I transfected GipZ lentiviral shRNA with packaging plasmids psPAX2 and VSVG into 293T cells. I also transfected LentiCRISPRv2GFP (lentiviral based GFP containing CRISPR from Adgene). The transfection efficiency is really good. But when I collected virus and infected into lung cancer stem cells, as well as lung cancer cell lines A549 and H1299, they infection/transduction is almost none. I tried this experiment in different ways: 1) transfecting in 6 wells plates and collecting 2 ml supernatant 2) Transfection in 10 cm plates, collection of supernatant and find the virus titer using qPCR. I had fairly a good virus titer (Ct of standard control is 12-15, Ct my viral lysates: around 22-24, no amplification in negative control) , but still I don't see the infection. To figure this out, I also tried transiently transfect these plasmids without packaging plastids into 293T cells and I could see a subtle knockdown in both ShRNAs and CRISPR transfected 293T cells. That means my plasmids are probably fine. Could you help me figure this out?
Thanks
Rajendra
The difference in cell types could be a major factor.
What's true for one cell type is not necessarily true for another.
Do you know the mycoplasma status of the hard-to-transfect cells?
Mycoplasma contamination is a known inhibitor of transfection.
Thanks Can. I concentrated lentiviral particles from the supernatant and transfected H1299 cells. They are infected now. I still need to determine if there is an enough knockdown. I first determined virus titer and then infected with higher titer.
I have a plan to infect into A549 and also in lung cancer stem cells (derived from the patients). lets see how it goes.
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