Determining protein function in cell culture using RNA interference

• US 20020182590A1
• Filed: 05/24/2002
• Published: 12/05/2002
• Est. Priority Date: 05/25/2001
• Status: Abandoned Application

• Current Assignee: Vanderbilt University
• Original Assignee: Vanderbilt University
• Inventors: Christensen, Michael, Strange, Kevin, Miller, David M. III
• Application Number:
  US10/155,909
• Publication Number:
  US 20020182590A1
• Time in Patent Office: Days
• US Class Current: 435/4
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: G01N 2333/195 from bacteria
• CPC Class Codes: G01N 2333/415 from plants
• CPC Class Codes: G01N 2333/43534 from Caenorhabditis
• CPC Class Codes: G01N 2333/43573 from Drosophila
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025

Abstract

The present invention addresses one of the major issues in molecular biology today, “functional genomics.” The inventors have provided methods that utilize the phenomenon of RNA interference as a tool for identifying cDNAs that encode proteins with assayable function.

Claims

1. A method for assigning functional properties to polypeptides comprising:

(a) obtaining a population of double-stranded (ds) RNA molecules, where each unique dsRNA molecule is segregated from other members of the dsRNA population;

(b) contacting one or more members of said dsRNA population with a host cell; and

(c) measuring one or more phenotypic parameters of the host cell of step (b),

wherein a change in a phenotypic parameter in the host cell of step (b), as compared to a host cell not contacted with dsRNA, assigns to a polypeptide or polypeptides encoded by the one or more members of said dsRNA population of step (b) a functional property.

2. The method of claim 1, wherein step (b) comprises contacting a first set of about 10 to about 100 different dsRNA sequences with said host cell.

3. The method of claim 2, further comprising:

(d) contacting each of the dsRNA sequences used in step (b) with a different host cell; and

(e) measuring one or more phenotypic parameters of each of the host cells of step (d),

wherein a change in a phenotypic parameter a host cell of step (d), as compared to a host cell not contacted with dsRNA, assigns to a polypeptide encoded by the dsRNA of step (d) a functional property.

4. The method of claim 1, further comprising the step, prior to step (a), of preparing dsRNA.

5. The method of claim 4, further comprising, prior to the step of preparing dsRNA, of preparing cDNA.

6. The method of claim 1, further comprising sequencing of one or more nucleic acids following assignment of a functional property.

7. The method of claim 1, wherein the host cell is bacterial, yeast, or mammalian.

8. The method of claim 1, wherein the phenotypic parameter is selected from the group consisting of cell growth, cell proliferation, apoptosis, Ca²⁺ signaling, ion channel activity, ion transporter activity, drug action, toxin action, metabolism, viral infectino, bacterial infection and stress response.

9. The method of claim 1, wherein said dsRNA population is derived from C. elegans, Drosophila or Arabidopsis.

10. The method of claim 2, wherein said method comprises concurrent testing of multiple sets of about 10 to about 100 dsRNA with individual host cells.

11. The method of claim 4, further comprising, prior to the step producing said dsRNA population, of performing a DNA or RNA subtraction with another population of DNA or RNA.

12. A method for identifying a functionally relevant polypeptides comprising:

(a) obtaining a population of double-stranded (ds) RNA molecules, where each unique dsRNA molecule is segregated from other members of the dsRNA population;

(b) contacting one or more members of said dsRNA population with a host cell; and

(c) measuring one or more phenotypic parameters of the host cell of step (b),

wherein a change in a phenotypic parameter in the host cell of step (b), as compared to a host cell not contacted with dsRNA, assigns to a polypeptide or polypeptides encoded by said one or more members of said dsRNA population of step (b) a relevant function.

13. A method for screening a population of nucleic acids for functionally relevant polypeptides comprising:

(a) obtaining a population of double-stranded (ds) RNA molecules, where each unique dsRNA molecule is segregated from other members of the dsRNA population;

(b) contacting one or more members of said dsRNA population with a host cell; and

(c) measuring one or more phenotypic parameters of the host cell of step (b),

wherein a change in a phenotypic parameter in the host cell of step (b), as compared to a host cell not contacted with dsRNA, assigns to a polypeptide or polypeptides encoded by said one or more members of said dsRNA population of step (b) a relevant function.