Multiplexed analysis of cellular responses using endogenous reporter genes

• US 20030170744A1
• Filed: 03/05/2003
• Published: 09/11/2003
• Est. Priority Date: 03/05/2002
• Status: Abandoned Application

• Current Assignee: Millipore Corporation
• Original Assignee: Millipore Corporation
• Inventors: Beske, Oren E.
• Application Number:
  US10/382,796
• Publication Number:
  US 20030170744A1
• Time in Patent Office: Days
• US Class Current: 435/7.2
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/6875 Nucleoproteins
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Abstract

System, including methods and compositions, for multiplexed analysis of cellular responses using endogenous, response-selective reporter genes.

Claims

1. A method of performing a multiplexed experiment on cells, the cells including an endogenous reporter gene for each of a plurality of responses, the endogenous reporter gene having a level of expression that changes substantially for only one of the responses, the method comprising:

exposing the cells to a candidate modulator; and

measuring the level of expression of the endogenous reporter gene for each of the responses to determine if the candidate modulator changes the level, thereby relating the candidate modulator to each of the responses.

2. The method of claim 1, wherein the cells include at least two different cell types, at least one of the responses being at least substantially restricted to less than all of the at least two different cell types.

3. The method of claim 2, each response being at least substantially restricted to one of the cell types.

4. The method of claim 1, wherein the cells includes primary cells.

5. The method of claim 1, wherein the cells express an exogenous receptor protein.

6. The method of claim 5, where the exogenous receptor protein is selected from the group consisting of cell-surface receptors and nuclear hormone receptors.

7. The method of claim 6, wherein the exogenous receptor protein is a G-protein coupled receptor.

8. The method of claim 1, wherein each of the responses is produced by contacting the cells with a different ligand.

9. The method of claim 8, wherein the different ligand binds specifically to a cell-surface receptor or a nuclear receptor.

10. The method of claim 1, further comprising the step of identifying the endogenous reporter gene for each response, before the step of exposing, by separately producing each response in a cell population.

11. The method of claim 10, wherein the step of identifying includes screening candidate genes using microarray hybridization.

12. The method of claim 1, wherein each response increases the level of expression of the corresponding reporter gene.

13. The method of claim 1, the endogenous reporter gene for each response being a plurality of genes.

14. The method of claim 1, wherein the step of measuring comprises

isolating RNA from the cells;

adding nucleic acid probes; and

treating the RNA and probes with a nuclease that degrades single-stranded RNA.

15. The method of claim 14, wherein the step of measuring includes hybridizing the treated RNA and probes to nucleic acids disposed on a solid support.

16. The method of claim 1, wherein the steps of exposing and measuring are performed a plurality of times in different wells of a microplate, the candidate modulator being different each of the times.

17. A method of performing a multiplexed experiment on cells, the cells including an endogenous reporter gene for each of a plurality of responses to different ligands, the endogenous reporter gene having a level of expression that changes substantially for only one of the responses, the method comprising:

exposing the cells to a candidate modulator; and

measuring the level of expression of the endogenous reporter gene for each of the responses to determine if the candidate modulator changes the level substantially, thereby relating the candidate modulator to each of the responses.

18. The method of claim 17, wherein at least a portion of the step of exposing is conducted while all of the different ligands are present.

19. The method of claim 17, each of the different ligands binding to at least one of a nuclear hormone receptor and a G-protein coupled receptor.