Claims
- 1. A method of identifying a cancer-related gene, said method comprising:
(a) maintaining cells in which tumorigenicity is dependent upon the expression of an inducible oncogene under conditions in which expression of said oncogene is not induced, wherein said cells are tumorigenic when said oncogene is expressed; (b) introducing into said cells a nucleic acid molecule that integrates into the genomes of said cells, thereby tagging the loci at which it integrates; (c) identifying a cell in which tumorigenicity has been induced by integration of said nucleic acid molecule; and (d) identifying, as said cancer-related gene, a gene that has been tagged in the cell of (c) by the integrated nucleic acid molecule.
- 2. The method of claim 1, wherein the cells of (a) comprise a mutation that renders them susceptible to tumorigenicity.
- 3. The method of claim 1, wherein the cells of (a) comprise a mutation in the INK4a/Arf gene.
- 4. The method of claim 3, wherein the cells of (a) are homozygous for said mutation.
- 5. The method of claim 1, wherein said oncogene is activated ras.
- 6. The method of claim 1, wherein said nucleic acid molecule comprises a retroviral vector sequence.
- 7. The method of claim 1, wherein said nucleic acid molecule is a Moloney murine leukemia virus.
- 8. The method of claim 1, wherein the cells of (a) are melanocytes.
- 9. The method of claim 1, wherein step (c) comprises introducing a cell produced in step (b) into a non-human mammal and monitoring said mammal for development of a tumor.
- 10. The method of claim 1, wherein step (c) comprises a soft agar assay of tumorigenicity.
- 11. The method of claim 1, wherein said cells comprise:
(a) a first expression construct comprising a gene encoding a reverse tetracycline transactivator operably linked to a tissue- or cell type-specific promoter or a general promoter; and (b) a second expression construct comprising said oncogene operably linked to a promoter that is regulated by said reverse tetracycline transactivator and tetracycline or a tetracycline analogue; wherein said first and second expression constructs are stably integrated into the genomes of said cells.
- 12. The method of claim 11, wherein said tissue or cell type-specific promoter is a tyrosinase promoter.
- 13. The method of claim 11, wherein said tetracycline analogue is doxycycline.
- 14. A method for testing the effect of a candidate cancer-related gene on tumorigenicity, said method comprising:
(a) providing a transgenic non-human mammal comprising cells into the genome of which are stably integrated:
(i) a first expression construct comprising a gene encoding a reverse tetracycline transactivator, the gene being operably linked to a tissue- or cell type-specific promoter or a general promoter, and (ii) a second expression construct comprising said candidate cancer-related gene operably linked to a promoter that is regulated by said tetracycline transactivator and tetracycline or a tetracycline analogue, and (b) observing said mammal in the presence of said tetracycline or tetracycline analogue for the development, maintenance, or progression of a tumor that is affected by the presence or absence of said tetracycline or tetracycline analogue.
- 15. The method of claim 14, wherein said candidate cancer-related gene was identified using the method of claim 1.
- 16. The method of claim 14, wherein said transgenic non-human mammal is a mouse.
- 17. The method of claim 14, wherein said tissue- or cell type-specific promoter is a tyrosinase promoter.
- 18. The method of claim 14, wherein said tetracycline analogue is doxycycline.
- 19. The method of claim 14, wherein said tumor is a melanoma tumor.
- 20. A cell comprising (a) a first expression construct comprising a gene encoding a reverse tetracycline transactivator operably linked to a tissue- or cell type-specific promoter or a general promoter, (b) a second expression construct comprising an oncogene operably linked to a promoter that is regulated by said reverse tetracycline transactivator and a tetracycline analogue, and (c) a retroviral vector, wherein said first and second expression constructs and said retroviral vector are stably integrated into the genome of the cell.
- 21. The cell of claim 20, wherein said cell is a tumor cell.
- 22. The cell of claim 20, wherein said retroviral vector is a Moloney murine leukemia virus vector.
- 23. A transgenic, non-human mammal comprising (a) a first expression construct comprising a gene encoding a reverse tetracycline transactivator operably linked to a tissue- or cell type-specific promoter or a general promoter, (b) a second expression construct comprising a candidate cancer-related gene operably linked to a promoter that is regulated by said reverse tetracycline transactivator and a tetracycline analogue, wherein said first and second expression constructs are stably integrated into the genome of cells of said mammal.
- 24. The mammal of claim 23, wherein said mammal is a mouse.
- 25. The mammal of claim 23, wherein said candidate cancer-related gene was identified using the method of claim 1.
- 26. A method of determining the efficacy of a candidate compound in treating cancer, said method comprising contacting the mammal of claim 23 with said candidate compound and observing the effect of said compound on tumor development, maintenance, or progression in said mammal.
- 27. The method of claim 26, wherein said cancer is melanoma.
- 28. A method of identifying a cancer-related gene, said method comprising:
(a) providing cells in which tumorigenicity is dependent upon expression of an oncogene; (b) maintaining said cells under conditions wherein said cells do not express said oncogene, said cells being tumorigenic when said oncogene is expressed; (c) introducing into said cells a nucleic acid molecule that integrates into the genomes of said cells, thereby tagging the loci at which said nucleic acid molecule integrates; (d) identifying a cell in which tumorigenicity has been induced by integration of said nucleic acid molecule; and (e) identifying, as said cancer-related gene, a gene that has been tagged in the cell of (d) by the integrated nucleic acid molecule.
- 29. The method of claim 28, wherein the cells of (a) comprise a mutation in the INK4a/Arf gene.
- 30. The method of claim 28, wherein said oncogene is ras.
- 31. The method of claim 28, wherein said integrated nucleic acid molecule comprises a retroviral vector sequence.
