Methods and materials using signaling probes

• US 8,916,503 B2
• Filed: 02/17/2005
• Issued: 12/23/2014
• Est. Priority Date: 02/18/2004
• Status: Active Grant

• Current Assignee: Secondcell Bio LLC
• Original Assignee: Chromocell Corporation
• Inventors: Shekdar, Kambiz, Sawchuk, Dennis J., Montez, Jason M.
• Primary Examiner(s): Riggs, Larry
• Assistant Examiner(s): DINES, KARLA A
• Application Number:
  US10/589,052
• Publication Number:
  US 20090106853A1
• Time in Patent Office: 3,596 Days
• Field of Search: 506/2
• US Class Current: 506/2
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2525/207 siRNA, miRNA
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels

Abstract

Methods of isolating cells or generating cell lines comprising the step of exposing the cells to signaling probes that produce a signal upon hybridization to a target sequence, as well as methods of quantifying the level of expression of an RNA of interest, methods for identifying genetic recombinational events in living cells and methods of generating a transgenic animal using the isolated cells. Methods for isolating a plurality of cells encoding a plurality of different RNAs associated with a same nucleic acid tag sequence, comprising the step of exposing the cells to a same signaling probe that produces a detectable signal upon hybridization to the same nucleic acid tag sequence, are also provided. Signaling probes and protease probes that form stem-loop structures, three-arm junction structures, and dumbbell structures may be used in the above methods.

Claims

1. A method of isolating a plurality of cells, wherein a subset of the cells expresses an RNA that is not expressed by another subset of the cells, comprising the steps of:

introducing into cells a plurality of DNAs encoding a plurality of different RNAs, wherein each DNA further encodes a nucleic acid tag sequence, and wherein at least a subset of the plurality of DNAs encodes the same nucleic acid tag sequence;

exposing the cells to a signaling probe that produces a detectable signal upon hybridization to said same nucleic acid tag sequence; and

isolating, in a single application, the cells that exhibit at least one selected level of fluorescence at a selected wavelength.

2. The method of claim 1, wherein the plurality of different RNAs forms an expression library.

3. The method of claim 1, further comprising the step of culturing the isolated cells.

4. The method of claim 1, wherein said plurality of DNAs encodes at least one antisense RNA, shRNA, or siRNA.

5. The method of claim 1, further comprising separately growing individually isolated cells to generate a plurality of separate cell lines.

6. The method of claim 1, further comprising pooling the isolated cells.

7. The method of claim 6, further comprising growing the pooled cells.

8. The method of claim 1, wherein the plurality of different RNAs are selected from the group consisting of RNAs in the same or a related biological pathway, RNAs that act upstream or downstream of each other, RNAs that have a modulating, activating or repressing function to each other, RNAs that are dependent on each other for function or activity, RNAs that are components of the same complex, and RNAs that encode proteins selected from the group consisting of proteins in the same or a related biological pathway, proteins that act upstream or downstream of each other, proteins that have a modulating, activating or repressing function to each other, proteins that are dependent on each other for function or activity, proteins that are components of the same complex, and proteins from the same protein family.

9. The method of claim 1, wherein the nucleic acid tag sequence comprises multiple target sequences, wherein one signaling probe hybridizes to each target sequence.

10. The method of claim 1, wherein the DNA encodes multiple nucleic acid tag sequences.

11. The method of claim 1, wherein the DNA encoding said nucleic acid tag sequence is:

(a) in frame with the DNA encoding said RNA;

or

(b) out of frame with the DNA encoding said RNA.

12. The method of claim 1, wherein the DNA further encodes a selection marker, and wherein the method further comprises the step of selecting the cells utilizing the selection marker after introducing the DNA into the cells but prior to exposing said cells to the signaling probe.

13. The method of claim 1, wherein said DNA is operably linked to a conditional promoter.

14. The method of claim 13, wherein the RNA encoded by the

DNA, or a protein encoded by the RNA, is damaging to the cell when expressed.

15. The method of claim 1, wherein each DNA of said plurality of DNAs further comprises a second DNA sequence encoding a second RNA, wherein said second DNA sequence is under the control of a conditional promoter.

16. The method of claim 15, wherein said plurality of DNAs encodes a plurality of variable test RNAs, and wherein said method further comprises the steps of:

assaying the isolated cells for the presence of the second RNA; and

identifying the test RNA that activates the conditional promoter in cells that express the second RNA.

17. The method of claim 15, wherein the second RNA, or a protein encoded by the second RNA, is lethal or damaging to the cell when expressed.

18. A method of isolating cells each expressing RNAs from two or more RNA expression libraries, comprising the steps of:

introducing into cells a plurality of DNAs encoding a first RNA expression library, wherein each DNA further encodes a first nucleic acid tag sequence, and wherein at least a subset of the plurality of DNAs encodes the same first nucleic acid tag sequence;

introducing into the cells a plurality of DNAs encoding a second RNA expression library, wherein each DNA further encodes a second nucleic acid tag sequence, and wherein at least a subset of the plurality of DNAs encodes the same second nucleic acid tag sequence;

exposing the cells to a first signaling probe that produces a detectable signal upon hybridization to said first nucleic acid tag sequence;

exposing the cells to a second signaling probe that produces a detectable signal upon hybridization to said second nucleic acid tag sequence; and

isolating, in a single application, the cells that exhibit at least one selected level of fluorescence at a selected wavelength for each of the first and second signaling probes.