Methods of monitoring production of gene products and uses thereof

• US 6,902,882 B2
• Filed: 05/15/2001
• Issued: 06/07/2005
• Est. Priority Date: 05/23/2000
• Status: Expired due to Fees

• Current Assignee: Kerong Gu
• Original Assignee: Kerong Gu
• Inventors: Gu, Kerong
• Primary Examiner(s): Shukla, Ram R.
• Application Number:
  US09/859,155
• Publication Number:
  US 20020006605A1
• Time in Patent Office: 1,484 Days
• Field of Search: 800/3, 424/9.1, 435/375, 435/320.1, 435/325, 435/4, 435/7.21, 435/7.6, 435/8, 435/455, 435/466, 435/468, 435/471, 435/483
• US Class Current: 435/4
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

This invention relates generally to the field of monitoring production of gene products. In particular, the invention provides methods of monitoring production of a gene product, methods of screening for modulators of production of a gene product, and methods of screening for cellular targets amenable to regulation by a treatment using a plurality of reporter gene systems. The methods described herein find uses in a number of fields such as drug discovery, agricultural or industrial production and environmental monitoring or protection.

Claims

1. A method of screening for a modulator of production of a target gene product, which method comprises:

a) constructing a first host cell containing a first reporter gene encoding a first flourescent protein operatively linked to a nucleotide sequence comprising at least the promoter portion of a target gene and constructing a second host cell containing a second reporter gene encoding second flourescent protein operatively linked to said nucleotide sequence to which said first reporter gene is linked, wherein expression of said first reporter gene results in a first flourescently detectable signal, expression of said second reporter gene results in a second flourescently detectable signal, said first and second flourescently detectable signals are distinct from each other but are integratable, and wherein said first and second host cells grown separately and then mixed to produce a third flourescently detectable signal distinct from said first and second flourescently detectable signals and detention of said third flourescent signal does not depend on the continued growth or viability of said first and second host cells;

b) detecting said third flourescently detectable signal from a mixture of said first and second host cells wherein neither type of said host cells is subjected to any desired treatment;

c) detecting said third flourescently detectable signal from a mixture of said first and second host cells wherein one and only one type of said host cells is subjected to a desired treatment prior to said mixing of said host cells; and

d) comparing said third flourescently detectable signals detected in steps b) and c), whereby said third flourescently detectable signal detected in step b) differs from that detected in step c) identifies said treatment as a modulator of production of said target gene product,

wherein the method is performed in vitro.

2. The method of claim 1, wherein the first and second host cells are identical strain or cells.

3. The method of claim 1, wherein the first and second host cells are identical strain or cells.

4. The method of claim 1, wherein the first and second host cells belong to two different species of the same or different genus.

5. The method of claim 1, wherein the host cells are selected from the group consisting of animal, plant, fungus, bacterium, viral infected and recombinant cells.

6. The method of claim 5, wherein the fungus cells are yeast cells.

7. The method of claim 6, wherein the yeast cells are S. cerevisiae cells.

8. The method of claim 5, wherein the animal cells are selected from the group consisting of fly, worm, insect, vertebrate and mammalian cells.

9. The method of claim 8, wherein the mammalian cells are human cells.

10. The method of claim 1, wherein the host cells are haploid or diploid cells.

11. The method of claim 1, wherein the host cells contain a missense, a nonsense or a null mutation in at least one endogenous gene.

12. The method of claim 1, wherein the fluorescent protein is selected from the group consisting of a green, a blue, a red and a yellow fluorescent protein.

13. The method of claim 1, wherein the nucleotide sequence comprises only the promoter portion of the target gene.

14. The method of claim 1, wherein the nucleotide sequence comprises the promoter portion and other transcriptional regulatory elements of the target gene.

15. The method of claim 1, wherein the nucleotide sequence comprises the promoter portion, other transcriptional regulatory elements and at least a portion of the coding sequence of the target gene.

16. The method of claim 15, wherein the portion of the coding sequence of the target gene directs post-translational protein processing, modification, cellular location, transportation or metabolism of the protein encoded by the target gene.

17. The method of claim 1, wherein the nucleotide sequence comprises the promoter portion, other transcriptional regulatory elements and entire coding sequence of the target gene.

18. The method of claim 1, wherein the nucleotide sequence comprises the entire target gene.

19. The method of claim 1, wherein the reporter gene operatively linked to the nucleotide sequence is integrated into the genome of the host cell or is maintained episomally in the host cell.

