cells.

(6) Slowly add the mixed liquid C to the cells, shake well, and culture in a 37℃ carbon dioxide incubator for 6-24 hours.

(7) After 6-24 hours of cell culture, remove the serum-free transfection solution and add normal culture medium to continue culturing.

Notes

During transfection, culture medium containing serum cannot be used, as serum has a great impact on transfection efficiency.
When plating before transfection, use culture medium without double antibodies, as the increase in resistance will affect transfection efficiency.
Perform transfection within 24 hours of plating.
Be gentle during the entire operation and avoid violent shaking.

* For research use only. Not intended for any clinical use.

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Comparison of Cell Transfection Methods