Gene transfection technology can be applied to: genome function research (gene expression regulation, gene function, signal transduction and drug screening research) and gene therapy research.
Operation method
Gene transfection (calcium phosphate-DNA co-precipitation method)
Principle
Nucleic acids in the form of calcium phosphate-DNA co-precipitates can make the DNA attached to the cell surface, which can be easily swallowed and ingested by the cell, or enter the cell through the gap opened during the contraction of the lipid phase of the cell membrane. Only 1%-5% of the DNA that enters the cell can enter the nucleus of the cell, of which less than 1% can be integrated with the cellular DNA and be stably expressed in the cell, and the frequency of gene transduction is about 10-4. With a transduction frequency of about 10-4 , this technique can be used for any DNA introduced into mammals for transient expression or long-term transformation. This method is the most common and preferred method for transfection of adherent cells.
Materials and Instruments
Cell samples Move 1. Preparation of donor DNA: Extracted according to the DNA extraction method described before, dissolved in TE at 40mg/L. 2. Culturing of recipient cells: Animal cell lines that do not contain human Alu sequence should be selected as recipient cells for the study of oncogene transfer. Such as mouse NIH3T3 embryonic fibroblast cell line, the cells have a certain tendency of spontaneous transformation, generally inoculate the cells one day before transfection, the inoculation density is 2×104 /cm2 ,with 10% fetal bovine serum containing DMEM solution, 37 ℃, 5% CO2 culture, to be cells accounted for 50-70% of the bottom area of the bottle, used for transfection test. 3、Preparation of DNA-calcium phosphate precipitates ① Prepare donor cell DNA and PSV2-neo plasmid vector DNA into 40mg/L DNA solution with TE, and add 220μl of plasmid DNA with gene neo (20mg/L) and 250μl of 2×HBS into 200μl of DNA solution from donor cell (PSV2-neo is used at the amount of 1～2mg/L). ② Take 500 μl of the above DNA solution and add it to a siliconized test tube, slowly add 3.1 ml, 2 mol/L CaCl2 and mix for 30 seconds. ③ Then mix and spin immediately, and let it stand at room temperature for 30 minutes, after the solution is mildly turbid, and then use it for transfection of recipient cells after blowing. 4、Transfection of recipient cells ①The recipient cells in the logarithmic growth period have accounted for 50-70% of the bottom of the bottle, replace the fresh medium once 4 hours before transfection, 5 ml per bottle (25 ml culture bottle). ② Aspirate 0.5 ml of DNA - calcium phosphate precipitate and add it to the cell bottle containing 5 ml of culture medium and shake well. ③ Place in 37℃ 5% CO2 incubation for 24h or longer, so that the cells can fully inhale the DNA - calcium phosphate crystallized particles. ④ Replace the fresh medium and continue to incubate for 24 h to induce the expression of transfected genes. Caveat The presence of impurities in DNA can interfere with transfection efficiency. Common Problems Calcium phosphate transfection is very inefficient and ineffective for many cell lines, and therefore does not meet the needs of many research endeavors. Currently, the most commonly used transfection reagents are cationic liposomes and cationic polymers, which have very similar characteristics to viruses in overcoming cellular barriers and can easily pass through cell membranes. Among them, cationic liposomes have high efficiency in gene transfection in vitro, however, in vivo, they are rapidly cleared by serum, accumulate in lung tissue, and induce strong anti-inflammatory responses, which leads to high levels of toxicity, thus limiting their application to a large extent. Due to the limitations of cationic liposomes, cationic polymer transfection reagents are gaining importance. For more product details, please visit Aladdin Scientific website.
HBS CaCl2 TE G418 (Neomycin G418) solution G418 Selection Medium
Culture Bottle