Method of screening for factors that modulate gene expression

• US 20030170656A1
• Filed: 06/18/2002
• Published: 09/11/2003
• Est. Priority Date: 10/16/1995
• Status: Abandoned Application

• Current Assignee: Chiron Corporation (Bayer AG)
• Original Assignee: Chiron Corporation (Bayer AG)
• Inventors: Escobedo, Jaime, Cen, Hui, Williams, Lewis
• Application Number:
  US10/174,152
• Publication Number:
  US 20030170656A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

The invention describes a method of screening for factors that modulate gene expression, by detection of factors including factors that stimulate or inhibit cell growth, differentiation or proliferation, factors involved in one or more signal transduction pathways, or factors involved in protein-protein interactions. Identification of such factors that modulate gene expression is accomplished by detection of an intracellular event mediated by the factor. The intracellular event can be an increase or decrease in transcription or translational activities. Detection of the intracellular event is accomplished by any highly sensitive assay targeting a transcription or translation product, including, for example, a bDNA assay to the nascent transcript induced by the factor.

Claims

1. A method of high throughput screening for a factor that modulates gene expression comprising the steps:

(a) providing a small amount of a candidate factor,

(b) providing a small amount of responding cells,

(c) contacting a responding cell with a candidate factor, wherein the responding cell is capable of responding to a factor that modulates gene expression by exhibiting an early intracellular event; and

(d) detecting directly the early intracellular event.

2. The method of claim 1, wherein step (d) comprises a detection facilitated by hybridization of a polynucleotide sequence to a target polynucleotide.

3. The method of claim 2, wherein the target polynucleotide is an mRNA transcript.

4. The method of claim 2, further comprising that the polynucleotide sequence that hybridizes to a target polynucleotide is selected from the group consisting of synthetic DNA, synthetic RNA, reverse transcription primers, polymerase chain reaction primers, and an RNAse protection assay probe.

5. The method of claim 4, wherein the synthetic DNA comprises branched DNA.

6. The method of claim 1, wherein step (a) comprises a candidate factor selected from the group consisting of a polypeptide factor, small molecule factor, and a polynucleotide factor.

7. The method of claim 6, wherein the polypeptide factor is encoded by a cDNA or a cRNA molecule.

8. The method of claim 7, wherein the cDNA or cRNA molecule is expressed in a producing cell.

9. The method of claim 6, wherein the small molecule factor comprises a small molecule selected from the group consisting of a small organic molecule, a peptide molecule, and a peptoid molecule.

10. The method of claim 6, wherein the polynucleotide factor comprises one selected from the group consisting of a ribozyme and an antisense oligonucleotide.

11. The method of claim 1, wherein step (c) comprises co-culturing a producing cell and a responding cell.

12. The method of claim 11, wherein the producing cell is transformed with a cDNA or a cRNA encoding a candidate polypeptide factor.

13. The method of claim 11, wherein the producing cell is a Xenopus oocyte.

14. The method of claim 11, wherein the responding cell is a mammalian cell.

15. The method of claim 1, wherein the candidate factor that modulates gene expression comprises a factor selected from the group consisting of a stimulatory factor, and an inhibitory factor.

16. The method of claim 15, wherein the stimulatory factor comprises a factor selected from the group consisting of a growth factor, a transcription factor, a differentiation factor, a developmental regulator, an apoptotic factor, an immunomodulatory factor, and an oncogenic factor.

17. The method of claim 2, wherein the target polynucleotide comprises a transcript of a gene encoding an inducible protein.

18. The method of claim 2, wherein the target polynucleotide comprises a transcript of a gene encoding a protein selected from the group consisting of a cytokine, a hematopoetic factor, a neuronal differentiation factor, a growth factor, a protein hormone, a repressor protein, and a tissue marker.

19. The method of claim 17, wherein the target polypeptide comprises a transcript of a gene encoding a protein selected from the group consisting of a DNA-binding protein, a disease marker, a growth marker, a differentiation marker, an apoptotic marker, a metastatic marker, a marker associated with a later onset of a disease, and an oncogene.

20. The method of claim 1, wherein step (a) comprises that the candidate factor is selected from the group consisting of a natural product of a producing cell, a product of expression by a producing cell that is transformed with a polynucleotide sequence encoding the candidate factor, and a small molecule derived from a small molecule library.

21. The method of claim 1 wherein step (b) comprises responding cells that are natural cells.

22. The method of claim 1, wherein step (b) comprises responding cells that are transformed with a reporter gene construct.

