Several commonly used transformed cells and transformation technology - Database & Sql Blog Articles

Several commonly used transformed cells and transformation techniques 1) Carcinogenic chemicals transformed cells: transformation of Syrian hamster embryonic cells
1. Materials: On the 13th day after pregnancy, take several littermates of embryos, remove head and viscera, cut them into rice size under aseptic conditions, and digest with 0.25% trypsin (containing 0.01% EDTA) for 30 minutes at 37 °C. Filtration, low-speed centrifugation, absorption of digestive juice, addition of nutrient solution, preparation of cell suspension, inoculation into 75cm/culture flask, inoculation density 10～20×106/75cm, culture at 37°C for 2~3 days, in smooth condition Underneath can quickly fill the surface of the bottle.
2. Storage: Choose a large number of well-preserved cells for cryopreservation and store them for later use.
3. Carcinogen treatment: Take frozen cells, thaw them, and inoculate them in 25ml culture bottles, each containing 100,000 to 300,000 cells. When the cells grow into the exponential phase, the carcinogen MNNG is added to the culture flask. Carcinogenic dose 1 ~ 3 μg / ml nutrient solution. Incubate for 12 to 48 hours in a 37 ° C incubator.
4. Low serum culture: Discard the MNNG solution, wash it with warm PBS for 1 to 3 times, add 20% calf serum, continue to incubate in a thermostat for 2 to 3 weeks, and then switch to culture with 5% serum. Low serum cultures are not conducive to normal cell growth, but transformed cells can still proliferate and form foci.
5. Focal formation and isolation: In the case of success, cells treated with carcinogens can produce varying amounts of foci between normal cells after 10 days.
2) Viral transformed cells
1. Blood preparation: Citric Acide or heparin anticoagulation 1 to 2 ml of blood, divided into tubes, 0.5 ml of whole blood per tube, placed in a refrigerator at 4 ° C for 30 minutes.
2. EBV virus transformation: Frozen 0.5 ml of whole blood was taken out from liquid nitrogen and rapidly thawed in water at 37 °C.
3. The thawed cells were mixed with 10 ml of serum-free RPMI 1640, centrifuged at 2500 rpm for 2 minutes, the supernatant was discarded, and resuspended in RPMI 1640 growth medium containing 20% ​​fetal bovine serum, cyan, streptomycin and gentamicin. cell.
4. Add 0.3 to 0.5 ml of EBV virus solution, and shake it at 37 ° C for 30 minutes to 2 hours, shaking from time to time to avoid clot formation.
5. The mixture was placed in a 50 ml culture flask, and an appropriate amount of the culture solution was added thereto, placed in a 5% CO2 incubator, and cultured at 100% temperature.
6. Add 2 ml of medium after one week.
7. Frequent microscopic examination of the culture, adding liquid or changing every 3 to 4 days, with the increase of the number of cells, can be divided into large bottles for cultivation.
3) Oncogene-transformed cells: genomic DNA transfection
1. Extract genetic DNA (including oncogenes).
2. DNA preparation: Take 50-100 micrograms of donor DNA, add 3M NaCl or sodium acetate to mix the final concentration to 0.3M.
3. Add 2 times the volume of absolute ethanol, centrifuge at 3000 rpm for 10 minutes, and remove the supernatant.
4. Add the transfection buffer. After the DNA is fully melted, add 2.5M CaCl2 to a final concentration of 125 mM and gently pipette three times with a pipette. Place at room temperature for 10 to 30 minutes, when the liquid transparency is reduced, and when there is a slightly turbid blue color, it can be used to transfect recipient cells.
5. Cells: Exponentially proliferating cells in a semi-confluent phase with good growth status were replaced with culture medium 1 hour before transfection.
6. Transfection: A DNA-calcium phosphate precipitate of 0.5 to 1 ml/vial was added to each vial and allowed to act at 37 ° C for 4 to 6 hours.
7. Culture: The culture solution was discarded, treated with 15% glycerol for 3 minutes, rinsed once in serum-free culture, and a new culture solution was added thereto, and cultured in a 37 ° C incubator for 24 hours.
8. Passage: Pass the 1:5 or 1:10 bottle to pass the passage, change the liquid every 2 to 3 days, close to the confluence, the serum dosage will be reduced to 5%, and culture for 2 to 3 weeks.
9. Detection: Observed day by day, after the foci appear, separated into another bottle for culture.