Cell transfection refers to the technique of introducing foreign molecules such as DNA and RNA into eukaryotic cells. With the continuous development of molecular biology and cell biology research, transfection has become a routine tool for studying and controlling the gene function of eukaryotic cells. It has become more and more widely used in biological experiments to study gene function, regulate gene expression, mutation analysis and protein production.
Technical content: Introduction of foreign molecules such as DNA, RNA, etc. into eukaryotic cells
Research content: research and control of eukaryotic cell gene function
Applications: Biological tests of gene function, regulation of gene expression, mutation analysis and protein production
Classification of cell transfection
1. Transient transfection: Exogenous DNA/RNA is not integrated into the host chromosome, so multiple copy numbers can exist in a host cell, resulting in high levels of expression, but usually only lasts for a few days, mostly used for promoters and other regulation Analysis of the original. Generally speaking, supercoiled plasmid DNA has higher transfection efficiency. The results are analyzed within 24-72 hours after transfection, and some reporting systems such as fluorescent protein and β-galactosidase are often used to help detection.
Purpose: Quick analysis
Screening method: antibiotic resistance
Expression duration: Dilute to 48-72 hours with cell division
Target DNA and host chromosome: unintegrated
2. Stable transfection: Exogenous DNA can be integrated into the host chromosome, or it may exist as an episome. Although the amount of linear DNA is lower than that of supercoiled DNA, the overall rate is high. The probability of integration of foreign DNA into the chromosome is very small, about 1,104 transfected cells can integrate, usually need to pass some selective markers, such as leucine transferase, hygromycin B phosphotransferase, thymidine kinase, etc. To obtain a homologous cell line that is stably transfected.
Objective: To obtain single cell clones stably expressing foreign genes
Screening method: antibiotic resistance
Duration of expression: replicates, transcribes, translates, and is stably inherited as the host cell’s own genome
Target DNA and host chromosome: unintegrated
Introduction of common cell transfection methods
1. DEAE-dextran method
It is one of the earliest reagents used to transfect mammalian cells. DEAE-dextran is a cationic polymer, which is bound to negatively charged nucleic acids and is taken up close to the cell membrane. DEAE-dextran transfection has been successfully used for transient start, but it is not reliable for stable transfection.
2. Calcium phosphate co-precipitation transfection method
Because reagents are easily available and cheap, they are widely used in the research of transient transfection and stable staining. The method is to first mix DNA and calcium chloride, and then add people to PBS to slowly form a DNA calcium phosphate precipitate, then add the suspension containing the precipitate to the cultured cells, and take people by endocytosis of the membrane DNA.
3. Liposome-mediated method (currently the most widely used)
Principle: After the cationic liposome reagent is mixed with DNA, a stable lipid bilayer complex is formed. The DNA is wrapped in the liposome. This lipid bilayer complex can be directly added to the cultured cells. The liposomes adhere to the cell surface and fuse with the cell membrane, and the DNA is released into the cytoplasm.
[Main application]: Instant transfection and stable transfection.
[Features]: The method is simple, it can carry large fragments of DNA, and it can be used for various types of naked DNA or RNA. It can transfect various types of cells without immunogenicity. Although it has high efficiency in in vitro gene transfection, in vivo, it can be cleared by serum and accumulate in lung tissue, inducing a strong anti-inflammatory response, resulting in high levels of toxicity, and its application is largely limited.
4. Physical methods (electroporation, microinjection and gene gun)
Introduction of experimental methods of liposome-mediated method
Cell transfection Lipofectamine 2000 transfection reagent: Taking HEK193 cells as an example:
The day before transfection, spread the cells in a Petri dish,
Add medium without serum (15ml);
After 24 hours, replace with 12ml of medium without serum and double antibody;
Dilute the required plasmid with 1.5ml of serum-free and double-antibody-free medium; take 40ul of Lipofectamine 2000 and add serum-free and double-antibody-free medium to dilute, and leave it at room temperature for 5min;
After 5 minutes, mix the plasmid and Lipofectamine 2000 together, put it in a petri dish after 20 minutes at room temperature, and change to complete medium for 48 hours after 4 hours;
Note: once every 24 hours, if the medium changes color, change the medium;
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