I have been having trouble getting a published TET-OFF cell culture line to work. I have been finding that the treated and not-treated cells look pretty much the same.
It occurred to me that streptomycin has a somewhat similar mode of action to tetracycline so I was wondering if the penicillin-streptomycin I had added to my culture (for anti-bacterial defense) might be interfering with the experiment?
Hello Andrew.
There have been many similar kinds of questions in RG threads. It seems to work OK in in-vivo system in conditional KO mice. In vitro, FBS may be blamed for the no specific activation of the system. Obviously, lots of antibiotics are used in animal industry to relieve health problems and just promote growth. Trace of antibiotics are even found in human milk samples. Tet-Off system in iv-vitro may be no good unless you order and use verified batches of Tet-free FBS for researches. It is unlikely caused by streptomycin.
Regards.
Tetracyclines are inhibitors of protein synthesis in vivo, but there is no publication establishing the same procedure for streptomycin beyond bacterial protein synthesis. Probably it is not interfering with your experiment.
The TET-OFF system is a Tetracycline-inducible system in which the induction occurs when Tetracycline (in this case it is Doxycyline) is REMOVED from the cell line. Tetracycline is a PKS1-type antibiotic compound which belongs to a chemically different class of compound than Streptomycin which is an aminglycoside antibiotic. Therefore it is highly unlikely that Streptomycin can be used instead of Tetracycline/Doxycycline.
Precisely one of the problems of tetracycline inducible systems is a system that acts as protein synthesis inhibitor.
So this may be an important experimental bias if the researcher is investigating protein synthesis.
Probably most of antibiotics may inhibit RNA or protein synthesis at certain concentration. However, PP and SM has been selected as components of supplements to nearly all culture media for animal and human cells as routine components. It would be doubtful to think that inhibition of protein synthesis by SM may caused Tet-Off system in all groups. Something present in the serum or oozed out of the culture container may mimic the roles of doxycycline via binding to Tet-Off fragment having many copies of elements in it.
Please remember that the antibiotics when used at the recommended concentrations have no effect on the protein biosynthesis of the mammalian system. The TET-OFF and TET-ON induction systems are based on the ability of the Doxycycline (TET analogue) molecule to induce the switching on of your gene of interest; and its role as an inducer is based on its chemical structure not its antibiotic mode of action. So Streptomycin should have no affect on the TET system.
What gene(s) are you switching on in your TET-OFF system? How is it detected?
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