- 32. The method of claim 28, wherein said cells are melanocytes.
- 33. The method of claim 28, wherein step (d) comprises introducing a cell produced in step (c) into a non-human mammal and monitoring said mammal for development of a tumor.
- 34. The method of claim 28, wherein step (d) comprises a soft agar assay of tumorigenicity.
- 35. The method of claim 28, wherein the cells of (a) comprise:
(a) a first expression construct comprising a gene encoding a reverse tetracycline transactivator operably linked to a tissue- or cell type-specific promoter or a general promoter; and (b) a second expression construct comprising said oncogene operably linked to a promoter that is regulated by said reverse tetracycline transactivator and tetracycline or a tetracycline analogue; wherein said first and second expression constructs are stably integrated into the genome of said cells.
- 36. A method of identifying a cancer-related gene, said method comprising:
(a) providing cells, wherein said cells were obtained by:
(i) altering the genome of a tumor cell such that at least one oncogene required for the tumorigenicity of said tumor cell is not expressed, or (ii) altering the genome of a tumor cell such that at least one tumor suppressor gene that prevents the tumorigenicity of said tumor cell is expressed; (b) introducing into said cells a nucleic acid molecule that integrates into the genomes of said cells, thereby tagging the loci at which said nucleic acid molecule integrates; (c) identifying a cell in which tumorigenicity has been induced by integration of said nucleic acid molecule; and (d) identifying, as said cancer-related gene, a gene that has been tagged in the cell of (c) by the integrated nucleic acid molecule.
- 37. The method of claim 36, wherein said tumor cell is a human tumor cell.
- 38. The method of claim 36, wherein said at least one oncogene is ras.
- 39. The method of claim 36, wherein said at least one tumor suppressor gene is an INK4a/Arf gene.
- 40. The method of claim 36, wherein said nucleic acid molecule comprises a retroviral vector sequence.
- 41. The method of claim 36, wherein step (c) comprises introducing a cell produced in step (b) into a non-human mammal and monitoring said mammal for development of a tumor.
- 42. The method of claim 36, wherein step (c) comprises a soft agar assay of tumorigenicity.
- 43. A method of identifying a gene related to a given phenotype, said method comprising:
(a) providing cells in which a given phenotype is dependent upon expression of a gene, (b) maintaining said cells under conditions wherein said cells do not express said gene, said cells comprising said given phenotype when said gene is expressed; (c) introducing into the cells of (b) a nucleic acid molecule that integrates into the genomes of said cells, thereby tagging the loci at which said nucleic acid molecule integrates; (d) identifying a cell in which said given phenotype has been induced by integration of said nucleic acid molecule; and (d) identifying, as said gene related to said given phenotype, a gene that has been tagged in the cell of (d) by the integrated nucleic acid molecule.
- 44. The method of claim 43, wherein said given phenotype is tumorigenicity or the ability to metastasize.
- 45. The method of claim 43, wherein said given phenotype is cell survivability.
- 46. The method of claim 43, wherein said nucleic acid molecule comprises a retroviral vector sequence.
- 47. A method of identifying a gene related to a given phenotype, said method comprising:
(a) providing cells, wherein said cells were obtained by:
(i) altering the genome of a cell exhibiting said given phenotype such that at least one gene required for said given phenotype is not expressed in said cell, or (ii) altering the genome of a cell exhibiting said given phenotype such that at least one suppressor gene that prevents said phenotype is expressed in said cell; (b) introducing into said cells a nucleic acid molecule that integrates into the genomes of said cells thereby tagging the loci at which said nucleic acid molecule integrates; (c) identifying a cell in which said phenotype has been induced by integration of said nucleic acid molecule; and (d) identifying, as said gene related to said given phenotype, a gene that has been tagged in the cell of (c) by the integrated nucleic acid molecule.
- 48. The method of claim 47, wherein said given phenotype is tumorigenicity or the ability to metastasize.
- 49. The method of claim 47, wherein said given phenotype is cell survivability.
- 50. The method of claim 47, wherein said nucleic acid molecule comprises a retroviral vector sequence.
- 51. A method of identifying a cancer-related gene, said method comprising:
(a) providing cells in which metastasis of said cells is dependent upon expression of a gene, (b) maintaining said cells under conditions wherein said cells do not express said gene, wherein said cells metastasize within a mammal when said gene is expressed; (c) introducing into said cells a nucleic acid molecule that integrates into the genomes of said cells, thereby tagging the loci at which said nucleic acid molecule integrates; (d) identifying a cell in which the ability to metastasize has been induced by integration of said nucleic acid molecule; and (e) identifying, as a cancer-related gene, a gene that has been tagged in the cell of (d) by the integrated nucleic acid molecule.
- 52. A method of identifying a cancer-related gene, said method comprising:
(a) providing cells, wherein said cells were obtained by:
(i) altering the genome of a cell exhibiting the ability to metastasize such that at least one gene required for said ability to metastasize is not expressed, or (ii) altering the genome of a cell exhibiting the ability to metastasize such that at least one suppressor gene that prevents said ability to metastasize is expressed in said cell; (b) introducing into said cells a nucleic acid molecule that integrates into the genomes of said cells, thereby tagging the loci at which said nucleic acid molecule integrates; (c) identifying a cell in which said ability to metastasize has been induced by integration of said nucleic acid molecule; and (d) identifying, as said cancer-related gene, a gene that has been tagged in the cell of (c) by the integrated nucleic acid molecule.
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Serial No. 60/279,506, filed Mar. 28, 2001.
Provisional Applications (1)
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Number |
Date |
Country |
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60279506 |
Mar 2001 |
US |