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help with retroviral infection of MEFs - (Jul/07/2008 )

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!


Did you use vsvg pseudotyped retrovirus?
Vsvg may induce cell fusion at high concentration.


Thanks, WHR. No, I didn't use Vsvg. I just used helper for transfection of Pheonix cells.


Did you stain the cells for senecence associated beta-galactosidase activity?


Have you tried not doing the second infection?


To WHR: Based on the morphology, those MEFs are obviously senescent. I didn't stain them with X-gal.
To Madrius: I always infected the cells twice. Do you think it's the infection causes the senescence?

I just wonder: Is there any report or article about effects of retroviral infection on MEFs or other cells?
Is 50% confluence of MEFs is too low for infection and leads to senescence?
Does anyone have a protocol for retroviral infection of MEFs including how seed and split the
MEFs for infection?
Thanks a lot!