Dear all,
I got these two plasmids from Addgene (#86849 and #138714) and tried to transduce into MDA-MB-231 cell line using the lentivirus. Therefore, I packaged these two plasmids in 293T cells with pMD2.G and pPAX2 and got the lentivirus. And I can see some very strong fluorescence in the 293T cells. However, when I tried to transduce my target cell, it did not show any fluorescence signal. Has anyone faced this problem? Thanks.
I am using 138714 PIP-FUCCI system without any problem. Check your virus preparation.
Di Xia
Hi Dr. Xia. Thank you for your answer.
Yes, I used a control virus. I packaged another plasmid that I have successfully packaged before. And it shows positive cells with green fluorescence when I transduced it into MDA-MB-231 cell line. I also tried to transduce the FUCCI lentivirus solution into 293T cell line but it did not show a significant signal.
Rahul Kumar
Hi Dr. Kumar. Thank you for your feedback.
I used pMD2.G and psPAX2 for packaging. Will that be a problem? May I know the packaging plasmids you used?
Actually, when I packaged this plasmid, it shows a strong fluorescence signal. But when I infected MDA-MB-231 with that lentivirus, it shows almost no signal. When I look at the cells under confocal microscopy, I can see some fluorescence signals and noisy background using really high laser parameters (Master gain 800V, pinhole 1AU, maximum laser power). I wonder if you have a similar observation?
Sounds like the problem was not specific to your cell line. Maybe the virus titer was too low? When you saw some fluorescence signals and noisy background, it could be just background or autofluorescence unless you have a direct comparison with un-transduced cells. You could also do a transient transfection (FUCCI plasmid only) to take a look at the signal in confocal.
Tianyi Wang
I am also using pMD2.G and psPAX2 plasmids for lentivirus packaging in 293T cells. The fluorescence signal in 293T cells suggest that your FUCCI plasmids are working fine, the low to nil signal in your transduction experiment suggest that your virus titer is very low. To rule out cell line specific discrepancy, you can infect 293T cells with your lentiviral suspension along with MDA-MB-231 cells, if you see signals in 293T but not MDA-MB then you can be sure that your virus preparation is good. Another tip is- incubate lentivectors at 30 degree and not 37 degree to prevent recombination of LTRs. Good Luck
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