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