I am currently using an inducible system for gene expression in mice and would like to know how long and at what serum concentrations doxycycline persists in the organism.
Dear Pedro:
How is the doxycycline prepared (do you add sucrose to the water?) and at what concentration is it added to the drinking water?
What is the average consumption per animal?
Depending on the system you are working with Tet-Off versus Tet-On you can expect different kinetics and sensitivity to Dox. Tet-Off generally requires a bit longer time to resume gene expression following withdrawl of Dox, while the Tet-On version generally has a bit better on-off kinetics. There are also different versions of tTA and rtTA that have different sensitivities to dox.
Persistance of doxycycline can vary depending on tissue of interest. We used a simple assay to look at doxycycline levels in rat serum. Basically we took a cell line transduced with a Tet-On GFP reporter and added different dilutions of rat serum. In parallel we added fixed amount of doxycycline to generate a rough standard curve of doxycycline versus GFP expression. This allowed for a rough estimate of serum/plasma dox levels. I believe there are also various methods to quantify dox in serum using HPLC. If you are working with a Tet-Off system the same type of assay could be used.
The best advice I can give is to do a pilot study to give dox in water for a fixed amount of time and then switch back to normal water and check periodically to see when dox levels have dropped to an undectable level (either by the cell based assay or by an HPLC method).
Good luck
Dear Pedro,
I don't know for your specific question but we had a different question. We used a doxycycline system to label progenitor and follow their fate during embryogenesis. So we treated E12.5, E14.5 and E16.5 mice for 2 days and then stop Doxo and see where labelled cells were located in the tissue (at birth). As a control we maintened doxo during the time periods. We could notice strong differences so I would assume that it is quite quickely metabolized.
Hope this could help a bit.
Best
Dear Pedro:
How is the doxycycline prepared (do you add sucrose to the water?) and at what concentration is it added to the drinking water?
What is the average consumption per animal?
Depending on the system you are working with Tet-Off versus Tet-On you can expect different kinetics and sensitivity to Dox. Tet-Off generally requires a bit longer time to resume gene expression following withdrawl of Dox, while the Tet-On version generally has a bit better on-off kinetics. There are also different versions of tTA and rtTA that have different sensitivities to dox.
Persistance of doxycycline can vary depending on tissue of interest. We used a simple assay to look at doxycycline levels in rat serum. Basically we took a cell line transduced with a Tet-On GFP reporter and added different dilutions of rat serum. In parallel we added fixed amount of doxycycline to generate a rough standard curve of doxycycline versus GFP expression. This allowed for a rough estimate of serum/plasma dox levels. I believe there are also various methods to quantify dox in serum using HPLC. If you are working with a Tet-Off system the same type of assay could be used.
The best advice I can give is to do a pilot study to give dox in water for a fixed amount of time and then switch back to normal water and check periodically to see when dox levels have dropped to an undectable level (either by the cell based assay or by an HPLC method).
Good luck
I agree with the notion that persistence of dox depends heavily on dose and tissue type. I don't know about in the drinking water, but I came across a paper a while back in dogs suggesting that a single injection of dox at 10mg/kg showed a serum half life of 12 hours (so "completely" eliminated by ~72-84 hrs). see http://www.ncbi.nlm.nih.gov/pubmed/111939
That being said, using 10X that dose (a single 100mg/kg injection) in mice with a Tet-on lacZ reporter (and looking in the inner ear), I still see reporter activity from this single injection up to (and perhaps later than) 4 weeks after the injection.
Dear Pedro, I just found your question. Might be a bit late, but anyway, it might still be useful for you . We have published the following data (measured by HPLC and Cell culture quantification using a tet-dependent luciferase cell line, the mouse serum was mixed to the culture medium- quite similar to what was mentioned by David Markusic ): "To avoid lethality, we applied a protocol of discontinuous treatment. Mice were given doxycycline for only 2 d and then pure water for 5 d a week before they were induced again. This kind of treatment leads to maximum
serum levels of 504 ng ml–1 (± 68 ng ml–1) doxycycline and complete clearance (o5 ng ml–1) within 24 h of doxycycline deprivation."
For details see: http://www.ncbi.nlm.nih.gov/pubmed/?term=18724376
In a different set of experiments the following data was found: The steady state levels of Dox in the serum of mice, fed with 200µg/ml Dox for one week, were around 580 ng/ml, if only 20µg/ml were used in the drinking water this resulted in 130 ng/ml in the serum. Upon withdrawal of Dox the serum is cleared with a half life time of about 6 hours.
published in: Schönig and Bujard, Series: Transgenic Mouse Methods and Protocols, Methods in Molecular Biology, Vol. 209
Hofker, Marten H.; Deursen, Jan (Eds.) 2002, Humana Press
regards -
kai
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