Non-invasive localization of a light-emitting conjugate in a mammal

• US 6,923,951 B2
• Filed: 12/16/2002
• Issued: 08/02/2005
• Est. Priority Date: 07/01/1994
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Contag, Christopher H., Benaron, David A., Contag, Pamela R.
• Primary Examiner(s): Graser, Jennifer E.
• Application Number:
  US10/319,542
• Publication Number:
  US 20030113264A1
• Time in Patent Office: 960 Days
• Field of Search: 424/9.1, 424/9.6, 424/9.61, 424/93.1, 424/93.2, 435/320.1, 435/325, 435/7.9, 435/968, 436/800
• US Class Current: 424/9.1
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

Methods and compositions for detecting and localizing light originating from a mammal are disclosed. Also disclosed are methods for targeting light emission to selected regions, as well as for tracking entities within the mammal. In addition, animal models for disease states are disclosed, as are methods for localizing and tracking the progression of disease or a pathogen within the animal, and for screening putative therapeutic compounds effective to inhibit the disease or pathogen.

Claims

1. A non-invasive method for detecting transformed eukaryotic cells in a mammalian subject, comprising:

administering to the subject eukaryotic cells transformed with a heterologous gene encoding a bioluminescent protein, wherein said subject comprises opaque tissue, and

measuring photon emission through opaque tissue of said subject wherein said photon emission is mediated by bioluminescent protein expressed from said heterologous gene.

2. The method of claim 1, wherein said measuring is done using a photodetector device.

3. The method of claim 2, wherein said measuring is carried out intensified charge-coupled photodetector device.

4. The method of claim 2, wherein said measuring is carried out with a cooled charge-coupled photodetector device.

5. The method of claim 1, wherein said measuring is carried out using fiber optic cables.

6. The method of claim 5, wherein said fiber optic cables terminate in a tightly-packed array.

7. The method of claim 5, wherein said fiber optic cables detect light from a limited defined region of the subject.

8. The method of claim 1, wherein said bioluminescent protein is encoded by lux or luc genes.

9. The method of claim 8, wherein said bioluminescent protein is encoded by lux genes.

10. The method of claim 9, wherein said heterologous expression further comprises expression of luxC, luxD, and luxE accessory genes.

11. The method of claim 9, wherein a substrate for the bioluminescent protein is provided.

12. The method of claim 11, wherein said substrate is an aldehyde.

13. The method of claim 8, wherein said bioluminescent protein is encoded by a luc gene.

14. The method of claim 13, wherein a luciferin substrate is provided for the bioluminescent protein.

15. The method of claim 1, further comprising constructing a photon emission image from said measured photon emission.

16. The method of claim 15, further comprising

acquiring a reflected light image of the subject, and

superimposing said image of photon emission on said reflected light image to form a composite image.

17. The method of claim 1, wherein said eukaryotic cell is a tumor cell.

18. The method of claim 1, wherein said eukaryotic cell is selected from the group consisting of primary culture cells, somatic cells, and lymphatic cells.

19. The method of claim 1, wherein said heterologous expression is regulated by an inducible promoter.

20. The method of claim 19, wherein said promoter is a Tet promoter.

21. The method of claim 1, wherein said heterologous gene expression is mediated by a constitutively active promoter.

22. The method of claim 21, wherein said constitutively active promoter is a CMV or SV40 promoter.

23. The method of claim 1, further comprising:

repeating said detecting at selected intervals,

wherein said repeating is effective to track localization of the expression of the heterologous gene in the subject over time.

24. The method of claim 1, further comprising

administering a compound to said subject, and

measuring photon emission from said subject after administration of said compound.

25. The method of claim 24, further comprising:

repeating at selected intervals said measuring after administration of said compound,

wherein said repeating is effective to track an effect of said compound on a level of expression of said heterologous gene in said subject over time.

26. The method of claim 1, wherein said method further comprises, prior to said detecting, placing the subject in a detection field of the photodetector device.

27. The method of claim 1, wherein photons that make up said photon emission are visible light photons.

28. The method of claim 1, wherein said measuring consists of measuring photon emission from within the subject with a photodetector device located outside of the subject.

29. The method of claim 28, wherein said measuring is done using a photodetector device.

30. The method of claim 29, wherein said measuring is carried out with an intensified charge-coupled photodetector device.

31. The method of claim 29, wherein said measuring is carried out with a cooled charge-coupled photodetector device.

32. The method of claim 28, wherein said measuring is carried out using fiber optic cables.

33. The method of claim 32, wherein said fiber optic cables terminate in a tightly-packed array.

34. The method of claim 32, wherein said fiber optic cables detect light from a limited defined region of the subject.

35. The method of claim 28, wherein said bioluminescent protein is encoded by lux or luc genes.

36. The method of claim 35, wherein said bioluminescent protein is encoded by lux genes.

37. The method of claim 36, wherein said heterologous expression further comprises expression of luxC, luxD, and luxE accessory genes.

38. The method of claim 36, wherein a substrate for the bioluminescent protein is provided.

39. The method of claim 38, wherein said substrate is an aldehyde.

40. The method of claim 35, wherein said bioluminescent protein is encoded by a luc gene.

41. The method of claim 40, wherein a luciferin substrate is provided for the bioluminescent protein.

42. The method of claim 28, further comprising constructing a photon emission image from said measured photon emission.

43. The method of claim 42, further comprising

acquiring a reflected light image of the subject, and

superimposing said image of photon emission on said reflected light image to form a composite image.

44. The method of claim 28, wherein said eukaryotic cell is a tumor cell.

45. The method of claim 28, wherein said eukaryotic cell is selected from the group consisting of primary culture cells, somatic cells, and lymphatic cells.

46. The method of claim 28, wherein said heterologous expression is regulated by an inducible promoter.

47. The method of claim 46, wherein said promoter is a Tet promoter.

48. The method of claim 28, wherein said heterologous gene expression is mediated by a constitutively active promoter.

49. The method of claim 48, wherein said constitutively active promoter is a CMV or SV40 promoter.

50. The method of claim 28, further comprising:

repeating said detecting at selected intervals,

wherein said repeating is effective to track localization of the expression of the heterologous gene in the subject over time.

51. The method of claim 28, further comprising

administering a compound to said subject, and

measuring photon emission from said subject after administration of said compound.

52. The method of claim 51, further comprising:

repeating at selected intervals said measuring after administration of said compound,

wherein said repeating is effective to track an effect of said compound on a level of expression of said heterologous gene in said subject over time.

53. The method of claim 28, wherein said method further comprises, prior to said detecting, placing the subject in a detection field of the photodetector device.

54. The method of claim 28, wherein photons that make up said photon emission are visible light photons.