High-throughput transcriptome and functional validation analysis

• US 6,924,109 B2
• Filed: 04/03/2002
• Issued: 08/02/2005
• Est. Priority Date: 07/30/1999
• Status: Expired due to Fees

• Current Assignee: AGY Therapeutics, Inc.
• Original Assignee: AGY Therapeutics, Inc.
• Inventors: Ye, Shiming, Gan, Li, McFarland, Keith Charles, Gonzalez-Zulueta, Mirella, Melcher, Thorsten
• Primary Examiner(s): Fredman, Jeffrey
• Assistant Examiner(s): Chakrabarti, Arun
• Application Number:
  US10/116,437
• Publication Number:
  US 20030082570A1
• Time in Patent Office: 1,217 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/325, 536/24.3, 935/6, 436/518, 424/94.67
• US Class Current: 506/4
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12N 15/1079 Screening libraries by altering the phenotype or phenotypic trait of the host reporter assays C12N15/1086
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2320/12 in functional genomics, i.e. for the determination of gene function
• CPC Class Codes: C12N 2330/30 chemically synthesised

Abstract

Methods for correlating genes and gene function are provided. Such methods generally involve selecting a candidate gene that appears to be correlated with a particular cellular state or activity and then validating the role of the candidate gene in establishment of such a cellular state or activity. Certain methods utilize RNA interference techniques in the validation process.

Claims

1. A method for validating the effect of a candidate gene that is expressed in a mammalian neural or glial cell of interest, said method comprising:

(a) producing a candidate dsRNA with a sequence identical to at least a portion of a said candidate gene;

(b) introducing said candidate dsRNA into a reference mammalian neural or glial cell; and

(c) validating the effect of said candidate gene by detecting an alteration in a cellular activity or a cellular state in said reference mammalian neural or glial cell, wherein said alteration is the result of specific attenuation of mRNA corresponding to said candidate in said reference mammalian neural or glial cell indicating that said candidate gene plays a functional role in mammalian neural or glial cells.

2. The method of claim 1, wherein said step of producing the candidate dsRNA comprises:

(i) producing a cDNA corresponding to said candidate gene from an mRNA of said mammalian neural or glial cell of interest; and

(ii) producing the candidate dsRNA from said cDNA.

3. The method according to claim 2, further comprising:

producing a plurality of candidate cDNAs from said mammalian neural or glial cell of interest

producing a plurality of candidate dsRNA with a sequence identical to at least a portion of said candidate cDNAs;

introducing each of the candidate dsRNA into a plurality of separate reference mammalian neural or glial cells having a gene expression similar said mammalian, neural or glial cell of interest; and

validating the effect of said candidate genes by testing for alterations in a cellular activity or a cellular state in said reference mammalian neural glial cell that result from attenuation of mRNA corresponding to said candidate cDNA in said reference mammalian neural or glial cell, wherein detection of said alterations is indicative that said candidate gene plays a functional role in said mammalian neural or glial cells of interest.

4. The method of claim 3, wherein said step of producing a plurality of candidate cDNAs comprises:

producing a double-stranded cDNA from mRNA by reverse transcription;

producing cDNAs of a similar length by digesting said cDNA with a restriction enzyme; and

producing a plasmid or PCR fragment from said cDNA after said digesting step.

5. The method of claim 4, wherein the candidate dsRNA is produced by transcribing said plasmid cDNA or PCR fragment.

6. The method of claim 4, wherein the restriction enzyme is selected from the group consisting of DpnI and RasI.

7. The method of claim 3, wherein said step of producing the plurality of candidate dsRNAs comprises:

selecting a candidate cDNA that is expressed at a detectably different level with respect to said reference mammalian neural or glial cell and said mammalian neural or glial cell of interest, and said reference mammalian neural or glial cell and said mammalian neural or glial cell of interest differ with a respect to cellular characteristic that is detectable by said step of testing for alterations in a cellular activity or a cellular state.

8. The method of claim 7, wherein the candidate cDNA is selected from a normalized library prepared from said reference mammalian neural or glial cells or said mammalian neural or glial cell of interest and is present in low abundance in the normalized library.

9. The method of claim 7, wherein the candidate cDNA is a differentially expressed cDNA gene selected from a subtracted library that is enriched for cDNAs that are differentially expressed with respect to said reference mammalian neural or glial cells or said mammalian neural or glial cell of interest.

10. The method of claim 7, wherein said step of selecting the candidate cDNA comprises:

preparing a tester-normalized cDNA library from test cells;

a driver-normalized cDNA library from control cells;

a tester-subtracted cDNA library which is enriched in one or more genes that are up-regulated with respect to the test cell and the control cell, and a driver-subtracted cDNA library which is enriched in one or more genes that are down-regulated with respect to the test cell and the control cell; and

selecting a cDNA from the normalized libraries by contacting cDNAs from the tester-normalized cDNA library with labeled probes derived from mRNA from test cells and contacting cDNAs from the driver-normalized cDNA library with labeled probes derived from mRNA from control cells under conditions whereby probes specifically hybridize with complementary cDNAs to form a first set of hybridization complexes and detecting at least one hybridization complex from the first set of hybridization complexes to identify a cDNA that is present in low abundance.

11. The method of claim 7, wherein said step of selecting the candidate cDNA comprises:

preparing a tester-normalized cDNA library from test cells, a driver-normalized cDNA library from control cells;

a tester-substracted cDNA library which is enriched in one or more genes that are up-regulated with respect to the test cell and the control cell, and a driver-substracted cDNA library which is enriched in one or more genes that are down-regulated with respect to the test cell and the control cell; and

selecting a cDNA from the substracted libraries by contacting cDNAs from the tester-substracted cDNA library and contacting cDNAs from the driver-substracted cDNA library with a population of labeled probes under conditions whereby probes from the population of probes specifically hybridize with complementary cDNAs to form a second set of hybridization complexes, and wherein the population of labeled probes is derived from mRNA from test cells and control cells; and

detecting at least one hybridization complex from the second set of hybridization complexes to identify a cDNA that is differentially expressed above a threshold level with respect to the subtracted libraries.

12. The method of claim 7, wherein the cellular characteristic is cell health, the test cell is a diseased neural or glial cell and the control cell is a healthy neural or glial cell, and the candidate gene is suspected of correlation with a disease.

13. The method of claim 12, wherein the test cell is obtained from a mammal that has had a stroke or is at risk for stroke.

14. The method of claim 7, wherein the cellular characteristic is cellular differention and the candidate gene is suspected of correlation with control of cellular differentiation.

15. The method of claim 7, wherein the candidate gene is endogenous to said mammalian neural or glial reference cell.

16. The method of claim 7, wherein the candidate gene is an extrachromosomal gene in said mammalian neural or glial reference cell.

17. The method of claim 12, wherein the said mammalian neural or glial reference cell is a neuroblastoma cell.

18. The method of claim 17, wherein said mammalian neural or glial reference cell has increased sensitivity by N-methyl-D-aspartate, β

-amyloid, peroxide, oxygen-glucose deprivation, or combinations thereof relative to a normal mammalian neural or glial cell.

19. The method of claim 18, wherein the detecting step comprises detecting a decrease in cellular sensitivity to N-methyl-D-aspartate, β

-amyloid peroxide, oxygen-glucose deprivation, or combinations thereof relative to a normal mammalian neural or glial cell.

20. The method of claim 3, wherein the detecting step comprises detecting modulation of ligand binding to a protein.

21. The method of claim 1, wherein the determining step comprises determining whether the protein encoded by the candidate gene binds to another protein to form a complex.