I want to control the timing and expression of two genes in the same cell line. I have already introduced one gene (EREG) in a Tet-ON system. To independently control the expression of the other interacting gene, I want it under a separate inducible control. Any suggestions?
Hi Bhuminder,
You can use the ER (Estrogen receptor) system [This is a mutated estrogen receptorwhich does not bind to estrogen, its physiological ligand, but binds with very high affinity to the synthetic ligand 4OHT (4-hydroxytamoxifen) and regulates the activity of its fusion partner in cis in a 4OHT-dependent fashion].
This system is similar to Tet-ON (where you use Tetracycline or Doxycycline for induction) the only difference is in TO system the expression will be induced upon treatment with Tet or Dox but in ER system the protein will be there but will become active only upon treatment with 4-OHT.
Good luck
Ram
Thanks Jonas, will it work for eukaryotic system as well? Have you tried it for your work?
This one looks interesting, thanks again Jonas. I will read these papers and ask for contact details of author on first paper, if needed.
Hi Bhuminder,
You can use the ER (Estrogen receptor) system [This is a mutated estrogen receptorwhich does not bind to estrogen, its physiological ligand, but binds with very high affinity to the synthetic ligand 4OHT (4-hydroxytamoxifen) and regulates the activity of its fusion partner in cis in a 4OHT-dependent fashion].
This system is similar to Tet-ON (where you use Tetracycline or Doxycycline for induction) the only difference is in TO system the expression will be induced upon treatment with Tet or Dox but in ER system the protein will be there but will become active only upon treatment with 4-OHT.
Good luck
Ram
Here is another inducible system to try.
http://www.systembio.com/lentiviral-technology/cumate-switch-vectors/sparq-lentivectors
I agree with Ram. I have used the ER system to induce gene expression in in vivo models and know of others who have used it in vitro who produced great results.
Question for Ram and Claire: is there a true ER inducible (expression) available? I am not looking at activity, and ER fusion seems like regulating the activity and not actual transcriptional upregulation! Thank you for the answers.
I would go with an ER or PR based system. They work well in mice. The problem might be the drug to induce the expression/translocation to the nucleus. If your cell type does react strongly to tamoxifen then the system may not work ver well at all. Many cells may have some form of ER expression and therefore will have some form of response to tamoxifen.
Hello Bhuminder,
You have also focused ultrasounds developped in-vivo on the little animal by Chrit Moonen Laboratory in US then Bordeaux (France) ; it is using hsp70 promoter induced by the beam of focused ultrasound, in other words by heat;
Dear Brian,
Cumate systems looks interesting. Worth trying. Thanks for sharing the information.
Bhumi
I was interested in trying an inducible cumate system as well, Bhuminder. Could you post your experience if you do go ahead with that option?
Dear All,
I would like to ask if anyone tried the cumate system. If yes can you share your opinion in it?
Recent paper on teh Cumate inducible system in vivo
https://ac.els-cdn.com/S0166685117300543/1-s2.0-S0166685117300543-main.pdf?_tid=spdf-d566c6b6-eab2-46a5-b2b4-3a6463ad240c&acdnat=1519839559_66576ace1ed46633a4c806c750c298e0
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