23. The method of claim 8, wherein the candidate factor is expressed on the surface of the producing cell.

24. The method of claim 8, wherein the candidate factor is secreted by the producing cell.

25. The method of claim 1, wherein the responding cell comprises a prokaryotic cell or an eukaryotic cell.

26. The method of claim 25, wherein the responding cell comprises a eukaryotic cell selected from the group consisting of a mammalian cell, a fungal cell, an insect cell, an avian cell, a worm cell, a fish cell, a crustacean cell, a reptilian cell, an amphibian cell, and a plant cell.

27. The method of claim 8, wherein the producing cell comprises a prokaryotic cell or an eukaryotic cell.

28. The method of claim 27, wherein the producing cell comprises a eukaryotic cell selected from the group consisting of a mammalian cell, a fungal cell, an insect cell, an avian cell, a worm cell, a fish cell, a crustacean cell, a reptilian cell, an amphibian cell, and a plant cell.

29. The method of claim 1, wherein the early intracellular event of step (d) comprises an increase or decrease in transcription.

30. The method of claim 15, wherein the inhibitory factor decreases a target polynucleotide amount, wherein the target polynucleotide comprises a transcript of a gene encoding a protein selected from the group consisting of a cytokine, a hematopoetic factor, a neuronal differentiation factor, a growth factor, a protein hormone, a repressor protein, and a tissue marker.

31. The method of claim 30, wherein the target polypeptide comprises a transcript of a gene encoding a protein selected from the group consisting of a DNA-binding protein, a disease marker, a growth marker, a differentiation marker, an apoptotic marker, a metastatic marker, a marker associated with a later onset of a disease, and an oncogene.

32. The method of claim 1 for use in high throughput screening for a receptor that binds a known ligand comprising the modification of steps (a) through (b) of:

(a) providing a small amount of a candidate receptor,

(b) providing a responding cell transformed with a polynucleotide sequence encoding a known ligand,

(c) contacting a responding cell that comprises a candidate receptor with a ligand to allow formation of a receptor/ligand specific binding pair that triggers a detectable early intracellular event in the responding cell,

(d) detecting the early intracellular event.

33. The method of claim 32, wherein the ligand comprises a molecule selected from the group consisting of a natural product of a producing cell, a product of expression by a producing cell that is transformed with a polynucleotide sequence encoding the ligand, and a small molecule.

34. The method of claim 32, wherein the responding cell is transformed with a polynucleotide encoding the candidate receptor.

35. The method of claim 34, wherein the polynucleotide comprises one selected from the group consisting of a cDNA molecule, a cRNA molecule, and a genomic DNA molecule.

36. The method of claim 34, wherein the polynucleotide is derived from a cDNA library of a life form selected from the group consisting of a mammal, a fungus, an insect, a worm, a bird, a fish, a crustacean, a bacterium, a reptile, an amphibian, and a plant.

37. The method of claim 36, wherein the mammal is a human.

38. The method of claim 1, wherein step (d) comprises that the early intracellular event is detected by one selected from the group consisting of a bDNA assay, a RNase protection assay, and RT-PCR.

39. The method of claim 32, wherein the intracellular event is detected by one selected from the group consisting of a bDNA assay, a RNase protection assay, and RT-PCR.

40. The method of claim 32, wherein the known ligand comprises a ligand selected from the group consisting of Noggin, Wnt, and Notch.

41. The method of claim 1 for use in high throughput screening for an unknown ligand that binds a known receptor comprising the modification of steps (a) through (b) of:

(a) providing a small amount of a candidate ligand,

(b) providing a responding cell transformed with a polynucleotide sequence encoding a known receptor,

(c) contacting a responding cell that expresses a known receptor with a candidate ligand to allow formation of a receptor/ligand specific binding pair that triggers a detectable early intracellular event in the responding cell,

(d) detecting the early intracellular event.

42. The method of claim 41, wherein the ligand comprises a molecule selected from the group consisting of a natural product of a producing cell, a product of expression by a producing cell that is transformed with a polynucleotide sequence encoding the ligand, and a small molecule.

43. The method of claim 41, wherein the candidate ligand is an antagonist to a receptor.

44. The method of claim 41, wherein step (a) comprises transforming a producing cell with a polynucleotide encoding a candidate ligand.

45. The method of claim 44, wherein the polynucleotide comprises a molecule selected from the group consisting of a cDNA, a cRNA and a genomic DNA molecule.

46. The method of claim 45, wherein the cDNA or genomic DNA molecule is derived from a sequence selected from a library derived from the group consisting of a mammal, a fungus, an insect, a worm, a bird, a fish, a crustacean, a bacterium, a reptile, an amphibian, and a plant.

47. The method of claim 46, wherein the library is derived from a mammal.

48. The method of claim 47, wherein the mammal is a human.

49. The method of claim 41, wherein the intracellular event is detected by a assay capable of detecting changes in transcription of a gene.

