In vivo reporter system
A construct system for expressing a reporter protein, as well as a transgenic animal and a screening method employing the same, are provided. In certain embodiments, the construct system is a three component system in which expression of a reporter protein from a reporter construct is induced by a transcription factor that is produced using two other constructs, each producing a different part of the transcription factor. The parts of the transcription factor are ligated together. Expression of the reporter only occurs in tissues in which both of the parts of the transcription factor are produced.
1. A construct system for expressing a reporter protein, comprising:
a) a reporter construct comprising:
i. an inducible promoter that is activated by a transcription factor; and
ii. a coding sequence encoding a reporter protein, wherein said coding sequence is in operable linkage with said inducible promoter;
b) a first transcription factor construct comprising:
i. a first tissue-restricted promoter; and
ii. a coding sequence encoding a first fusion protein comprising a first portion of a transcription factor and a first subunit of a split intein, wherein said coding sequence is in operable linkage with said first tissue-restricted promoter; and
c) a second transcription factor construct comprising:
i. a second tissue-restricted promoter; and
ii. a coding sequence encoding a second fusion protein comprising a second portion of said transcription factor and a second subunit of said split intein, wherein the second subunit of said split intein specifically binds to the first subunit of the split intein and said coding sequence is in operable linkage with said second tissue-restricted promoter;
wherein the interaction between the first and second fusion proteins encoded by the first and second transcription factor constructs is solely mediated by the first subunit of the split intein and the second subunit of the split intein,
wherein the first and second tissue-restricted promoters are different and wherein expression of said first and second fusion proteins in a cell results in ligation, by a protein-splicing reaction mediated by said first and second subunits of said split intein, of said first and second portions of said transcription factor to produce said transcription factor and provides expression of the reporter protein only in tissues in which expression of the promoters overlap.
2. The construct system of claim 1 , wherein said system provides for expression of said transcription factor only in regions in which expression of said promoters overlaps.
3. The construct system of claim 1 , wherein said transcription factor has a portion of the GAL4, VP16 or the tetracycline activator.
4. The construct system of claim 1 , wherein the promoter that is activated by said transcription factor comprises at least one binding site for GAL4 or the tetracycline activator.
5. The construct system of claim 1 , wherein at least two of said constructs are present on the same vector.
6. The construct system of claim 1 , wherein the constructs are present on different vectors.
7. The construct system of claim 1 , wherein the reporter protein is optically detectable.
8. The construct system of claim 1 , wherein the reporter protein comprises a luciferase.
9. The construct system of claim 1 , wherein the reporter protein comprises a fluorescent protein.
10. An isolated cell or a cell of a non-human organism comprising the constructs system of claim 1 .
11. The isolated cell or cell of a non-human organism of claim 10 , wherein said cell is an animal cell.
12. The isolated cell or cell of a non-human organism of claim 11 , wherein said cell is a mammalian cell.
13. The isolated cell or cell of a non-human organism of claim 10 , wherein said first and second fusion proteins are expressed in said cell, thereby resulting in the expression of said reporter protein.