Factors and precautions affecting cell transfection efficiency
Transfection is a specialized technique for introducing exogenous genes into cells. Divided into physical mediation methods: electroporation, microinjection and gene gun; chemical mediation methods: such as classical calcium phosphate coprecipitation, lipofection, and a variety of cationic material-mediated techniques; Mediation methods: There are relatively primitive protoplast transfections, and various virus-mediated transfection techniques are more common nowadays. 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 process 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 influence transfection efficiency, from cell type, cell culture conditions and cell growth status to operational details of the transfection method. Here are some of the six major factors affecting cell transfection: 1, serum A. DNA-cationic liposome complexes cannot form serum when formed because serum can affect the formation of complexes. B. Generally, cells can be tolerated for several hours without serum. The culture medium for transfection may or may not contain serum, but serum was once thought to reduce the transfection efficiency. Serum was added to the transfection medium. The conditions need to be optimized. C. For cells that are sensitive to serum deficiency, a nutrient-rich serum-free medium can be used, or serum can be used in the transfection medium. Dedicated transfection reagents are recommended for adherent cells that are sensitive to serum deficiency. If necessary, you can use the serum-free medium instead of PBS to wash the cells twice, pay attention to the light when washing, slowly add liquid to the edge, and then do not blow the cells, but turn the plate to let the liquid roll on the cell surface. If the washing is too strong, the cell loses a part. After adding the liposome, the cells are affected more and the dead cells will increase. 2, antibiotics (PS) Antibiotics, such as penicillin and streptomycin, are media supplements that affect transfection. These antibiotics are generally non-toxic to eukaryotic cells, but cationic liposome reagents increase the permeability of the cells, allowing antibiotics to enter the cells. This reduces the activity of the cells, resulting in low transfection efficiency. Therefore, antibiotics should not be used in transfection media, and antibiotics should be avoided even when cell plating is performed prior to preparation for transfection. In this way, it is not necessary to rinse the cells before transfection. For stable transfection, do not use penicillin and streptomycin in competitive media, a competitive inhibitor of ICIN-selective antibiotics. In addition, in order to ensure healthy growth of cells in serum-free medium, a smaller amount of antibiotics than serum-containing medium is used. 3, cell status This is very important. Don't rush to make progress. Make sure the cells are in optimal growth state. There is literature that the passage should not exceed 17 generations. The cell state is best when the cells are regenerated for about 3 generations. Do not use cells that have been passed down for many generations, and the morphology of the cells will change. Most established cell lines are aneuploid, and primary cultures include mixtures of cells expressing different combinations of genes. Cell cultures are evolved in the laboratory for months and years after undergoing mutations, total chromosome recombination or changes in gene regulation. This can lead to changes in cell behavior associated with transfection. If this change is discovered over time, melting a fresh tube of cells may restore the original transfection activity. Therefore, if a reduction in transfection efficiency is observed, try to transfect freshly cultured cells to restore optimal results. Alternatively, several cell lines derived from screened, transfected cell lines with higher efficiency are now available. 4, cell plating density The optimal cell density for transfection varies depending on the cell type or application. Because the transfection reagent is toxic to cells, the cells are too small and are prone to death. Generally, when transfected, the adherent cell density is 70%-90%, and the suspended cell density is 2-4×106 cells/ml, ensuring that the cells are not overgrown or in a stationary phase during transfection. Because transfection efficiency is sensitive to cell density, it is important to maintain a basic passaging step between experiments. An increase in the number of plated cells can increase transfection activity and cell yield. The degree of cell fusion must be 90% to be done. 5. Promoter selection The promoter of choice required to achieve high transfection activity depends on the cell line selected and the protein to be expressed. The CMV promoter can achieve high expression activity in most cell types. Compared with other promoters, such as SV40 and RSV (Rousus sarcoma virus), it has the highest activity in BHK-21. These three viral promoters have lower expression levels in T cell-derived cell lines, such as Jurkat. The addition of PHA-L and PMA to the medium after transfection activates the CMV promoter in Jurkat cells, whereas single PMA is sufficient to activate the CMV promoter in KG1 and K562 (human myeloma leukocytes). Expression of the SV40 promoter is increased when it contains a large T antigen (present in COS-1 and COS-7) because the large T antigen can stimulate extrachromosomal synthesis. 6, the amount of DNA High quality DNA is essential for efficient transfection. Transfected plasmids must be of high purity, high concentration, and no endotoxin. The concentration should not be lower than 0.35ug/ul. The expression of the product was highest in 48 hours, and the highest in 72h. North Pacific Squid Ring,Seafood Squid Ring Frozen,Frozen Food Squid Ring Zhoushan Xifeng aquatic co lid. , https://www.xifengaquatic.com
For more experimental projects, please contact us:
Enterprise QQ
Phone / 020-32053431
mailbox:
Official website:
Address: D506, Guangzhou International Business Incubator, Science City, Luogang District, Guangzhou 