I am using lentiviral transduction system. I produce virus in HEK by calcium phosphate method. However, I don't have fluorescent marker. The only marker in my plasmid is neomycin. When I transduce the cells, they start to die 24 hours?, however, there is no problem with untransduced controls. Thus I cannot proceed to neomycin selection. I don't think it might be related to polybrene concantration because I always use the same concantration with this cell line before. So what do you think about the problem? What can be done to solve it?
Agree with all the above troubleshooting - just one more to add: After harvesting the virus, give a quick and short spin to precipitate any invisible debris from the supernatant followed by a filtration step through a syringe fitted with a 0.45 micron filter to make sure that toxic proteins are not carried over to your target cells for transduction. You have not mentioned whether you use the supe directly or ultracentrifuge it to make concentrated stocks. IF you do the latter, it is always advisable to resuspend the viral pellet in PBS rather than medium. Best of luck.
When you say untransduced controls you mean without any virus added at all? The only reason I am asking is that depending on your controls you can't say whether the virus is the problem or whether it is the protein you are trying to overexpress (or knockdown). It may be an issue with the specific protein being expressed rather than the virus itself. You need to transduce cells with the empty viral vector as a control to see if the virus is what is killing your cells. If these cells survive then this suggests it is the protein you are overexpressing/knocking out that is toxic to the cells.
Hi
What cells are you trying to transduce, I agree with the previous comment, certain cells particularly primary cells maybe sensitive to high titres, also try without the polybrene, 24hrs is quite short to be getting a transgene effect in your cells, Regards
Hi!
I agree with previous the answers.I may add that if the cells you are trying to transduce are human, they require even lower titer in respect to murine cells! If your titer is too high, culture start suffering /dying usually shortly, exactly in the time you mentioned(24 hours).
Agree with all the above troubleshooting - just one more to add: After harvesting the virus, give a quick and short spin to precipitate any invisible debris from the supernatant followed by a filtration step through a syringe fitted with a 0.45 micron filter to make sure that toxic proteins are not carried over to your target cells for transduction. You have not mentioned whether you use the supe directly or ultracentrifuge it to make concentrated stocks. IF you do the latter, it is always advisable to resuspend the viral pellet in PBS rather than medium. Best of luck.
Dear Ahmet,
Neomycin resistance gene is not a marker, it is for selection of infected cells from non-infected (transduced) ones. And it should be used 24 hours after transduction. If you see that culture is dying at the moment you should add anti-eukaryotic antibiotic it means toxicity started before 24 hours. This cannot be due to toxicity of target protein - too early, lentiviral production takes at least 2-3 days before it achieves a significant level in media and longer before it becomes toxic with intracellular expression. It doesn't mean that you can work without control - empty virus transduction. But it means transduction conditions are toxic - virus was not diluted to appropriate titre, polybrene was toxic for this cell culture, sterility was not perfect, toxic proteins from HEK media - less likely, etc.
Hi,
I agree with all above comments and troubleshootings. But one more notice; Antibiotic tolerance of different cells are various. So please check the suitable neomycin concentration for your cell lines and do some test before lentivirus transduction. In my experience, toxicity of lentivirus is not the major problem.
Hi,
agree with the comments above. What kind of cells are your recipient cells?
Suggestion:
1. do serial dilution of virus soup you´re using for infection as recommended by Al
2. start with G418 selection not earlier than 48hrs after infection
Dear Juraj,
48 hours to start selection of transduced clones is possible for cultures with low proleferative potential with half-double time 36-42 hours - insulin-dependend cells, for example. If you start selection pressure for fast-proliferating culture 24 hours later when it achieved complete monolayer/suturated suspension the sensitivity of its cells passed their log phase long ago to G418 (puromicin, hygromicin, etc.) will be already different, much lower than 24 hours after transduction. The result will be respective - more cells will survive the selection pressure but less of them will be with transferred gene expression. That is why MTT assay for in vitro toxicity/cell survival curves which gives IC50 for tested medicine/agent is always being performed 24 hours after the passage. One can shift it 4-6 hours depending on the culture half-double period, but longer shifts simply will bring false results, maybe 200-400 percent different from what you will later see in vivo for ID50.
Cheers,
E.F.
Hi Ahmet,
What is the concentration of the virus? If it is unconcentrated virus than the problem could be the media and serum used for the HEK cells is not compatible with cells you transduced (some cells are extremely sensitive to changes in media conditions). This could be determined by simply mimicking the transduction conditions on the cells (use the media from HEK cells and polybrene, no virus) and see how the cells do. If you used concentrated virus than the problem is the problem could be toxicity due to the higher concentration of VSVg and debris in the concentrated viral supernatant. Centrifuging out the debris right before transducing the cells can help (just a quick spin in a minifuge and then take the supernatant). Also, try titrating the virus, you might find a lower concentration of virus will work much better (your cells will still be transduced but with less hits per cell, so the cells are less likely to die). You can also try doing a complete media change on the cells 3hrs after you add the virus….this could help with unconcentrated (sensitivity to a different media) and concentrated virus (sensitivity to VSVg and concentrated debris). Just remember the most important rule for a successful transduction is to keep the cells happy. Hope this helps….Good Luck.
Karen
PS. If the cells are sensitive to polybrene use protamine sulfate (titers are the same or a tiny bit lower …but cells particularly stem cells are much happier with protamine sulfate).
Hi Ahmet,
Virus toxicity after transduction may be caused by the protein you are trying to express, either the cells you are transducing don’t regularly express that protein or the expression of the protein at high levels is a problem. You can try transducing different cells, some cell types may not mind the protein as much as others. You could try titering the virus to determine if there is a point at which you can express the protein without killing the cells. Other options are to use a less toxic form of the protein or to use a switch type mechanism (or a repressed promoter) in which the protein is not expressed until you turn it on.
Hope this helps,
Karen
Agree with the old posts, it could be due to the nature of your transgene.
Another parameter to check is the optimale concentration of G418 to use because it is not always the same for all the cell lines.
When we do Neo transductions with a new a cell line, or even with a new batch of G418, we realize a range of different concentrations and select the lowest concentration where all the cells die (generally between 0.8-1.7mg/ml).
Hope this help you,
Best
Nicolas
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