20. The method of claim 19, wherein the reporter gene operatively linked to the nucleotide sequence is maintained episomally in a plasmid in the host cell.

21. The method of claim 1, wherein the first and second reporter genes are expressed at the same or different levels.

22. The method of claim 21, wherein the different expression levels of the first and second reporter genes are caused by the different cell ploidities or by different copy numbers of the reporter genes contained in the host cells.

23. The method of claim 1, wherein the nucleotide sequence is endogenous or exogenous to the host cell.

24. The method of claim 1, wherein the host cells used are yeast cells and the reporter genes are operatively linked to a nucleotide sequence of a non-yeast gene.

25. The method of claim 24, wherein the yeast cells are S. cerevisiae cells.

26. The method of claim 1, wherein the signal detection is real-time and/or in-situ detection.

27. The method of claim 1, wherein the signal detection is real-time and/or in-situ detection.

28. The method of claim 1, wherein the first host cell is treated, the third detectable signal detected in step c) moves toward the first detectable signal indicates that test substance enhances production of the target gene product controlled by the nucleotide sequence.

29. The method of claim 1, wherein the first host cell is treated, the third detectable signal detected in step c) moves toward the second detectable signal indicates that test substance inhibits production of said target gene product controlled by the nucleotide sequence.

30. The method of claim 1, wherein a plurality of treatments is screened for simultaneously.

31. The method of claim 1, wherein the treatment is a physical, chemical or biological treatment.

32. The method of claim 31, wherein the chemical treatment is effected by a test substance.

33. The method of claim 1, wherein a first plurality of host cells are constructed so that each of said first plurality of host cells contains a first reporter gene encoding a first flourescent protein operatively linked to a different nucleotide sequence of a plurality of nucleotide sequences, a second plurality of host cells are constructed so that each of said second plurality of host cells contains a second reporter gene encoding a second flourescent protein operatively linked to a different nucleotide sequence of said plurality of nucleotide sequences to which said first reporter gene is linked in said first plurality host cells, and the method is used to identify a modulator of production of a plurality of gene products.

44. The method of claim 33, wherein the first plurality of host cells, the second plurality of host cells, or the mixture of the first and second plurality of host cells are placed and assayed in an array on a solid surface.

46. The method of claim 44, wherein the solid surface is selected from the group consisting of silicon, plastic, glass, polymer, ceramic, photoresist and rubber surface.

47. The method of claim 46, wherein the polymer is selected from the group consisting of cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride and polypropylene.

48. The method of claim 46, wherein the silicon surface is a silicon dioxide or a silicon nitride surface.

49. The method of claim 44, wherein the array is made in a chip format.

50. The method of claim 44, wherein the solid surfaces are in the form of tubes, beads, discs, silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon membrane, other porous membrane, non-porous membrane, a plurality of polymeric pins, or a plurality of microtitre wells.

51. The method of claim 1, wherein more than two different reporter genes are used.

52. The method of claim 1, wherein the first and second reporter genes are used duplicatively as the reporter gene in a yeast two-hybrid system, and the method is used for screening for a treatment that modulates the protein-protein interaction assessable by said yeast two-hybrid system.

53. The method of claim 44, wherein the reporter genes used in the plurality of host cells further comprise a molecular tag capable of identifying the host cell containing the reporter gene.

59. The method of claim 1, wherein the first cell and the second host cell is the same cell.

60. The method of claim 1, wherein prior to or concurrently with measurement of the third detectable signal, the growth of the first and second host cells is stopped.

34. A method of screening for a cellular target nucleotide sequence wherein said cellular target nucleotide sequence is regulated by treatment, which method comprises:

a) constructing a first host cell containing a first reporter gene operatively linked to a target nucleotide sequence and constructing a second host cell containing a second reporter gene encoding a second flourescent protein operatively linked to said target nucleotide sequence to which said first reporter gene is linked, wherein expression of said first reporter gene results in a first flourescently detectable signal, expression of said second reporter gene results in a second flourescently detectable signal, said first and second flourescently detectable signals are distinct from each other but are integratable, and wherein mixing of said first and second host cells are growth separately and then mixed to produce a third flourescently detectable signal distinct from said first and second flourescently detectable signals and detection of said third flourescently signal does not depend on the continued growth or viability of said first and second host cells;

b) detecting said third flourescently detectable signal from a mixture of said first and second host cells wherein neither type of said host cells is subjected to any desired treatment;

c) detecting said third flourescently detectable signal from a mixture of said first and second host cells wherein one and only one type of said host cells is subjected to a desired treatment prior to said mixing of said host cells; and

d) comparing said third flourescently detectable signals detected in steps b) and c),

whereby said third flourescently detectable signal detected in step b) differs from that detected in step c) identifies said target nucleotide sequence as amenable to regulation by said treatment.

wherein the method is performed in vitro.

