I usually collect 48h and 72h after transfection. It also helps to do an AM and a PM collection of a half-volume each to prevent viral re-infection of the producer cells. In other words, if you're collecting 8mL at 48h and 72h, try collecting 4mL at 36h, 48h, 60h, and 72h (obviously within reason, I'm not suggesting you come in to the lab in the middle of the night; just one collection when you get in and one when you leave for the day).

What transfection reagent do you use? We use Trans-IT LT1 from Mirus. Expensive but it works really well. We use a 10:9:1 plasmid ratio for that.

In my opinion, you need to troubleshoot with different packaging/envelope plasmids.

Your 293FT cells were GFP positive after 24 hours transfection. This indicates that your expression plasmid is fine but it doesn't tell you if viral particles are made successfully and this could be improved by the choice of packaging/envelope plasmids.

What you did is alright! (transfection ratio, collection method/time points)

Have you used the sup to transduce other cell lines (293FT or 3T3) which are known to be easily transduced in case the problem is due to your desired cell line?

Thank you. 

I'm currently trying with two new plasmids (packaging and envelope). But it seems that it still not working (I tried 1:1, 1:5, 1:10, 1:20 with fresh media/virus containing media...)

I will try to infect a 293FT cells also, but even if my cells are difficult to infect I think I still should be able to observe even just a small fluorescence in some cell, but nothing. 

Let me know if the sup works on 293FT cells. Some cells do require double/triple spinfection or use of fibronectin.

What percentage of FBS supplemented medium you use to culture 293FT cells after transfection? We replace the medium 12-16 hours post transfection with 2% (instead of 10%) FBS supplemented DMEM medium to collect viral particles. We found high percentage of FBS affect the secretion of viral particles by transfected 293FT cells.

I'm late to answer but I found where the problem came from couple of days right after. So to avoid someone facing the same problem here is the answer:

The commercially available shRNA I used wasn't a classical construct and the GFP gene wasn't located between the 5'- 3' but outside. So with this type of construct I should expect fluorescence in 293T cells (which I had) but not in the cells of interest after infection. The only way to know if the infection worked is to run a western blot.

I hope it'll help...

Sylvain Ferrandon I am new in lentivirus infection and having the same problem with a lentivirus from a paper (figure 1 of this paper). Article Development of an All-in-One Inducible Lentiviral Vector for...

What do you think could be the reason? thank you.

Hi everyone,

On similar lines to the original question in this thread by Sylvain, we are facing similar issues - our HEK293T cells after primary transfection for lentivirus production are positive for fluorescence but when we take supernatant from these cells to transduce our MCF7 cells, they show no expression. Annoyingly, our system used to work really well until quite recently but stopped working all of a sudden. To troubleshoot, we have tried different generation (II and III) packaging, different passage HEK293T cells, different test fluorescent proteins (GFP, mVenus, mCherry) and sequenced the transfer plasmid for our gene of interest. We have tried everything we can think of but our failed system refuse to buzz. This is really frustrating as the whole pipeline was working before and now it has changed its mind. We feel like there is some issue with virus production but not sure why it happened, how to check for it and how to sort this problem. We are unable to make any retrovirus which was a norm in our lab.

Any suggestions would be really helpful.

Thanks so much in advance.

Hello,

-What I would advice you first would be to either to sequence all the different plasmids (not only the tranfer plasmid) OR to directly make yourself new stocks of plasmids by doing maxi/midi prep to start with fresh material.

-It sounds like you tried it already but just to make sure I would thaw old frozen vials of 293T cells from the time that it was working.

-Maybe you also can buy new stock of polybrene, in case you're still using the same one.

-Have you tried to infect 293T cells instead of MCF7. They're usually a good control for infection as they have an extreme high rate of protein expression.

Are you sure your fluorescence microscope works properly? (I know sounds goofy but...)

Hi Sylvain,

we have already tried everything you had suggested and still having the same issue. But thanks any way.

Best wishes,

Nitin

Hi,

We have had several issues in our lab at different points in time. One issue was that a mutation was acquired in the packaging plasmid that reduced packing efficiency for one lab member. Another issue that I think is more interesting but harder to control for: the 293T cells themselves. We have found vastly different efficiencies between different 293T lines. I suggest getting lines from as many different sources as you can and compare them in tandem.

Good luck,

Dave

Hi Nitin!!

We are having the same problems you describe above. Did you finally found a solution?

Thank you,

Catalina