Hi,
I have been trying to transduce A549 and Calu-3 cells using the Lenti-X Tet-One inducible expression system (TakaraBio). My gene of interest is inserted in a pLVX-TetOne-Puro plasmid and has been validated by transfection and Western blot.
After several steps of optimization, I finally succeeded to select transduced cell lines using 4ug/mL puromycin. However, validating the induced expression of my proteins-of-interest (adding 0.5 ug/mL Doxycyline and incubating 48 hours) from the transduced cell lines was not successful by western blot.
How can it be that I am able to select cells with puromycin (I had a control with untransduced cells which all died) but can't induce expression of the proteins-of-interest? I'm using normal FBS and know that it can interfere with inducible systems, but rather in terms of leading to basal expression with no doxy....
Epigenetic silencing might be an issue. I recommend reading Stepanenko & Heng 2017 (10.1016/j.mrrev.2017.05.002) and associated literature. We also touched on some of these topics in Lungu-Mitea & Lundqvist 2020 (10.1007/s00204-020-02783-6).
Best regards
Hi, I would like to do a PCR detecting the DNA of your interested gene in those selected transduced cell lines.
Note: I guess "My gene of interest is inserted in a pLVX-TetOne-Puro plasmid and has been validated by transfection and Western blot" means protein expression of your inserted gene could be induced by adding 0.5 ug/mL Doxycyline.
It's possible that the gene expression is very low which can not detected by WB but enough for drug selection. I was also stuck with similar problems.
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