35. The method of claim 34, wherein the treatment is a physical, chemical or biological treatment.

36. The method of claim 34, wherein the treatment is effected by a bioactive substance.

37. The method of claim 34, wherein the target nucleotide sequence is screened against a plurality of treatments simultaneously.

38. The method of claim 36, wherein the target nucleotide sequence is screened against a plurality of bioactive substances simultaneously.

39. The method of claim 34, wherein a first plurality of host cells are constructed so that each of said first plurality of host cells contains a first reporter gene encoding a first flourescent protein operatively linked to a different target nucleotide sequence of a plurality of target nucleotide sequences, a second plurality of host cells are constructed so that each of said second plurality of host cells contains a second reporter gene encoding a second flourescent protein operatively linked to a different target nucleotide sequence of said plurality of target nucleotide sequences to which said first reporter gene is linked in said first plurality of host cells, and the method is used to identify at least one target nucleotide sequence within said plurality of target nucleotide sequences that is amenable to regulation by said treatment.

40. The method of claim 39, wherein the plurality of target nucleotide sequences are endogenous to the host cells and the host cells are derived from a target organism.

41. The method of claim 40, wherein the plurality of target nucleotide sequences comprises an ensemble of the transcriptional regulatory elements of the target organism sufficient to model the transcriptional responsiveness of the target organism to the treatment.

42. The method of claim 41, wherein the ensemble comprises a majority of all different transcriptional regulatory elements of the target organism.

43. The method of claim 39, wherein a plurality of target nucleotide sequence is screened against a plurality of treatments simultaneously.

45. The method of claim 39, wherein the first plurality of host cells, the second plurality of host cells, or the mixture of the first and second plurality of host cells are placed and assayed in an array on a solid surface.

54. The method of claim 45, wherein the reporter genes used in the plurality of host cells further comprise a molecular tag capable of identifying the host cell containing the reporter gene.

55. The method of claim 45, wherein the solid surface is selected from the group consisting of silicon, plastic, glass, polymer, ceramic, photoresist and rubber surface.

56. The method of claim 45, wherein the array is made in a chip format.

57. The method of claim 45, wherein the solid surfaces are in the form of tubes, beads, discs, silicon chips, microplates, polyvinylidene difluoride (PVDF) membrane, nitrocellulose membrane, nylon membrane, other porous membrane, non-porous membrane, a plurality of polymeric pins, or a plurality of microtitre wells.

58. The method of claim 34, wherein more than two different reporter genes are used.

61. A method of screening for a modulator of production of a target gene product, which method comprises:

a) constructing a first host cell containing a first reporter gene encoding a first flourescent protein operatively linked to a nucleotide sequence comprising at least the promoter of a target gene and constructing a second host cell containing a second reporter gene encoding a second flourescent protein operatively linked to said nucleotide sequence to which said first reporter gene is linked, wherein expression of said first reporter gene results in a first flourescently detectable signal, expression of said second reporter gene results in a second flourescently detectable signal, said first and second flourescently detectable signals are distinct from each other but are integratable, and wherein said first and second host cells belong to the same genus, said first and second host cells are grown separately or together, and mixing of said first and second host cells results in a third flourescently detectable signal distinct from said first and second flourescently detectable signals;

b) detecting said third flourescently detectable signal from a mixture of said first and second host cells wherein neither type of said host cells is subjected to any desired treatment;

c) detecting said third flourescently detectable signal from a mixture of said first and second host cells wherein one and only one type of said host cells is subjected to a desired treatment prior to said mixing of said host cells; and

d) comparing said third flourescently detectable signals detected in steps b) and c), whereby said third flourescently detectable signal detected in step b) differs from that detected in step c) identifies said treatment as a modulator of production of said target gene product,

wherein the method is performed in vitro.