Experimental principle:
Transfection is a specialized technique for introducing exogenous genes into cells. With the deepening of research on gene and protein function, transfection has become a basic method often involved in laboratory work.
Conventional transfection techniques can be divided into two broad categories, one is transient transfection and the other is stable transfection (permanent transfection). The former exogenous DNA/RNA does not integrate into the host chromosome, so multiple copy numbers can exist in one host cell, resulting in high levels of expression, but usually only last for several days, and are mostly used for analysis of promoters and other regulatory elements. In general, supercoiled plasmid DNA transfection efficiency is high, and within 24 to 72 hours after transfection (depending on various constructions) analysis results, often using some reporting systems such as fluorescent protein, beta-galactosidase Wait to help detect. The latter is also called stable transfection, and the foreign DNA can be integrated into the host chromosome or as an episome. Although linear DNA has a lower transfer rate than supercoiled DNA, but the integration rate is high, the probability of integration of foreign DNA into the chromosome is very small. About 1/104 of the transfected cells can be integrated, usually through some selective markers, such as aminopropyl transfer. The enzyme (APH; neomycin resistance gene), hygromycin B phosphotransferase (HPH), thymidine kinase (TK) and the like were repeatedly screened to obtain a stably transfected homologous cell line.
Transfection can be broadly classified into three types: physical mediation, chemical mediation, and biological mediation. Electroporation, microinjection, and gene guns are examples of physical introduction of genes into cells; many chemically mediated methods, such as classical calcium phosphate coprecipitation, lipofection, and multiple cationic species The technology; biologically mediated methods, with relatively primitive protoplast transfection, and nowadays more common viral-mediated transfection techniques.
The ideal cell transfection method should have the advantages of high transfection efficiency and small cytotoxicity. Virus-mediated transfection technology is currently the most efficient method of transfection, and has the advantage of low cytotoxicity. However, the preparation procedure of the virus transfection method is complicated, and it is often highly selective to cell types, and it is difficult to popularize in a general laboratory. Other physical and chemically mediated transfection methods have their own characteristics.
It should be pointed out that no matter which transfection technique is used, it may be necessary to optimize the transfection conditions in order to obtain optimal transfection results. There are many factors that affect transfection efficiency, from cell type, cell culture conditions and cell growth status, to operational details of transfection methods.
Common transfection methods and comparison
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. 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:
Transfection method | principle | application | Characteristics |
Calcium phosphate method | The calcium phosphate DNA complex is adsorbed on the cell membrane and is endocytosed by the cell. | Stable transfection | Not suitable for primary cells. Easy to operate but poorly reproducible. Some cells are not suitable. |
DEAE-dextran method | The complex formed by the positively charged DEAE-dextran interacting with the negatively charged phosphate backbone of the nucleic acid is endocytosed by the cell. | Transient transfection | Relatively simple, repeatable results But it has certain toxic side effects on cells. In addition to serum when transfected |
Electroporation | High pulse voltage disrupts cell membrane potential and DNA is introduced through pores formed in the membrane | Stable transfection Transient transfection | All cells Wide applicability but high cell fatality The amount of DNA and cells needs to be optimized according to different cell types. |
Virus-mediated method |
| Stable transfection Transient transfection | Can be used for cells that are difficult to transfect, primary cells, cells in vivo, etc. |
Retroviral | Specific host cell | But carrying a gene can't be too large, cells need to be in a splitting phase. Safety factors need to be considered. | |
Adenovirus | Integrate foreign genes into chromosomes by infecting host cells | Transient transfection | Can be used for cells that are difficult to transfect, need to consider safety factors |
Cationic liposome method | The positively charged liposome forms a complex with the negatively charged phosphate group of the nucleic acid and is endocytosed by the cell. | Stable transfection Transient transfection | Wide applicability of all cells High transfection efficiency and good repeatability, but serum is required for transfection Transfection effect varies greatly with cell type |
Biolistic particle transfer method | The DNA is precipitated with microscopic heavy metal particles, and the coated particles are projected into the cells by a ballistic device, and the DNA is gradually released in the cells and expressed. | Transient transfection | Can be used for: human epidermal cells, fibroblasts, lymphocyte lines and primary cells |
Microinjection | Direct injection of DNA into target nuclei by micromanipulation | Stable transfection Transient transfection | Limited number of transfected cells for engineering or transgenic, animal embryonic cells |
In addition to the above traditional methods, in recent years, some cationic polymer gene transfection technologies have been introduced internationally, which are favored by researchers because of their wide range of applications, simple operation, low cytotoxicity and high transfection efficiency. The transfection performance of the polymers (Dendrimers) and Polyethylenimine (PEI) is the best, but the structure of the dendrimer is not easy to be further modified, and the synthesis process is complicated. Polyethyleneimine is an organic macromolecule with a high cationic charge density, separated by two carbon atoms, that is, every "third atom is a protonated amino nitrogen atom, making the polymer network at any pH. It can act as an effective "proton sponge". This polycation can transfer various reporter genes into various species of cells, which is better than lipid polyamides. After further modification, Its transfection performance is better than that of dendrimers, 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.
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 conducive to the formation of small transfection complexes, thereby improving transfection efficiency, but at the same time increasing cytotoxicity. The ultra-highly branched, more flexible PEI derivatives contain additional secondary and tertiary amine groups, which were found to be less toxic in dyeing experiments but more efficient in transfection.
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 degradable chemical bond under physiological conditions is hydrolyzed intracellularly, and the cross-linked polymer is decomposed into a non-cytotoxic small molecule PEI. Thus, the transfection reagent of the structure can obtain high transfection efficiency and low cells in vitro. Toxicity, its degradability is also of great significance for in vivo applications.
