problem with retroviral infection of MEFs

[wantz722]

Hi Guys,

I has trouble with retroviral infection of MEFs for several monthes and really hope get some help here.
I have tried infecting MEFs with a MSCV-shRNA-GFP retroviral construct. My purpose is to knock down of a gene by retroviral-directed shRNA. However, after infection, I frequently saw a significant portion of MEFs become senescent(flat, big, multi-nucleus,etc.). Those MEFs are only in passage 3 or 4 and are not supposed to be so senescent. It's also not a phenotype of the knockdown since the empty vector control has the same phenomenon. However, MEFs in the same passage from the same batch without infection doesn't have the senescence. Therefore, I think sth during the retroviral infection induces the senescene. However, I don't know what causes the senescence. Could you give me any possible reason? Thanks. Below is my protocol for retroviral infection of MEFs:

Day 1.
Morning: (1) Transfect Pheonix Packaging Cell with retroviral vector
by Lipofectamine 2000.
(2) Seed one vial of Passage 2 MEFs to 3 10-cm plates
Day 2. early afternoon: Split 1 10-cm plate of MEFs into 2~3 6-well plates.
Day 3. Morning(48hrs after transfection): Collect viral supernatant from Pheonix
cell and infect MEFs for 1st time.
Afternoon: repeat 2nd infection about 6 hours after 1st infection

Note: 1. MEFs are in 45~75% confluence during the 1st infection.
2. I use Polybrene at at a final concentration of 5ug/ml.
3. Some times, I use viral supernatant stock which is collected around
72-82hrs after transfection and has been kept at -80C until usage.

Day 4. Morning: change medium for infected MEFs.
Day 5. Split infected MEFs at 1:2 or 1:3 for either NO drug selection or 2ug/ml
puromycin selection

On Day 3 afternoon, during the 2nd infection, I could see MEFs change their morphology(become more flat) and some MEFs look like senescent. After split on Day 5, senescent cells become more obivious! (See picture below：red arrow indicates examples of senescent MEFs in passge 4)

Occasionally, there is only few senescent MEFs after infection. However, most time there is a lot of senescent cells no matter I use fresh viral supernatant or frozen viral stock.

Do you have any idea what causes this unexpected senescence of MEFs infected by retrovirus? Thanks a lot!

[pinkyjun]

Hi there,
I had the same problem twice already. I was using 10ug/ml polybrene and I thought this was causing the senescence/ cell fusuion. But obviously 5ug/ml does the same.
Did you actually find a solution to this senescence issue? If you have I would be glad to hear it.
I will next try using fresh serum during infection as I think the media from the packaging cells is a bit too much used.
Cheers,