Methods and materials using signaling probes

• US 20090106853A1
• Filed: 02/17/2005
• Published: 04/23/2009
• Est. Priority Date: 02/18/2004
• Status: Active Grant

• Current Assignee: Secondcell Bio LLC
• Original Assignee: Chromocell Corporation
• Inventors: Sawchuk, Dennis J., Shekdar, Kambiz, Montez, Jason M.
• Granted Patent: US 8,916,503 B2
• Time in Patent Office: Days
• US Class Current: 800/21
• 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

The present invention relates to methods of isolating cells or generating cell lines using signaling probes that produce a signal upon hybridization to a target sequence. Other methods that utilize the signaling probe include methods of quantifying the level of RNA expression, methods for identifying genetic recombinational events in living cells and methods of generating a transgenic animal using the isolated cells. The invention also provides protease probes. Signaling probes and protease probes that form stem-loop structures, three-arm junction structures, and dumbbell structures are provided.

Claims

1. A method of isolating cells expressing an RNA comprising the steps of:

providing cells potentially expressing an RNA;

exposing the cells to a signaling probe that produces a detectable signal upon hybridization with said RNA; and

isolating the cells that produce the signal.

25. The method of claim 1 or 19, further comprising the step of culturing the isolated cells.

46. A cell obtained by the method of claim 1 or 19.

49. The cell of claim 46, wherein the cell is an embryonic stem cell.

50. A method for generating a transgenic or chimeric animal comprising the step of using the embryonic stem cell of claim 49 to produce said transgenic or chimeric animal.

2-18. -18. (canceled)

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

introducing into cells a plurality of DNA that results in the expression of a plurality of endogenous RNA, wherein at least a subset of the plurality of endogenous RNA are different from each other;

exposing the cells to a plurality of different signaling probes, wherein the signaling probes produce a detectable signal upon hybridization to one or more RNAs of the plurality of endogenous RNA; and

isolating the cells that produce the signal.

21. The method of claim 19, wherein the plurality of RNA form an expression library.

20. (canceled)

22-24. -24. (canceled)

26-33. -33. (canceled)

34. A method of isolating cells with reduced expression of a protein comprising the steps of:

introducing into cells a DNA encoding an antisense RNA or an shRNA that reduces expression of said protein;

exposing the cells to a signaling probe that produces a detectable signal upon hybridization to said antisense RNA or shRNA; and

isolating the cells that produce the signal.

35-37. -37. (canceled)

39-40. -40. (canceled)

41. A method of identifying a compound that modulates expression of an RNA, comprising the steps of:

adding a test compound to cells expressing said RNA;

exposing the cells to a signaling probe that produces a detectable signal upon hybridization with said RNA;

comparing the signal produced by cells exposed to the test compound to the signal produced by cells not exposed to the test compound;

wherein an increase or decrease in signal produced by the cells exposed to the test compound as compared to the signal produced by the cells not exposed to the test compound indicates that the compound is a compound that modulates expression of said RNA.

42. The method of claim 41, wherein the RNA is encoded by a DNA that is introduced into the cells.

43. (canceled)

44. A method for identifying a genetic recombinational event in living cells comprising the step of:

exposing a cell to a signaling probe that produces a detectable signal upon hybridization with an RNA transcribed from a recombined sequence,

wherein detection of a cell producing the signal indicates that the cell comprises the genetic recombinatorial event.

45. (canceled)

47-48. -48. (canceled)

51-102. -102. (canceled)

103. A library of mammalian cell lines comprising at least 1,000 cell lines each comprising a stably integrated expressed sequence.

109. The library of claim 103, wherein each cell line comprises a variable library sequence.

110. The library of claim 109, wherein the variable sequence of said expression library is selected from the group consisting of genomic, genomic untranslated, genomic translated, gene, cDNA, EST, oligo, random, RNA, protein, protein domain, peptide, intronic, exonic, tag, or linker sequence, or combination thereof or recombination thereof, or one or more of the unmodified, mutagenized, randomized, shuffled or recombined sequences.

114. The library of claim 103, wherein the library is used in a cell-based screening assay.

104-108. -108. (canceled)

111-113. -113. (canceled)

115. (canceled)

116. A method of identifying a compound that mediates or improves the introduction of signaling probes into cells comprising the steps of:

exposing cells to a signaling probe in the presence of a test compound, wherein the cells comprise a target sequence and wherein the signaling probe produces a signal upon hybridization with the target sequence; and

detecting the signal produced by the cells,

wherein an increase in signal produced by the cells exposed to the test compound as compared to cells not exposed to the test compound indicates that the test compound is a compound that mediates or improves the introduction of signaling probes into cells.