I use pWPI plasmid to overexpress two proteins in neurons. I did it once, and everything worked beautiful. But now I am not capable of overexpressing the proteins, even tough I use the same plasmids´ stock as the first time.
I produced the lentivirus by transfecting the plasmids in HEK293FT cells, together with another two for the packaging machinery and the capsid of the viruses. I use CaCl2 for transfection, and I change the media after 6 hours. Then I collect the viruses 24-48 hours after transfection (I also harvest these cells, lise them and do western blot to check for the expression of my protein). Finally I use the media with the viruses to infect other cells.
By western blot I see huge expression of the protein in transfected cells, but none in the infected ones. I use GFP expression to monitor the infection and it is fine, because I see many green cells.
The upsetting point is that I used these plasmids before and they worked very well. Now, I have checked and changed the transfection protocol, the helper plasmids and the rates of infection, and I always get the same result: beautiful transfection with green cells and a lot of protein overexpressed, beautiful infection by GFP expression, but none overexpression of my protein with the viruses.
Could anyone give me some advice? Thanks!
Hi:
I think you could use your virus to infect the 293/293T cells to test whether the virus is ok since the 293 cells are easy to transduce.
Do you do the ultracentrifuge to concentrate the lentivirus, we always do it (from 30ml to 300ul).
of course, you would need to confirm whether the plasmid is ok. redo the midi and seq.
Hi, your Western blots look Good (transfected ones). Protein-GFP expression is positive/fluorescent in 1-2 cells only in the picture shown. The other green cells are not fluorescent, looks like background.
If your target cells are transfectable you don't have to make the virus you can use the transfection directly into the target cells for overexpression i.e. using Lipofectamine 3000. I had a similar problem where HEK293FT cells were perfectly transfectable but the virus never worked (the problem was solved eventually by purchasing a fresh batch of HEK cells). However, in order to move the project forward, I resorted to direct transfection to target cells without the production of the virus and was successful.
It may be that the packaging of this virus failed, and your target gene was not packaged onto the virus. You can try a special viral transfection reagent.
https://transfection.bocsci.com/product/lentiviral-transfection-reagent-338867.html?nid=3731
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