Claims
- 1. A method for analyzing in a cell for the effect on expression of an expression inhibiting nucleic acid, where the nucleic acid interacts with mRNA using an expression construct expressing a fusion protein of the small enzyme donor (ED) fragment of β-galactosidase with a polypeptide, where said expression inhibiting nucleic acid affects the activity of β-galactosidase resulting from said ED forming a functional enzyme with the large enzyme acceptor (EA) fragment of β-galactosidase, said method comprising:
maintaining a cell comprising said expression construct and said expression inhibiting nucleic acid providing said EA to any of said fusion protein produced in said cell to form β-galactosidase, and a β-galactosidase substrate that produces a detectable product; and determining the activity of said functional enzyme by use of said detectable product, whereby the activity of said functional enzyme is related to said effect on expression.
- 2. A method according to claim 1, wherein said effect on expression is the inhibition of expression by a DNA molecule.
- 3. A method according to claim 1, wherein said nucleic acid is an RNA molecule
- 4. A method according to claim 3, wherein said RNA molecule is dsRNA
- 5. A method according to claim 4, wherein said dsRNA is RNAi.
- 6. A method according to claim 1 wherein said cell is grown in the presence of a candidate compound.
- 7. A method according to claim 1, wherein said cell is a mammalian cell.
- 8. A method for analyzing in a cell the effect on expression of an expression inhibiting RNA where the RNA interacts with mRNA using an expression construct expressing a fusion protein of the small enzyme donor (ED) fragment of β-galactosidase with a polypeptide, where said effect on expression affects the activity of said ED in forming a functional enzyme with the large enzyme acceptor (EA) fragment of β-galactosidase, said method comprising:
maintaining a cell comprising said expression construct and said expression inhibiting RNA; providing said EA to any of said fusion protein produced in said cell to form β-galactosidase, and a β-galactosidase substrate that produces a detectable product; and determining the activity of said functional enzyme by use of said detectable product, whereby the activity of said functional enzyme is related to said transcription in said cell.
- 9. A method according to claim 8, wherein said RNA is double stranded RNA
- 10. A method according to claim 9, wherein said RNA is RNAi.
- 11. A method according to claim 8, wherein said expression inhibiting RNA is added to said cell.
- 12. A method according to claim 8, wherein said expression inhibiting RNA is transcribed in said cell.
- 13. A method according to claim 8, wherein said substrate produces a fluorescent product.
- 14. A method according to claim 8, wherein said cell is a cell line.
- 15. A method according to claim 8 wherein said cell is grown in the presence of a candidate compound.
- 16. A method according to claim 8, wherein said cell is lysed prior to said determining and said determining is of said lysate.
- 17. A method according to claim 8 wherein said expression inhibiting RNA inhibits expression of a transcription factor.
- 18. A system for determining in mammalian cells the effect of an expression inhibiting dsRNA on expression of a first protein where the dsRNA interacts with mRNA, employing a fusion protein comprising a β-galactosidase enzyme donor (“ED”) fused to a second protein, where said first and second proteins are related in that the level of expression of said first protein fusion protein are interrelated, said determining comprising measuring the β-galactosidase activity of said fusion protein in the presence of an enzyme acceptor (“EA”) capable of being complemented by said ED of said fusion protein to form a functionally active β-galactosidase enzyme, said system comprising:
(1) a vector comprising a first transcriptional and translational regulatory region functional in said host cell, (2) an ED sequence encoding said ED joined to a multiple cloning site (“mcs”) under the regulation of said transcriptional and translational regulatory region; the same or different vector as (1) comprising a second transcriptional regulatory region functional in said host cell and a gene encoding said inhibiting dsRNA under the regulation of said transcriptional regulatory region; (3) an enzyme acceptor protein; (4) a gene when inserted in said mcs in reading frame with said ED sequence expresses a biologically active protein and an ED capable of complementing said EA; (5) host cells in which said transcriptional and translational region is functional; and (6) substrate for said β-galactosidase enzyme that upon hydrolysis produces a detectable signal.
- 19. A system according to claim 18, wherein said first and second transcriptional regulatory regions have the same transcription factors.
- 20. A system according to claim 19, wherein said first and second transcriptional regulatory regions have different transcription factors.
- 21. A system according to claim 18, wherein said host cell expresses EA.
- 22. A kit for use in a method according to claim 1 comprising: an expression construct of the small enzyme donor fragment of β-galactosidase fused to a protein of interest, an expression inhibiting double stranded RNA for said protein of interest, and at least one of an enzyme acceptor fragment of β-galactosidase or a β-galactosidase substrate producing a detectable product.
Priority Claims (1)
Number |
Date |
Country |
Kind |
PCT/US02/27497 |
Aug 2002 |
WO |
|
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of provisional application 60/316,428, filed Aug. 30, 2001 and 60/353,086, filed Jan. 30, 2002, and a continuation of full application Ser. No. 10/229,747 filed Aug. 27, 2002 and PCT application PCT/US02/27497 filed Aug. 27, 2002 the contents of each of which is incorporated herein by reference.
Provisional Applications (2)
|
Number |
Date |
Country |
|
60353086 |
Jan 2002 |
US |
|
60316428 |
Aug 2001 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
10229747 |
Aug 2002 |
US |
Child |
10702232 |
Nov 2003 |
US |