The recombinants of foreign genes and λ - phage DNA are introduced into human Escherichia coli and mammalian cells
Calcium phosphate DNA coprecipitation, referred to as phosphoric acid precipitation, can transfer the recombinants of foreign genes and λ - bacteriophage DNA into human beingsEscherichia coliandMammalian cell。[1]
Calcium phosphate precipitation methodreagentEasy to obtainPriceCheap and widely usedinstantaneousTransfection and stabilizationtransfectionThe research ofDNAMixed with CaCl2,thenAdd to PBS to form DNA calcium phosphate slowlyprecipitateFinally, the suspension containing precipitation is added to the cultured cells and absorbed through the endocytosis of cell membraneDNA。
Calcium phosphate also seems to inhibitserumNeutralize nuclease activity in cells to protect foreign DNA fromdegradation.
nucleic acidwithcalcium phosphate-DNAWhen the formation of coprecipitate occurs, it can make DNA adhere tocell surfaceIt is conducive to cell ingestion, or enters the cell through the gap opened when the cell membrane lipid phase shrinks. Only 1% - 5% of the DNA entering the cell can enter the nucleus, and less than 1% of the DNA can be integrated with the cell DNA, stably expressed in the cell, and the frequency of gene transductionabout10, this technology can be used for any DNA introductionMammalian cellResearch on temporary expression or long-term transformation.
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This method is applicable to wall stickingCell transfectionIs the most commonly used and preferred method.
Solution preparation
2× HBS1.63gNaCl；1.19g Hepes；0.023gNatwoPOfour.2H2O；Add water to 100ml (pH7.1), filter, and store at<4 ℃.
G418 (neomycin G418) solution 1g G418 is dissolved in 1mmol/L Hepes buffer solution, added with water to 10ml, filtered and sterilized, and stored at<4 ℃.
Be carefulReceptor cellDo the pre experiment first, and select the lowest concentration that can kill more than 50% of the cells in 10-14 days.
Operation steps
(1) Donor DNA preparation: extract DNA according to the DNA extraction method described previously, dissolve it in TE, 40mg/L.
(2) Culture of receptor cells: for the study of oncogene metastasis, we should choose the cells without human Alu sequenceAnimal cellAs receptor cells.For example, the mouse NIH-3T3 cell line has a certain tendency of spontaneous transformation. Generally, the cells are inoculated one day before transfection. The inoculation density is 2 × 10 cells/cm, and the dosage of 10%fetal bovine serumOfDMEM medium，37℃、5% COtwoCulture, when the cells account for 50% - 70% of the bottle bottom area, it is used for transfection test.
Preparation of DNA calcium phosphate precipitate
The donor cell DNA and PSV-2neo plasmid carrier DNA were prepared into a 40 mg/L DNA solution with TE, and the 200 ul donor cell DNA solution was added with 220 ul plasmid DNA solution (20 mg/L) of neomycin gene and 2 x HBS 250 ul (1-2mg/L PSV2 neo).
Take 500ul of the above DNA solution and add it to silicificationtest tubeMedium, slowly add 3.1ml CaCltwo(2mol/L) for 30s.
Then immediately mix and spin, and let stand at room temperature for 30min. After the solution is slightly turbid, it is used for transfection of receptor cells after blowing.
Transfected receptor cells
Will be inLogarithmic growth periodThe recipient cells that have accounted for 50% - 70% of the bottom of the bottle should be replaced with fresh culture media 4 hours before transfection. Each bottle of 5ml (25mlCulture flask）。
Suck 0.5DNA-calcium phosphate precipitation, add it into a cell flask containing 5ml of culture medium and shake well.
Set at 37 ℃, 5% COtwoIncubate for 24 hours or longer to allow cells to fully inhale DNA calcium phosphate crystal particles.
Replace the fresh medium and continue to culture for 24 hours to induce the expression of transfected genes.
Replace G418 with a concentration of 800mg/L to select the medium for screening.At the same time, there were control cells that could not be transfected.
After 3-5 days of culture, most of the control cells died. At this time, the transfected cells were replaced with G418 with a concentration of 200 mg/L to select the medium, and the medium was replaced every 3-4 days.
Two weeks later, the control cells died, and drug-resistant cell clones appeared in the transfected cell bottles. After they were enlarged, they were cloned and expanded.Can be establishedTransformed cellAnd further identification.
In this experiment, PSV neo and DNA are added to the foreign DNA to co transfect the receptor cells, so that the receptor cells can obtain neomycin (neo) gene resistance, so that even if there is no obvious "transformation focus" of the oncogene, the anti neomycin label of the transferred foreign gene can be measured.Moreover, the receptor cells transfected with neomycin gene can be used to screen the transformed cells through G418 selective medium to establish cell lines.For the purpose of obtaining "transformation focus", Uoyi does not need to add PSV2 neo DNA, but only needs to import the genomic DNA extracted from cancer cells into receptor cells, as follows.
3. Genomic DNA transfection · method 2
Prepare phosphoric acid for transfection.NaCl 8.0g，Hepes5.0g；Na2HPO40.099g；Warm the water to 1000ml.The pH is very important. It must be controlled at 6.95 ± 0.65, sterilized, and stored at - 20 ℃ for standby.
Extract the genomic DNA of cancer cells according to the method described above.
Take DNA50-100ug of donor gene, add 3mmol/L NaCl or sodium acetate, make the final concentration to 0.3mmol/L, and mix well.
Add 2 times the volumeAnhydrous ethanolCentrifuge at 3000 r/min for 10 min and discard the supernatant.
Add transfection buffer, and clamp it in 2.5mmol/L CaCltwo, the final concentration is 125mmol/L, blow it with a straw for three times, and leave it at room temperature for 10-30min. When the transparency of the liquid decreases and a slight turbid blue appears, it can be used for transfection of cells.
The logarithmic growth phase cells in good growth condition and in half confluence stage were taken, and the culture medium was renewed once 4 hours before transfection.
Transfection.Add 0.5-1ml of DNA calcium phosphate precipitate to each bottle, and let it sit at 37 ℃ for 4-6 hours.
Cultivation.Discard the culture medium and treat it with 15% glycerol for 3min,Serum free mediumRinse once, reduce the serum dosage to 5% when it is close to convergence, and culture for 2-3 weeks.
testing.Observe day by day. After the "transformation focus" appears, clone, isolate, and expand the culture to establish the transformation cell line.
