Inducible Promoters/Repressors for Tissue-Specific Expression? - (Jun/10/2008 )
I keep hearing about Tet-on/off systems, and I'm wondering if something like that will work for me. Basically, the idea is that we have a tissue-specific promoter construct for expressing GFP. However, we want to turn off the expression (i.e. inactivate the promoter) until a certain time. Is this what the Tet-on system is used for? How would this work? Is it just a matter of inserting some repressor DNA sequence in our plasmid, and then bathing cells in tetracycline until we want expression? If I'm way off-base here, I apologize. I hope the essence of my question can be understood. Thanks.
-brightfield-
QUOTE (brightfield @ Jun 10 2008, 07:19 AM)
I keep hearing about Tet-on/off systems, and I'm wondering if something like that will work for me. Basically, the idea is that we have a tissue-specific promoter construct for expressing GFP. However, we want to turn off the expression (i.e. inactivate the promoter) until a certain time. Is this what the Tet-on system is used for? How would this work? Is it just a matter of inserting some repressor DNA sequence in our plasmid, and then bathing cells in tetracycline until we want expression? If I'm way off-base here, I apologize. I hope the essence of my question can be understood. Thanks.
For tet system, you need to insert tetracycline response element (TRE) in your expression vector also containing your promoter.
In addition, you also need to express in your cell/organism tet-on or off transactivator protein.
Then you add or remove tetracycline, respectively, from the medium/feed and you get the expression of your gene under your tissue-specific promoter.
There are other inducible systems with less work such as tamoxifen-inducible, ecdysone-inducible, and a small twist on tet, that is doxycycline-inducible.
-cellcounter-
QUOTE (cellcounter @ Jun 10 2008, 01:38 PM)
QUOTE (brightfield @ Jun 10 2008, 07:19 AM)
I keep hearing about Tet-on/off systems, and I'm wondering if something like that will work for me. Basically, the idea is that we have a tissue-specific promoter construct for expressing GFP. However, we want to turn off the expression (i.e. inactivate the promoter) until a certain time. Is this what the Tet-on system is used for? How would this work? Is it just a matter of inserting some repressor DNA sequence in our plasmid, and then bathing cells in tetracycline until we want expression? If I'm way off-base here, I apologize. I hope the essence of my question can be understood. Thanks.
For tet system, you need to insert tetracycline response element (TRE) in your expression vector also containing your promoter.
In addition, you also need to express in your cell/organism tet-on or off transactivator protein.
Then you add or remove tetracycline, respectively, from the medium/feed and you get the expression of your gene under your tissue-specific promoter.
There are other inducible systems with less work such as tamoxifen-inducible, ecdysone-inducible, and a small twist on tet, that is doxycycline-inducible.
Thanks, for your reply. I think the dox-inducible is the Tet-On system that Clontech sells. It seems like the right choice for me. Would you happen to know if anyone has ever tried this approach - combining inducibilty with a tissue-specific promoter? A cursory pubmed search didn't give me any results. Also, the tech support woman at Clontech said she had never heard of someone doing this. It seems possible, though, that it could work.
-brightfield-
QUOTE (brightfield @ Jun 11 2008, 11:34 AM)
Thanks, for your reply. I think the dox-inducible is the Tet-On system that Clontech sells. It seems like the right choice for me. Would you happen to know if anyone has ever tried this approach - combining inducibilty with a tissue-specific promoter? A cursory pubmed search didn't give me any results. Also, the tech support woman at Clontech said she had never heard of someone doing this. It seems possible, though, that it could work.
Oh, a huge number of inducible-tissue specific expression mice have been made using this system, while I don't know what organism you work on, but it would work on any, in fact better than mice.
See link no.1, and go to page no.9 of this pdf article, there is a schema for this.
http://search.vadlo.com/b/q?sn=158621799&a...cific&rel=0
Edit: And by the way, look up the12 mice in the jax database (link no. 2-3.. upto 12) in Vadlo results.
Also check out some of these journal articles:
http://www.google.com/search?hl=en&q=i...tissue-specific
..
-cellcounter-