Hi all, I'm trying to produce lentiviral particles using TRIPZ vector (13kb) and second-generation packaging system (as recommended). My transfection step, using calcium phosphate, worked because over 90% of the HEK293T cells are green. The problem is that I could not observe any GPF positive cell (target cells) under a fluorescence microscope and hence I think transduction has not occurred or I didn’t have any virus to start with. When I run the target cells into a flow, I see slight shift in green channel (5-10%). Four days post transduction, my target cells died (the plasmid contains a sequence for pro-death protein, but it’s an inducible system, so the insert should not be expressed unless I add Doxycycline into the media). p.s ( I had this vector working 8 months ago but not anymore) can somebody explain to me what's wrong?
Thanks for your time.
In my experience, Packaging with Calcium transfection in HEKs works very well, and since you see fluorescence, that's probably not the issue. The confluency of the HEKs is very important for the yield of the virus, so make sure your cells are 80-90 % confluent. Do you use polybrene during the infection step? This should help as well.
Also, I would recommend that you concentrate your virus before use. You can either use a classic ultracentrifugation method, or you can use the LentiX Concentrator (Clontech) which I find really convenient. Hope this helps
In addition to the above mentioned comments. previous experience suggests that making new plasmid stocks, or trying different ones, of the accessory plasmids sometimes leads to identification of the confounding issue.
Thanks all for your help.
@Antonio Ruiz-Vela, I don't have problem transfecting the cells, they are almost all green using calcium phosphate protocol. I used to use Lipofectamine 2000, but it's a bit expensive and gives same result as CaP. I'm expressing BIM, I also have mutant for that which is not supposed to kill. I have also tried using tetracycline free serum thinking that my plasmid might be leaky but I didn't get any green cells either (after transduction)
@Thibaut Eguether, I do use polybrene in my transduction step. I also will try to concentrate the virus, I ordered the LentiX Concentrator. Will see next week.
@Erik Martin, I did use different plasmids , I did even re-do the maxiprep and start from fresh prep and fresh cells and the result was always the same.
Hi All.
I followed the advice of Thibaut Eguether and used the LentiX Concentrator (Clontech) and I measured of infectious virus titer using Leni-X Gostix (clontech). The result is in the image attached. Before (is before concentrating the viral solution) and after is after the concentration. The presence of the test band (T) indicates that I got >5 x 10^5 IFU/ml. I transduced the cells today and I will know in 48h the tranduction efficiency.
Update:
48h post transduction, no green cells were detected. I was able to get green cells using another vector as control for my transduction (8kb of length) but not with my 13kb vector. This has nothing to do with my gene of interest because I wasn't able to get green cells using the same 13kb vector without the insert.
One possibility that I can think of is a mutation in the psi packaging signal that results in the generation of an empty viral particles (I don't even know if this will be possible)!!
What are you thoughts?
I would definitely sequence your plasmid... Maybe you have a mutation in your GFP as well.
I have the same problem. I transduction 293T cells using lentivirus it is good , GFP positive and cells are healthy. but using same lentivirus transduction PBMC then induced into macrophage and DC, the cells all died after 4 days.
Junjie Shao Abbas Hadji Did you figure out and solve your problem, having the same issues with my NK cells. Do you have any suggestions?
just changed the plasmids, using another lentivirus plasmids, it works. Didem Tecimel
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