Doxycycline, a modified form of tetracycline is regularly used to induce gene expression or suppress gene expression in xenografted cells transfected with TET-ON or TET-OFF systems. It is usually provided by drinking water. Can anyone help me understand how will it reach the xenografted cells or how long will it take?
Actually curious to know how it's efficiency might be affected if the cells are injected directly into the mammary ducts. (mammary intra-ductal xenograft model)
When doxycycline is provided orally, it is absorbed from the upper small intestines and rapidly distributed to almost all tissues and body fluids. It will rich high concentrations in kidneys, liver, lungs, spleen and even in bone tissue. Likewise, it will also be distributed into tumor xenografts through the supporting tumor vasculature. Doxycycline delivered into tumor tissue will first appear in the tumor interstitial fluid (liquid component of the space between tumor cells) and from there it will penetrate through cell membranes into tumor cells (this penetration is well supported, for example, by the activity of doxycycline against strictly intracellular microorganisms).
The question about the time from doxycycline ingestion to the observable effect on the gene expression includes two components: pharmacokinetics (how long will it take for doxycycline to get to tumor cells) and kinetics of induction/repression (time needed from intracellular delivery to the effect on the gene expression). The total time depends on tissue type and it has been reported to take 4-24 hours for most tissues (for reference see Kistner A. et al. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. PNAS 1996; 93: 10933-38).
Thank you very much Roman.
So, if you were to guess what will be the possible effect on cells injected within intact mice mammary ducts what would be your answer?
Thank you again.
Hello,
I performed xenograft experiment on TET-on cells and I orally administrated doxycycline to the mice. I injected the cells subcutaneously and the exogenous gene was strongly induced.
Doxycycline is well distributed in the mouse tissues to reach the injected cells. The time as clearly explained by Roman will depend on different factors.
Good luck,
Ivano
You are welcome Sandipto. In very early stages the implanting cells will be "sitting" in the lumen of lactiferous ducts or lactiferous sinuses. At that early stage of tumor xenograft development, the cells will depend on nutrients supplied by diffusion from the lumen environment. We know that doxycycline is secreted into breast milk; therefore we can reasonably expect the presence of doxycycline in small amounts of ductal fluid in non-lactating animals as well. As tumors grow and develop their own vasculature, they will receive doxycycline from blood delivered to tumor tissue.
Hi Sandipto
Just want to add to the above, it might be worth checking the MDR1 status of your zenograft cells. I think that doxycycline might be a substrate for P-gp and while enough might be getting into the mouse it could potentially not accumulate in your zenograft cells.
Dear Ivano, I'm using a Tet on system in a xenograft model. I'd be interested to hear how much doxy you were giving orally- &was it by garage or in drinking water? I guess the latter would give some variability
Another factor to consider in Tet-on systems is the cell line you're using. We've done Tet-on systems on several occasions (http://altogenlabs.com/rna-interference-rnai-services/generation-of-tet-inducible-knockdown-cell-lines/) but their responsiveness differs, so as Roman said, there will be pharmakinetic and repression factors affecting the overall result.
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