Hi Vasco,

If by "on/off" you mean all-or-nothing, then no. In my experience, tetracycline regulated expression is quite sensitive to dose and expression time. There are many such systems, but they broadly fall into two categories: "tet-on," and "tet-off."

- For tet-on systems, higher doses of tetracycline and/or longer treatment periods definitely correlate with higher expression of your inducible gene product. You might also observe weak "leaky" expression depending on the sensitivity of your detection method.

- For tet-off systems, withdrawing tet will indeed lead to maximal expression, but you can apply varying levels of tetracycline to tamp it down.

Also note that, even in clonal cell lines, you can observe some heterogeneity in terms of cell-by-cell expression levels.

We use doxycyline (a longer-lived tetracycline derivative) for our experiments. I'd recommend starting at as low as 1ng/mL in cell culture, and testing doses as high as 2ug/mL. As far as time course, we treat for 1-3 days depending on the cell line and experiment. However, this will always depend on your system, so carefully characterize the induction dynamics in your cells.

Also, it is important to compare cells with your inducible gene, and also cells without the inducible gene, because you'll want to characterize doxycycline-related effects on phenotype/gene expression absent your transgene. Sometimes high doses of doxycycline can be toxic or otherwise influence cell phenotypes.

For additional reading, I recommend this short article from Addgene as a starting point: https://www.addgene.org/tetracycline/

Good luck!

Thanks for the reply Brian! That was really helpful!

And exaclty, I meant "all or nothing". A co-worker of mine had some brief past experience with the tet-on system and described it as a "all or nothing" system. I'll try to recover her data and cells to have a closer look at.

Btw, most tet systems currently use doxy correct? Is this due to longer half-lives or to higher affinities that correlate with lower doses of doxy vs tet to achieve the same expression levels?

Again, thanks!

Glad to help. Perhaps your colleague was using a system I'm unfamiliar with. It would certainly be worthwhile to figure it out, but generally tet/dox-dependent systems yield graded responses to the drug.

Doxycycline is a preferred tetracycline derivative because it is resistant to acidic degradation yet still biologically active (1,2; this was discovered in the late 1950s, and is true for all 6-deoxy tetracycline derivatives). It has generally been the most widely adopted tet-derivative for cell biology/genetic engineering, and appears more potent in cell culture applications than tetracycline (3).

Here are a a couple of references on the matter:

1. Stephens et al., J. Am. Chem. Soc., 1958, 80 (19), pp 5324–5325

2. Stephens et al., Journal of the American Chemical Society 85.17 (1963): 2643-2652

3. Krueger et al., BioTechniques, 2004, 37(4):546, 548, 550

Cheers

Thank you so much Brian! :)

In our lab, we have been using the Tetracycline-regulated expression system for many years. We optimized the ‘Tet on’ system to obtain a tightly regulated Tet-inducible expression system, which has been tested in several different experimental studies (Arthritis & Rheumatism, 2005, Vol. 52(9), p 2906 – 2916; Arthritis Research and Therapy. Jan. 2007 9(1) R7; Yousaf & Gould, The Tetracycline responsive system, Methods Mol Biol 2017, 1651, 159-172, https://www.ncbi.nlm.nih.gov/pubmed/28801906). We routinely used our usual tissue culture fetal bovine serum in Tetracycline-regulated expression studies without any problem. In the absence of doxycycline, we did not observe background expression (leaky expression) of the target gene. Increasing concentrations of doxycycline led to a dose-dependent increase in expression of the target protein.