Biological assays using coded RNA reporters

• US 6,977,152 B2
• Filed: 09/09/2002
• Issued: 12/20/2005
• Est. Priority Date: 09/07/2001
• Status: Expired due to Fees

• Current Assignee: Millipore Corporation
• Original Assignee: Vitra Bioscience, Inc.
• Inventors: Beske, Oren E., Foulkes, J. Gordon
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US10/238,914
• Publication Number:
  US 20030082608A1
• Time in Patent Office: 1,198 Days
• Field of Search: 435/6, 536/23.1, 536/24.3
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

Systems for multiplexed analysis of gene expression using coded RNA reporters. The coded RNA reporters are expressed from a library of reporter genes, with each reporter gene directing expression of a distinguishable coded RNA reporter. Each RNA reporter thus reports activity of a corresponding reporter gene. The distinguishable reporter RNA species allow otherwise similar reporter genes to be functionally linked to different effectors within a cell reporter library. Multiplexed analysis of reporter gene activity is carried out by measuring the level of each RNA reporter within the cell reporter library, thus providing an ability to analyze the effect of a biological modulator on many effectors.

Claims

1. A method for multiplexed analysis of reporter gene activity, comprising:

introducing a first reporter gene into a first cell population;

introducing a second reporter gene into a second cell population, wherein the first and second reporter genes express distinguishable first and second RNA reporters, respectively; and

measuring the levels of the first and second RNA reporters associated with the first and second cell populations, respectively.

2. The method of claim 1, further comprising isolating the first and second RNA reporters as a mixture from the first and second cell populations.

3. The method of claim 2, wherein the first and second cell populations are combined before isolating the first and second RNA reporters.

4. The method of claim 2, further comprising incubating the first and second cell populations prior to the steps of isolating and measuring to allow transcription of the first and second reporter genes.

5. The method of claim 1, wherein the first cell population expresses an exogenous first effector, and the second cell population expresses an exogenous second effector.

6. The method of claim 5, wherein the first and second effectors are both receptor proteins.

7. The method of claim 6, wherein the first and second effectors are both membrane-associated receptor proteins.

8. The method of claim 7, wherein the first and second effectors are both G-protein coupled receptor proteins.

9. The method of claim 6, wherein the first and second effectors are both nuclear receptor proteins.

10. The method of claim 6, wherein the first and second effectors are both receptor kinases.

11. The method of claim 1, wherein the first and second reporter RNAs have a common region and a distinguishing region.

12. The method of claim 11, wherein the distinguishing region includes between about 30 and 70 nucleotides.

13. The method of claim 1, wherein the first and second reporter genes have at least substantially identical regulatory regions.

14. The method of claim 1, wherein the first and second reporter genes have at least substantially different regulatory regions.

15. The method of claim 1, wherein the step of measuring the level of RNA reporters is carried out by hybridization to identifier sequences that include polynucleotide regions that are at least substantially complementary or at least substantially identical to the RNA reporters.

16. The method of claim 15, wherein at least some of the identifier sequences are associated with coded carriers in a nonpositional array.

17. The method of claim 15, further comprising labeling the isolated RNA reporters or a complementary transcript with a fluorescent tag, prior to the step of measuring.

18. The method of claim 17, further comprising illuminating the isolated RNA reporters or the complementary transcript with light capable of inducing fluorescence emission from the fluorescent tag, following the step of labeling.

19. The method of claim 1, further comprising exposing the first and second cell populations to a biological modulator, prior to the steps of isolating and measuring.

20. The method of claim 1, wherein the first and second reporter genes include distinguishing code regions having the same length but different nucleotide sequences.

21. The method of claim 1, further comprising introducing at least one small interfering RNA (siRNA) into at least one of the first and second cell populations, prior to the step of measuring.

22. The method of claim 1, further comprising:

introducing a third reporter gene into a third cell population, wherein the first, second, and third reporter genes express distinguishable first, second, and third RNA reporters, respectively; and

measuring the level of the first, second, and third RNA reporters associated with the first, second, and third cell populations, respectively.