Hi all,
I have been trying to produce viral particles containing pLKO-tet-on plasmid in HEK cells. I am using Addgene's 3rd generation packaging system (pMDLg/pRRE, pRSV-Rev, pMD2.G) I followed exactly the same protocol which was mentioned in Clontech. Instead of Fugene, I used PEI in 1:3 ratios (12ug plasmid: 36 ul PEI). However I don't have any viral packaging. Because after puromycine selection, all my cells are dying. I will be really grateful if you could give me some suggestions.
Viral packaging is only one of several things that could go wrong. Note however that it is possible that route of delivery (different transfection reagents etc..) may affect your packaging. We did at one point use some other Fugene (Fugene HD I think) and failed to obtain functional viral particles as assessed by our inability to obtain puro resistant cells (with PLVX system). Switching back to Fugene 6 apparently fixed the problem (n=1). Note that we didn't do all the controls (were 293FT properly transfected etc...), so take this with a grain of salt.
Good luck!
Dear Sebastien,
Thank you so much for your answer! I have a GFP control and all the cells are green after transfection. Based on this, I could tell that transfection itself is working. I tried two different pLKO-shRNA plasmids, they all worked fine. Whenever I have inducible plasmid, I am having problems.
Hi Aysegul,
Good work including the GFP control for the transfection. Also, the fact that you are able to confer puro-resistance using the other plasmids indicates that it's probably not an issue with overdosing the cells.
I can't say for sure what problem you are having exactly, however, I can hopefully give some advice based on my own foray into using inducible RNAi from an all-in-one lentiviral plasmid.
One thing to note about these all-in-one inducible lentiviral vectors is that they are quite large (usually over 10kb). I'm actually using a vector right now that's 13kb, quite monstrous. While many people don't seem to have trouble getting decent lentiviral titer with 10kb vectors, I definitely noticed a difference in the transduction efficiency when I tried making particles from these larger vectors compared to the old pLKO.1-TRC vector, which is only 7kb or so. Basically, while your transfection is clearly working, you might not be getting great lentiviral titers.
To overcome that problem in my lab, I just took a brute force approach and transfected more 293T cells in hopes of getting more virus and transfected a smaller number of target cells. However, before you try something like that, I would at least consider the other possibility below.
One other thing you might try is waiting for 48 hours (instead of the typical 24hrs) after transduction before adding puromycin to the cells. I am not sure if this will be reproducible in your lab, but in our lab we have found that many more of the cells survive selection if we give them an extra day to express the puromycin.
I notice, though, that you say "all" your cells are dying upon puro selection; do you not notice any resistant cells at all after a few days?
Hope this helps.
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