西亚试剂：：Stable Transfection (Electroporation)

 
Stable Transfection (Electroporation)
Outline:
Electroporation can be used for both transient and stable transfection of mammalian cells. Cells are placed in suspension in an appropriate electroporation buffer and put into an electroporation cuvette. DNA is added, the cuvette is connected to a power supply, and the cells are subjected to a high-voltage electrical pulse of defined magnitude and length. The cells are then allowed to recover briefly before they are placed in normal growth medium.

Supplies & Equipment:
BioRad gene pulser with capacitance extender
Electroporation cuvettes with 0.4 cm interelectrode distance 
Reagents:
sterile 1 x HeBS buffer (Hepes buffered saline)
G 418 (Geneticin) stock solution
Procedure:
DNA preparation
Per sample, linearize overnight 10 - 20 µg plasmid DNA (containing the gene of interest). Although circular DNA can be successfully transfected, this linearization prevents mammalian cells from randomly digesting the plasmid before incorporation in genome.
Phenol-Chloroform-Ethanol-precipitate the DNA. This concentrates and sterilizes the DNA. If the plasmid does not contain a selectable marker gene, then cotransfect with 1 - 2 µg of pSV2-neo.
Resuspend the DNA in 800 µl 1 x HeBS buffer and wait for 1 hour for the DNA to completely resuspend at room temperature.
Electroporation
Harvest, count and wash 3 - 40 x 106 cells which are growing in log phase (best if passaged 1 - 2 days before) with cold 1 x HeBS. Resuspend these cells in the 0.8 mL of 1 x HeBS which contains the DNA. Allow to sit at room temperature for 15 minutes.
Gently transfer the DNA / cell suspension to an electroporation cuvette and electroporate at appropriate voltage and 960 µF capacitance. Record time constant. Leave the mixture in the cuvette for 10 minutes at room temperature.
Transfer the cells with a sterile pipette to T75 flask containing culture media with 10% FBS.
Selection (e.g. with G418)
At 48 hours switch to media containing 10% FBS and G418 (make from 10 mg/ml stock in HANKS). Concentration needed may vary:
500 - 1000 µg / ml for COS-cells
500 - 1000 µg / ml for CHO-cells
300 - 500 µg / ml for 293-cells
Keep 2 weeks (10 - 20 days) under selective conditions, changing media (as needed) every 3 - 5 days.
Cloning
Harvest and count survivors. Dilute cells with selective media and 10% FBS to a concentration of 1 cell per 200 µl (or 100 cells per 20 ml). Some cells (e.g. COS) require conditioning of media by non-transfected cells growing at high density.
Using a multipipettor, plate 100 µl of cells per well (0.5 cell per well) into two 96-well plates. Freeze down the remaining parental transfected cells (~ 1 Mio cells / viol) in culture media + 10% DMSO.
After approximately another 2 weeks some wells turn yellow, indicating growth. For cloning, less than 50% of the wells should be positive. Check expression of plasmid DNA.
Expand the best 10 - 30 clones, from 6 well plates to T75 flasks, maintaining selective conditions.
Check expression repeatedly. Some cells need to stay all the times under selective pressure, so make sure that at least some transfected cells are either frozen down immediately after selection (parental cells), or kept in selective media.
Time Required: five weeks