6 replies to this topic

[Ambinlab]

Dear All,

Finally I could package 5 plasmids into a tet-on inducible lentivirus. I tried to concentrate the supernatant from 293T transfected with 5 plasmids using ultracentrifugation. I resuspended the pellet into 100ul media and use half of it to infect HEK293s to see if there is any virus. Apparently there was no virus! So I used 11ml of the supernatant (which was supposed to contain low titer virus) and put it on another "clean" plate of 293T. The next day, I could see some cells expressing GFP (my vector has GFP expressed constitutively), but the majority of them were either sick or were already floating!! So that means my lentivirus induces death for the cells?
I am wondering if anybody had experienced cell death upon virus infection on 293T. If no, then that is only my virus, which means it is of no use (((((
I cloned a tumor suppressor on this lentivirus but because it is inducible I doubt that the death is because of my transgene. I am just worried that the lentivirus is toxic for the cells.

[drwho]

just a general comment, I do not have specific experience with viruses in HEK cells.  However all viruses will be toxic to cells if there are enough of them!  Do you know  the concentration you viruses so you can check your MOI (multiple of infection).  If your MOI is very high you will get non specific death due to large numbers of viruses clogging up the cells.

If you do not know the virus number, try doing various dilutions of your stock virus and see if your cells are more happy.

Hope this helps

[Ambinlab]

drwho

thanks for the reply. However I donot think that would be the case as I used unconcentrated virus and the sup should not have many viruses. I tried it again, and now even my 293Ts could not get infected!!! (ie no cells with expression of GFP)

Have you ever packaged lentivirus with 5 plasmids? What ratio of these plasmids will you suggest?

[drwho]

No sorry I have not done anything like that, I have only done rather simple shRNA plasmid packaging.  This was a very long time ago we tend to buy particles in now!

Could you go into a bit more detail, about exactly what you have done and I will see if I can spot any issues.  But I can not promise anything

[Ambinlab]

drwho,

thanks for offering help. I have moved one step forward and now I can see some cells expressing GFP. However, it seems to be autofluorescence cuz the same cells glow red too!!!!!

Is there any way to get rid of autofluorescence so that I can figure out which cells are really expressing my virus? I used unconcentrated virus so even if I can find very few cells expressing GFP, I can further concentrate the virus and get the higher efficiency.

Thanks

[zodiac1505]

I have posted some info in one of your other posts. When you use the sups to infect 293 cells, the viral titers might be too low for infection. Since you didn't see any green cells even with your ultra centrifuged and concentrated samples, I am thinking that your viral production was too low. You might want to try out different transfection reagents as they vary in their efficiency.

As I mentioned in my other post, Fugene HD works really well and we used to get really good titer viral preps (1x10e8 since we were using them to make transgenic mice). When you are doing in vitro infections, the viral titers don't have to be that high.

Still you should do a titration once you have the viral sample to figure out the number of particles you have. For this you plate out 293 cells in a 6 well plate (around 400000 cells per well) and infect them the next day with 1 micro liter, 1:10 and 1:100 dilutions of your prep. You can then count the number of green cells 2 days later under a microscope or do FACS to figure out the viral titer.

We have never had problems with cells dying from the virus unless it is too concentrated just like drwho said.

[drwho]

This may be a typo you said you are seeing red fluorescence?  This may be really simple but are you using the right filters on your microscope.  If you are using GFP (Green fluroescent protein) you should not be seeing red as you mentioned above.  

GFP excites at ~395nm and emits at 509 just double check this