High throughput assay for identification of gene expression modifiers

• US 20020164636A1
• Filed: 05/06/2002
• Published: 11/07/2002
• Est. Priority Date: 05/04/2001
• Status: Active Grant

• Current Assignee: Health Research Incorporated, The Research Foundation for The State University of New York (State University of New York)
• Original Assignee: The Research Foundation for The State University of New York (State University of New York)
• Inventors: Hangauer, David G., Stewart, Carleton C., Pruitt, Steven C., Mielnicki, Lawrence Mark
• Granted Patent: US 7,083,919 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

The present invention provides a method for screening of a large number of compounds for their ability to modulate the expression of genes. The method uses gene trap technology and comprises the steps of transfecting a population of cells with a gene-trap vector, sorting cells according to their level of fluorescence, distributing sorted cells into pools and expanding the pools to obtain a sufficient number of cells representing each trapped gene to permit distinction of the effect of a test compound over controls, exposing the cells to the test compounds and identifying compounds which alter the fluorescence distribution pattern of cells using FACS analysis.

Claims

1. A method of screening a plurality of test compounds to identify one or more compounds that affect expression of at least one trapped gene comprising the steps of:

a) providing a gene-trap vector encoding a fluorescent reporter protein, wherein the fluorescent reporter protein directly or indirectly produces fluorescence;

b) stably transfecting a population of cells with the gene-trap vector, wherein expression of the reporter protein is indicative of the expression of the trapped gene;

c) sorting the population of stably transfected cells by FACS into groups according to expressed levels of reporter protein;

d) distributing cells from each group into one or more pools, wherein each pool represents more than one trapped gene;

e) expanding the cells from each pool, wherein the expansion is such that a sufficient number of cells representing each trapped gene are produced to permit distinction of the effect of a test compound on the expression of the trapped gene over a control;

f) placing the expanded cells into individual wells;

g) incubating the wells from each pool in the absence or presence of a plurality of test compounds to generate control wells and test wells respectively, wherein the plurality of test compounds are added to the same wells well or separate wells;

h) conducting FACS analysis on the test wells and control wells to generate fluorescence distribution patterns for the cells in the test wells and control wells;

i) comparing the fluorescence distribution patterns of each test well with the control well; and

j) identifying the test well in which the fluorescence distribution pattern differs from the control well, wherein identification of a different fluorescence distribution pattern is indicative of a test compound affecting the expression of one or more trapped gene represented in the test well.

2. The method of claim 1, wherein the reporter protein is the green fluorescent protein.

3. The method of claim 1, wherein the reporter protein is the enhanced green fluorescent protein.

4. The method of claim 1, wherein the number of cells in each group represents about 500 trapped genes.

5. The method of claim 1, wherein the cells from each pool are expanded at least five fold.

6. The method of claim 5, wherein the cells from each pool are expanded at least ten fold.

7. The method of claim 1, further comprising the step of cloning and sequencing the trapped gene.

8. The method of claim 1, wherein sorting parameters for FACS are adjusted to accommodate for heterogeneity in fluorescence of the reporter protein due to cell cycling.

9. The method of claim 1, wherein the cell stream velocity of cells in the flow cytometer is less than 25 meters per second.

10. The method of claim 9, wherein the cell stream velocity of cells in the flow cytometer is less than 15 meters per second.

11. The method of claim 10, wherein the cell stream velocity of cells in the flow cytometer is about 5 meters per second.

12. The method of claim 1, wherein the background fluorescence is reduced by illuminating the sample with white light.

14. The method of claim 13, wherein the reporter protein is the green fluorescent protein.

15. The method of claim 13, wherein the reporter protein is the enhanced green fluorescent protein.

16. The method of claim 13, wherein the number of cells in each group represents about 500 trapped genes.

17. The method of claim 13, wherein the cells from each pool are expanded at least five fold.

18. The method of claim 17, wherein the cells from each pool are expanded at least ten fold.

19. The method of claim 13, further comprising the step of cloning and sequencing the trapped gene.

20. The method of claim 13, wherein the cell stream velocity is less than 15 meters per second.

21. The method of claim 20, wherein the cell stream velocity is about 5 meters per second.

13. A method of screening a plurality of test compounds to identify one or more compounds that affect expression of at least one trapped gene comprising the steps of:

a) providing a gene-trap vector encoding a fluorescent reporter protein, wherein the fluorescent reporter protein directly or indirectly produces fluorescence;

b) stably transfecting a population of cells with the gene-trap vector, wherein expression of the reporter protein is indicative of the expression of the trapped gene;

c) sorting the population of stably transfected cells by FACS into groups according to expressed levels of reporter protein;

d) distributing cells from each group into one or more pools, wherein each pool represents more than one trapped gene;

e) expanding the cells from each pool, wherein the expansion is such that a sufficient number of cells representing each trapped gene are produced to permit distinction of the effect of a test compound on the expression of the trapped gene over a control;

f) placing the expanded cells into individual wells;

g) incubating the wells from each pool in the absence or presence of a plurality of test compounds to generate control wells and test wells respectively, wherein the plurality of test compounds are added to the same wells well or separate wells;

h) conducting FACS analysis on the test wells and control wells to generate fluorescence distribution patterns for the cells in the test wells and control wells;

i) comparing the fluorescence distribution patterns of each test well with the control well; and

j) identifying the test well in which the fluorescence distribution pattern differs from the control well, wherein identification of a different fluorescence distribution pattern is indicative of a test compound affecting the expression of one or more trapped gene represented in the test well,

wherein the sorting parameters are adjusted to compensate for heterogeneity in the reporter protein fluorescence due to cell cycling and wherein the cell stream velocity during FACS analysis is less than 25 meters per second, and wherein background fluorescence is reduced by illuminating white light on the sample.

22. A method of identifying multiple modulators of a known trapped gene comprising the steps of

a) obtaining a clone of stably transfected cells in which the known gene has been trapped by a gene-trap vector encoding a fluorescent reporter protein has been inserted, wherein the fluorescent reporter protein directly or indirectly produces fluorescence;

b) placing cells from the clone into multiple wells;

c) incubating the wells in the absence or presence of a plurality of test compounds to generate control wells and test wells respectively, wherein the plurality of test compounds are added to the same wells well or separate wells;

d) conducting FACS analysis on the test wells and control wells to generate fluorescence distribution patterns for the cells in the test wells and control wells;

e) comparing the fluorescence distribution patterns of each test well with the control well; and

f) identifying the test wells in which the fluorescence distribution pattern differs from the control well, wherein identification of a different fluorescence distribution pattern is indicative of a test compound affecting the expression of the trapped gene,

wherein the sorting parameters are adjusted to compensate for heterogeneity in the reporter protein fluorescence due to cell cycling, wherein the cell stream velocity during FACS analysis is less than 25 meters per second, and wherein background fluorescence is reduced by illuminating white light on the sample.

23. The method of claim 22, wherein the reporter protein is the green fluorescent protein.

24. The method of claim 23, wherein the reporter protein is the enhanced green fluorescent protein.

25. The method of claim 22, wherein the number of cells in each group represents about 500 trapped genes.

26. The method of claim 22, wherein the cells from each pool are expanded at least five fold.

27. The method of claim 26, wherein the cells from each pool are expanded at least ten fold.

28. The method of claim 22 wherein the cell stream velocity is less than 15 meters per second.

29. The method of claim 22 wherein the cell stream velocity is about 5 meters per second.