Hi everyone,
I am trying to produce eGFP lentiviral vector by transducting HEK 293T with calcium phosphate method. I transduce cells when they are at 70 % confluent (following this method https://www.jove.com/video/54176/optimal-lentivirus-production-cell-culture-conditions-necessary-to) using a ratio of transfer plasmid 2,5 : packaging plasmid 2 : envelope plasmid 1.
When titering the virus in the target cell line (primary fibroblasts) I see toxicity at highest concentrations (1:10 and 1:25) but I don't see any GFP expression. While at lower concentrations cells are morphologically fine.
Does anyone has some advice?
Also I have a curiosity, since in every protocol I have read, HEK cells are not lysed after the transduction in order to withdraw the virus. Can someone explain how do these cells secrete the virus in the culture media without suffering some type of toxicity?
I am sorry if these are very basic questions but I am new to this!
Thak you
Hi Simona,
Check if your lentiviral backbone is properly transfected into HEK293t. It should not be difficult since you can track eGFP expression.
Since your target cells are suffering from high MOI transduction (even in absence of eGFP), I would say that you are most likely producing empty lentiviral particles (probably indicating that your packaging plasmids transfection are fine).
Most of the times when lentivirus are not produced is because there's something wrong in the vectors.
Besides checking eGFP expression after transfection, re-prep all your plasmids and run them on a gel to verify their integrity.
The lentiviral backbones are sometimes subject to rearrangement if they are not propagated in the correct E.Coli strain (NEB Stable, or Invitrogen Stbl2-3). This happened to me a couple of time (nice eGFP after transfection but no virus production). It turned out that some of the vectors just spontaneously lost a portion of the LTR, making them useless as lentiviral backbones.
Hope it helps
Good luck
Francesco
I agree with everything Francesco said, we also had sporadic issues with lentiviral vector rearrangements which were resolved by switching to the Stbl2 or 3 bacteria for growing up plasmid stocks. The main other thing to look out for is the health of your HEK 293t cells. These need to be low passage number and you need to make sure they don't get over confluent . Unhappy cells will really drop the amount of lentivirus you produce. The cells should not lyse with lentivirus. The virus is secreted into the media.
Greg Towers Lab has a lentivirus protocol which includes images of how confluent your cells should be and culture conditions. Whilst they use Fugene rather than calcium chloride the results should be similar. You can find their protocol here: https://www.ucl.ac.uk/towers-lab/sites/towers-lab/files/gregsretroprep.pdf
You transformed your plasmid and packaged them in 293T cells, but then you were having problems during your infection/transduction of your target cells? One of the easy things to try is to see if you have GFP fluorescence in the 293Ts.
An important consideration is that the expression levels in your target cells is not high enough to get a clear signal. Your choice of promoter you use for your expression can be a problem. A CMV promoter may not work as well in comparison to a EF1a, CAGG, or UBC promoter in primary fibroblasts. You can also try to see if you can detect expression by western blot using an antibody for GFP or your protein of interest.
Thak you a lot!
We checked the integrity of the GFP plasmid before using it because we had problems amplyifing it in different basteria strains. At the end, we run a gel and see that the plasmid didn't suffer rearrangement.
About checking the GFP expression in HEK293T, we see cells that express GFP 30h after transfection (foto attached), this is why it seems so strange to me that we don't see GFP expression in fibroblasts.
Regarding the fact that the promoter we are using could not work well in primary fibroblasts, the lentiviruses we are trying to produce have already been used in different papers on mouse embryonic fibroblasts, so I didn't think it could be a problem, but I will consider it!
Anyway I will prepare new plasmids as Francesco told, and see if there is any difference.
Again, thak you!
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