50. The method of claim 49, wherein the assay comprises one selected from the group consisting of a bDNA assay, a RNase protection assay, and RT-PCR.

51. The method of claim 1 wherein the factor causes regulation of transcriptional activity of a gene.

52. The method of claim 51, wherein step (c) further comprises contacting a candidate factor to be tested for its ability to regulate transcriptional activity with a responding cell that comprises a regulatory sequence subject to transcriptional regulation, and further wherein step (d) further comprises detecting up-regulation or down-regulation activity of the regulatory sequence.

53. The method of claim 51, wherein the responding cell is capable of responding to a factor that modulates gene expression by exhibiting an early intracellular event.

54. The method of claim 51, wherein the responding cell is not transformed with a reporter gene sequence connected to a regulatory sequence subject to transcriptional regulation.

55. The method of claim 51, wherein the responding cell is transformed with a reporter gene sequence connected to a regulatory sequence subject to transcriptional regulation.

56. The method of claim 55, wherein the detection of up-regulation or down-regulation is performed by detection of reporter gene expression.

57. The method of claim 56, wherein the reporter gene comprises one selected from the group consisting of luciferase, secreted alkaline phosphatase, β

-galactosidase, CAT, and GFP.

58. The method of claim 56, wherein the detection of reporter gene expression is accomplished by a nucleic acid hybridization assay.

59. The method of claim 57, wherein the reporter gene expression is detected by one selected from the group consisting of a bDNA assay, a RNase protection assay and RT-PCR.

60. The method of claim 52, wherein the regulatory sequence comprises one selected from the group consisting of a promoter, an enhancer and a repressor.

61. The method of claim 51, wherein the candidate factor comprises one selected from the group consisting of a natural product of a producing cell or virus, a product of expression by a producing cell or virus that is transformed with a polynucleotide sequence encoding the candidate factor, and a small molecule.

62. The method of claim 52, wherein the regulatory sequence comprises a regulatory sequence derived from a gene selected from the group consisting of a viral gene, a bacteriophage gene, a prokaryotic gene and an eukaryotic gene.

63. The method of claim 62, wherein the eukaryotic gene comprises one selected from the group consisting of cytokines, hematopoetic factors, neuronal differentiation factors, growth factors, differentiation factors, protein hormones, transcription factors, repressor proteins, DNA-binding proteins, tissue markers, cancer markers, disease markers, ob protein, A20 protein, ICAM, c-fos protein, and any inducible protein.

64. The method of claim 62, wherein the eukaryotic gene comprises one selected from the group consisting of a mammalian gene, a fungal gene, a worm gene, an insect gene, an avian gene, a fish gene, a crustacean gene, a reptilian gene, an amphibian gene, and a plant gene.

65. A growth factor discovered by the method of claim 1.

66. A differentiation factor discovered by the method of claim 1.

67. An inhibitory factor discovered by the method of claim 1.

68. A ligand discovered by the method of claim 1.

69. A receptor discovered by the method of claim 1.

70. A hormone discovered by the method of claim 1.

71. A cytokine discovered by the method of claim 1.

72. A transcription factor discovered by the method of claim 1.

73. An antagonist to a receptor discovered by the method of claim 1.

74. A polynucleotide comprising a nucleotide sequence encoding the growth factor of claim 65.

75. A polynucleotide comprising a nucleotide sequence encoding the differentiation factor of claim 66.

76. A polynucleotide comprising a nucleotide sequence encoding the inhibiting factor of claim 67.

77. A polynucleotide comprising a nucleotide sequence encoding the ligand of claim 68.

78. A polynucleotide comprising a nucleotide sequence encoding the receptor of claim 69.

79. A polynucleotide comprising a nucleotide sequence encoding the hormone of claim 70.

80. A polynucleotide comprising a nucleotide sequence encoding the cytokine of claim 71.

81. A polynucleotide comprising a nucleotide sequence encoding the transcription factor of claim 72.

82. A polynucleotide comprising a nucleotide sequence encoding the antagonist to a receptor of claim 73.

83. A polypeptide comprising a portion of the sequence of SEQ ID NO. 2, and exhibiting growth factor activity as demonstrated by induction of c-fos transcription.

84. The polypeptide of claim 83, wherein the induction of c-fos transcription is at least about 5-fold induction above normal levels.

85. A polypeptide having the sequence of SEQ ID No. 2.

86. A polypeptide comprising a molecule selected from the group consisting of an analog, a derivative, and a variant of the polypeptide of claim 85.

87. A polynucleotide sequence encoding the polypeptide of claim 86.

88. A polynucleotide sequence of SEQ ID No. 1 connected to a heterologous polynucleotide sequence.