I have a Lenti-FasL plasmid (CMV promoter) I bought from GeneCopoeia, and I am trying to produce Lentiviral particles containing the human FasL plasmid.
For that I am using 293FT cells with Lipofectamine 2000 or LTX.
In all of my attempts, the 293FT cells die after a few hours, probably because they express the FasL gene and kill each-other (they naturally express Fas- receptor).
Does any of you has some experience with making these kind of lenti-particles? any insights?
my plasmid is from Genecopia: http://www.genecopoeia.com/product/search/detail.php?prt=1&cid=&key=L0009
Thank you!
If you have a way to assay for FasL production, I would test to see if that's the case for the cell death (as you suspect), and adjust your next plasmid concentration accordingly. I would also test a control with just the transfection reagent, in case it has any cytotoxic effects as well. If it does, then you might want to transition to a 293-specific transfection reagent (https://altogen.com/product/293t17-transfection-reagent-emb-kidney-crl-11268/). Otherwise, I would look at previous studies with FasL (Article Transfection of apoptosis related gene Fas ligand in human h...
) and look at what effects others have noted. It seems like apoptosis is the right effect of the transfection.
Ted Bremmer, thank you for your answer!
My suspicion that the 293 cells are dead because of the FasL is because I see this behavior only when I transfect the cells with my plasmid. This doesn't happen when I add only the trasnfection reagent.
I also know that there is a problem of transfecting these cells with FasL as noted in this paper:
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9614583
However, I did see some papers noting that they succeeded in making these lenti-viral particles in the same method I use.
for example:
http://www.academicjournals.org/journal/AJB/article-abstract/C52021F24032
This is the reason I am looking for some insights from top researchers such as yourself.
From the first paper you mentioned, it seems that 293T cells are Fas receptor positive, so your production of FasL induces apoptosis. From the second paper, it seems that you aren't actually using the same method that they did; they produced the lentiviral particles with three plasmids (pWPLXd, pMD2G, and psPAX2). The plasmid you're using (at least judging from the link http://www.genecopoeia.com/product/search/detail.php?prt=1&cid=&key=L0009 you wrote in your initial question) does not encode the packaging/envelope proteins necessary for lentiviral production, and only encodes the mRNA for FasL. I might not have found all of the information about your plasmid, but check again and see if you're actually making lentiviral particles and not just the Fas ligand.
Hello Ted Bremmer,
Thank you again for your reply.
I am familiar with the fact that the 293 cells have Fas receptor, this is exactly the problem.
As for the packaging plasmids, I add the packaging and envelope plasmids by using ViraPower mix (https://www.thermofisher.com/order/catalog/product/K497500) so this side is covered.
Right now I am working on cloning a lenti-plasmid with a TetOn system in order to induce the FasL gene only in my target cells. This is the only solution I found.
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