I got some problems with growing a GFP recombinant adeno-virus in HEK-293T. I used the stock virus left by a previous lab member. The virus can infect 293T and express GFP, which can be detected as early as 24h. I let the infection last 3-4 days and harvest the virus when 90% cells showed CPE. Then I used the harvested virus to do the infection study again, however, the retrieved virus seems not be able to infect the cells. I think the problem may be caused by 293T. My labmate used HEK-293A, but currently we don't have that cell line. Any thoughts?
HEK293T contains the SV40 large T as well as E1A and E1B, so your viral vector should grow also in these cells. And the fact that you saw CPE indicates that you had viral production. How did you harvest the virus? Normally I collect the cells and lyse them in a small volume of serum-free media by 3-4 freeze thaw cycle. After spinning down the cell debris, the supernatant should contain your viral vector.
293A cells are immortalized by addition of E1 gene expressing E1a and E1b. 293T cells are immortalized by addition of SV40 large T antigen. The expression of E1 gene products are required for adenovirus production. That's why you're having problems producing new virus. You need to track down some HEK-293A (or the HEK-293AD daughter line) for your experiments.
Thank you Eric. I can't find any HEK-293A or HEK-293AD right now. But I do have HEK-293 in our lab. I'm wondering does HEK-293 have E1a and E1b ? I'm new to cell culture.
HEK293T contains the SV40 large T as well as E1A and E1B, so your viral vector should grow also in these cells. And the fact that you saw CPE indicates that you had viral production. How did you harvest the virus? Normally I collect the cells and lyse them in a small volume of serum-free media by 3-4 freeze thaw cycle. After spinning down the cell debris, the supernatant should contain your viral vector.
As Paola says, RAds should grow fine in HEK293T's. Did you titre your harvested virus on 293T's? If so, what was the titre, and what MOI did you use for your infection study?
Paola is correct, 293 cells were originally immortalised in the 70s by the addition of sheared Ad5 DNA to human embryonic kidney cultures, and as others have pointed out above the part of the Ad genome responsible for transformation was the LHS of the Ad genome (which includes E1A and E1B amongst other things). It's the E1A region that does most of the work complementing the missing sequnces in your AdGFP. Also, as Paola points out it looks like you're getting a productive infection occurring, suggesting that E1A is allowing replication of and expression from your AdGFP.
I would be suspicious that perhaps the large T is interfering with some step of virus production, especially as Large T and E1A share overlapping functions (ie they're both strong viral transactivators and both bind RB and p300/CBP). Thus I would strongly recommned just using the vial labelled 'HEK-293' that you talk about. As for 293A's, they're essentially the same cells as 293's but they've been selected for the ability to adhere better to plastic. Also, with respect to the viral harvest protocol, we do it pretty much the same as Paola, but with Tris-HCL pH7.4 instead of SFM.
Good luck.
We follow the same procedure as Paola outlined. Did you see any GFP signal on infection with your harvested virus?. If you can see a small GFP signal just blindly passage your cells after 5-6 days eventually virus will pick up. However if you are not seeing any GFP signal then you need to go back to your stock virus.
GOOD LUCK
Thanks everyone. I really appreciate all your suggestions. When I harvested the virus, I collected the cells and froze them in -80°c. Then I thawed the culture media in 37°c water bath. After that, I lysed cells by ultrasonication (40 kHz 10s, 3 times in ice water). The cell debris was removed by centrifugation (2000 rpm , 5min). I’m not sure whether this will make any differences with the 3-4 freeze thaw method mentioned by Paola, as both methods are used in our lab. The E1A gene of the virus I used was replaced by a green fluorescent protein (GFP) gene. I think Alasdair’s comments are very helpful. I’m trying to grow some HEK-293 cells and will see how it works. BTW, how long does it take for the HEK-293 to regrow in the flask after taking out from the liquid N2. I seeded the cells about 5 days, haven’t see many cells attach to the flask.
Hi Eric... it sounds like your cells are dead unfortunately :(
293s grow like weeds. Lets say you froze about 1 - 2 million cells in a vial, then got them up and put them in a T25 or T75, then I'd expect them to be confluent in 2 to 3 days maximum. Although they should be grown in DMEM (+serum), they're pretty robust so even if you used the wrong media they would most likely have grown. You don't have any selective antibiotics in your media (eg Geneticin/G418) do you? One other thing I should mention, if you're a lab on a budget then you may be re-seeding cell lines into the same flask that you split them from.. don't do this with 293s - they hate it.
I'd find another vial of 293s if I were you :)
Good luck
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