Hi Nicholas,

I cannot be 100% sure, but from what you describe it looks like your transduction is not working at all. Lifted cells expressing eGFP might as well be a microscopy artifact (dead autofluorescent cells).

Have you checked eGFP expression by FACS upon excluding dead cells by either using DAPI or DRAQ7 incorporation?

HEK293T are very easy to transduce with eGFP encoding lentiviruses. My suggestion is to check by restriction digestion all your vectors (including packaging) before attempting virus production again. Also make sure that your packaging vectors are compatible with your lentiviral backbone and that no re-arrangement are present in the latter.

Hope it helps

Francesco

Which cell did you used for checking the MOI, 293T? If your vector construction is working on other cells then I suggest you to use a new vial of HEK293T, in my experience I have used Collagen IV coating for various cell types including primary cells. Hope you will be able to obtain good transduced cells.

Hey Nicholas,

I agree with Francesco Aulicino. It could be an autofluorescence artifact. Another explanation could be that the very few cells, which are efficiently transduced, die shortly after. Meaning they have enough time to express eGFP but die then very quickly because of transduction dependent toxicity. Considering the very low MOI this could explain why you see only very few eGFP+ cells. Best, Marcel

It's been my experience that once confluent, HEK cells tend to easily lift off the plate and then will eventually die without attachment. This is especially true after undergoing stress usually associated with any transduction/transfection protocol. We've had luck with coating the culture plates with PEI solution (0.1% PEI in borate buffer) for HEK cell plating.

Thank you everyone for your responses.

I have been seeding the HEK293T cells on plates pre-treated with 0.1% poly-L-lysine in PBS. I allow the plates to dry for 1 - 2 h before seeding the cells. The cells are at ~80% confluence at the time of transduction.

To make the issue more confounding, I've noticed the balled up fluorescent cells only show up via microscopy. If I perform flow cytometry, there are no eGFP-positive cells.

The primary difference from traditional lentiviruses is that we are using a lentivirus pseudo-typed with an alternate glycoprotein on the surface. The HEK293T cells I am using overexpress the appropriate receptor and I have validated this by flow cytometry. The collaborator we received the plasmids and cells from have observed the same phenomenon when they transduce the cells with the same virus.

Marcel Klingenberg-Ernst, is there a way to minimize transduction dependent toxicity? I have attempted to purify the virus by passing it through a 0.45 µm filter, then concentrated it in an ultracentrifuge and resuspend in PBS. I assume this leads to removal of cellular toxins released in the production process, but is that incorrect? Are there other methods?