Stable Clones by Retrovirus

Generate retrovirus

1. Transfect 293T cells (40-60% confluent) with 3 plasmids: 9 ÎŒg of retroviral plasmid (e.g. pBABE-ING2), 4.5 ÎŒg of GAG-POL plasmid and 4.5 ÎŒg of VSV plasmid using GeneExpresso Max transfection reagent.

2. (Optional) The next day, change the medium, but only replace 4-5 ml DMEM-10% FCS (to keep virus concentrated). The medium change is required to remove a growth inhibitory factor produced by 293 cells upon transfection.

3. 2 days after tranfection, collect the supernatant (medium) with a 10 ml syringe, and add 4-5 ml fresh DMED.

4. Filter the medium through 0.45ÎŒm filter.

5. Aliquote 2 ml each and freeze @ -80oC or use fresh.

6. The next day, harvest the virus as day 3.

Transduction

1. Plate of 293FT cells should be around 40-50% confluent (MMLV based retrovirus cannot enter non-dividing cells).

2. For a 10-cm dish, remove media and add 6 ml of new medium containing 64 ÎŒg Polybrene (16 ÎŒl of stock solution 4 mg/ml at -20oC).

3. Thaw the virus in the medium at 37oC quickly, and add 2ml into each plate (final polybrene conc. is 8 ÎŒg/ml).

4. After 4-6 hrs, add 4 more ml media.

5. After 24 hrs, change media/and split if necessary.

Stable line selection

1. 24 hrs after the first media change (2 days after transduction), start selection with proper drugs (2 ÎŒg/ml puromycin (1 to 1000 dilution from 2 mg/ml stock).

(1) Split cells if necessary, cells need to be dividing for puro to be active.

(2) Puro can be added 24 hrs or 48 hrs after transduction.

(3) 1 mock tranfected plate can be used to ensure that the puro did its job.

2. Change media every 1-2 days/split cells when necessary.

3. In 2-3 days all non-transduced cells should have been killed, high yield expression stable clones can be selected by FACS sorting.