Background[1-3]
Calcium phosphate method cell transfection kit is used to transfect cells using the calcium phosphate method and is suitable for transfection of most adherent cells. The calcium phosphate method cell transfection kit is improved on the basis of the traditional calcium phosphate cell transfection method, which improves the transfection efficiency and reduces the toxicity. Using the calcium phosphate method to transfect cells not only allows transient expression, but also allows selection of stable strains.
The calcium phosphate precipitation method is a transfection method based on calcium phosphate-DNA complex to introduce DNA into eukaryotic cells. Calcium phosphate is considered to be beneficial in promoting the binding of exogenous DNA to the surface of target cells. The calcium phosphate-DNA complex adheres to the cell membrane and enters the target cell through pinocytosis. The transfected DNA can be integrated into the chromosome of the target cell to produce stable clones with different genotypes and phenotypes.
Calcium phosphate transfection technology was pioneered by Graham et al. in 1973 and is the most widely used method to transfect mammalian cells in the laboratory. Although calcium phosphate transfection is a simple gene therapy method, it is greatly affected by precipitation conditions (such as pH value, temperature, concentration of salt and DNA, and time control of precipitation and transfection, etc.), and calcium phosphate is easily lost. live, so the process is difficult to standardize.
The principle of calcium phosphate transfection method is to mix DNA, calcium chloride and phosphate buffer to precipitate to form extremely small insoluble calcium phosphate particles containing DNA. These calcium phosphate particles will adhere to the cell membrane and enter the cells through pinocytosis, thereby achieving the purpose of transfecting foreign plasmids into the cells. Calcium phosphate transfection method is mainly used for lentivirus packaging. Since lentivirus packaging requires a large amount of plasmid and a large amount of liposomes, it may affect the cell state. The calcium phosphate precipitation method is widely used because the reagents are easy to obtain and cheap.
Apply[4][5]
Used for the study of cytotoxicity and apoptosis of modified montmorillonite and modified calcium phosphate transfection vector
Use cell activity reduction experiment, LDH release detection, apoptosis degree detection, caspase-3 enzyme activity detection, apoptosis-related gene transcription activity detection, and related gene protein level and mRNA level detection to measure PSAN-MMT.
Nontoxic MMT was used as a control against PSAN-MMT. Both PSAN-MMT and MMT reduce cell activity and increase LDH release in a dose-dependent manner. This effect is obvious at a concentration of 1 g/L, but the effect of PSAN-MMT is significantly lower than that of MMT. The cells were also treated at a concentration of 1 g/L to detect the degree of apoptosis. The degree of apoptosis of cells treated with PSAN-MMT was still lower than that of cells treated with MMT. The caspase-3 enzyme activity was tested and the results were still consistent with the previous results.
It was found that high concentrations of PSAN-MMT and MMT can both increase the transcriptional activity of p53. MMT increases the transcriptional activity of p53 to a greater extent than PSAN-MMT. PSAN-MMT and MMT can also upregulate the protein expression level of p53 and p53, The level of p21 mRNA is also lower in PSAN-MMT than in MMT. This study found that PSAN-MMT, like MMT, is non-cytotoxic, and speculated that the mechanism of apoptosis caused by PSAN-MMT and MMT at high concentrations is the activation of some pro-apoptotic genes such as p53 and p21. It can be considered that oligostyrene-cyanoethylene-modified montmorillonite can be safely used as a carrier for controllable macromolecule drugs and gene therapy drugs.
Study on protamine-modified calcium phosphate transfection vector In order to study whether protamine modification can improve the calcium phosphate transfection effect and what effect it has on calcium phosphate particles, pEGFP-N1 green fluorescent plasmid was used as an indicator. Cells were stained to study the changes in transfection efficiency, atomic force microscopy was used to detect the morphological changes and particle size of calcium phosphate particles, and the MTT method was used to detect the inhibitory effect of protamine-modified calcium phosphate particles on cell activity.
Calcium phosphate synthesized by co-precipitation method was used as a comparison with protamine-modified calcium phosphate. In 293 FT, HEK 293 and NIH 3T3 cells, the transfection efficiency of protamine-modified calcium phosphate was significantly higher than that of calcium phosphate synthesized by co-precipitation method. At the same time, stearic acid showed that protamine-modified calcium phosphate was It can still have good transfection effect 7 days after synthesis, indicating that protamine may be able to improve the stability of calcium phosphate. Atomic force microscopy photos show that the particle size of protamine-modified calcium phosphate particles is significantly smaller than that of calcium phosphate synthesized by co-precipitation method, and there is no obvious difference in the degree of inhibition of cell activity between the two.