Experimental Materials:
15ml centrifuge tube, culture dish, dropper, alcohol lamp, culture flask, culture solution, fetal bovine serum, transfection reagent, plasmid, PBS, 75% alcohol, 0.25% trypsin, ultra clean bench, carbon dioxide incubator, inverted Microscope, microscope, centrifuge, constant temperature water bath, refrigerator (4 ° C, -20 ° C, -70 ° C), waste liquid tank, etc.
Experimental procedure (taking liposome transfection as an example):
The liposome (LR) reagent is a mixture of cationic liposomes DOTMA and DOPE (1:1). It is suitable for transfecting DNA into suspension or adherent culture cells and is one of the most transfection methods under current conditions. The transfection rate is higher than that of the calcium phosphate method, which is 5-100 times higher than that, and can transfect DNA and RNA into various cells.
When transfecting with LR, it is first necessary to optimize the transfection conditions, and the amount and time of action of the batch of LR for transfecting a particular cell should be found, and it is necessary for each batch of LR. First fix the amount of DNA and the time that the DNA/LR mixture interacts with the cells. DNA can be started from 1-5ug and incubation time 6h. According to these two parameters, the corresponding LR requirement curve is drawn, then LR and The optimal dose of both DNA determines the time of transfection. Because LR has certain toxicity to cells, the transfection time should not exceed 24h.
First, the operation steps of method one
1. A 6-well culture plate was taken, and 2 ml of a medium containing (1-2) x 10 5 cells was added to each well, and 40%-60% confluence at 37 ° C and 18% CO 2 medium.
2. Preparation of transfection solution: The following two solutions (the amount used for transfecting one empty cell) were prepared in a polystyrene tube.
Solution A: Dilute the DNA in serum-free medium to a concentration of 1-10 ug, with a final dose of 100 ul.
Solution B: Dilute LR with serum-free medium to a final concentration of 2-50 ug, with a final dose of 100 ul.
Gently mix the A and B solutions and set them at room temperature for 10-15 minutes. Later, microturbidity will appear, but it does not hinder transfection.
3. Transfection preparation: rinse 2 times with 2 ml serum-free medium, and then add 1 ml serum-free medium.
4. Transfection: The A/B complex was slowly added to the medium, shaken, placed in a 37 ° C incubator for 6-24 h, aspirate the serum-free transfection solution, and exchanged for normal medium to continue the culture.
5, the rest of the processing: such as observation, screening, testing, etc. are the same as other transfection methods.
6, note: do not add serum when transfected, serum has a great impact on transfection efficiency.
Second, the rapid lipofection method of operation (method 2):
1. Cells were seeded at 5 x 105 cells/well in 6-well plates for 24 h to achieve a 50%-60% plate bottom area.
2. Prepare a DNA-liposome complex in a test tube.
a. Dilute PSV1-neo plasmid DNA or donor DNA in 1 ml serum-free DMEM.
b. Rotate for 1 s, then add the liposome suspension and rotate.
c. Leave at room temperature for 5-10 min to allow DNA to bind to the liposomes.
3. Discard the old solution in the cells, wash the cells once with 1 ml of serum-free DMEM, discard them, add 1 ml of DNA-liposome complex directly to each well, and incubate at 37 ° C for 3-5 days.
4. Add DMEM containing 20% ​​fetal bovine serum to each well and continue to culture for 14-24 hours.
5. Aspirate the DMEM-DNA-liposome mixture into fresh DMEM containing 10% fetal bovine serum, 2 ml/well, and culture for another 24-48 h.
6. Collect cells by cell scraping or digestion for analysis and identification.
Third, stable lipofection method:
1. Inoculate the cells as described above, and the cells can be used for transfection with a cell length of 50%.
2. Preparation of transfected cells by DNA-liposome complexes.
3. Add 1 ml of DMEM containing 20% ​​fetal bovine serum to each well and incubate at 37 ° C for 48 h.
4. DMEM was aspirated, the cells were diluted with G418 selective medium, and the cells were allowed to grow for a certain period of time, and the transfected clones were screened by the cell clone screening method.
Invitrogen's Lipofectamine 2000 was transfected into a single layer of adherent cells in a 24-well plate. (For other methods, please refer to the manufacturer's protocol)
1. One day before transfection, 0.5-2×105 cells were seeded in 24-well culture plates, and 500 ul of complete medium containing no antibiotics was added to ensure that the cells confluence reached 90-95% during transfection.
2, prepare the composite
a. Dilute 0.8 ug of DNA into 50 ul of serum-free antibiotic-free medium and mix gently.
b. Dilute 2ul Lipofectamine2000 in 50ul serum-free antibiotic-free medium, gently rub and incubate for 5 minutes at room temperature. Note: Must be done within 25 minutes.
c. Mix them after 5 minutes, gently knead and incubate for 20 minutes at room temperature.
3. Aspirate the medium from the plate and wash the cells twice with PBS or serum-free medium (preferably).
4. Add the complex (total volume 100 ul) to the culture well, and shake the culture plate back and forth to make it evenly distributed.
5. After incubating the cells in the incubator for 4-6 hours, the serum-containing medium can be replaced to remove the complex (or not).
6, 24 ~ 48h can be observed after the transfer of gene expression.
7. Stable transfection: After changing the serum-containing medium for 24 hours, the cells were passaged at 1:10 (or higher ratio), and after 1 day, the screening medium was replaced and screened.
8, optimization: to ensure that the cell confluence rate of 90 ~ 95% (higher); DNA / Lipofectamine2000 ratio of 1: 0.5 ~ 1:5, the general cell 1: 2 ~ 3.
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