Compositions and methods for isolating genes comprising subcellular localization sequences

• US 20020151051A1
• Filed: 03/26/2002
• Published: 10/17/2002
• Est. Priority Date: 03/27/2001
• Status: Abandoned Application

• Current Assignee: Abmaxis, Inc. (Merck & Co., Inc.)
• Original Assignee: Abmaxis, Inc. (Merck & Co., Inc.)
• Inventors: Li, Sheng Feng
• Application Number:
  US10/108,118
• Publication Number:
  US 20020151051A1
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: C07K 14/82 Translation products from oncogenes
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/033 containing a motif for targeting to the internal surface of the plasma membrane, e.g. containing a myristoylation motif
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12N 15/625 containing a sequence coding for a signal sequence
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2799/021 for the expression of a heterologous nucleic acid

Abstract

The present invention provides an expression vector and library thereof suited for categorizing and identifying genes comprising subcellular localization sequences. The invention vectors are particularly suited for isolating extracellular membrane bound, extracellular or secreted proteins. The present invention also provides kits and eukaryotic host cells comprising the invention vectors. Further provided by the invention are methods of using the subject vectors for cloning genes encoding proteins that are preferentially located in certain subcellular locations. Also included is a method of determining the subcellular location of a protein.

Claims

1. A selectable fusion gene comprising a subcellular localization sequence fused in-frame with a defective oncogene that lacks a functional subcellular localization sequence, wherein the selectable fusion gene when expressed in a cell confers cell transformation.

2. The selectable fusion gene of claim 1, wherein the cell transformation is characterized by a phenotypic change selected from the group consisting of formation of cell foci, reduced requirement of serum for cell growth in vitro, and loss of anchorage dependence.

3. The selectable fusion gene of claim 2, wherein the loss of anchorage dependence is further characterized by cell growth in soft agar.

4. The selectable fusion gene of claim 1, wherein the functional subcellular localization sequence is required for the cell transforming activity of the oncogene.

5. The selectable fusion gene of claim 1, wherein the subcellular localization sequence encodes a signal peptide.

6. The selectable fusion gene of claim 1, wherein the subcellular localization sequence encodes a membrane anchorage domain.

7. The selectable fusion gene of claim 1, wherein the subcellular localization sequence encodes a nuclear localization sequence.

8. The selectable fusion gene of claim 1, wherein the defective oncogene is a defective v-sis that lacks a fundamental subcellular localization sequence.

9. The selectable fusion gene of claim 1, wherein the defective oncogene is selected from the group consisting of defective ras, src, v-fos, hedgehog, Wnt1, FGF-8, FGF-9, Mob-5, WISP-1, Int2, and matrix metalloproteinase genes.

10. An expression vector, comprising:

(a) a cloning site;

(b) a region encoding a defective oncogene lacking a functional subcellular localization sequence;

wherein upon inserting in the cloning site a gene fragment comprising a subcellular localization sequence, in-frame with the defective oncogene, expression thereof confers cell transformation.

11. The expression vector of claim 10, wherein the gene fragment comprising a subcellular localization sequence is inserted in-frame with the defective oncogene, expression thereof confers cell transformation.

12. The expression vector of claim 10, wherein the functional subcellular localization sequence is required for the cell transforming activity of the oncogene.

13. The expression vector of claim 10, wherein the cloning site of (a) and the region of (b) are arranged from 5′

to 3′

.

14. The expression vector of claim 10, wherein the region of (b) and the cloning site of (a) are arranged from 5′

to 3′

.

15. The expression vector of claim 10, wherein the cloning site is a multiple cloning site.

16. The expression vector of claim 10, wherein at least one nucleotide is added or subtracted to the cloning site to facilitate the expression of gene fragment in multiple reading frames.

17. The expression vector of claim 15, wherein the multiple cloning site contains an excisable stop codon.

18. The expression vector of claim 10, further comprising at least two origins of replication, wherein at least one first origin facilitates replication in an expression cell type, and at least one second origin facilitates replication in an amplification cell type.

19. The expression vector of claim 10, further comprising at least one gene encoding a selectable marker.

20. The expression vector of claim 18, wherein the expression cell type is eukaryotic and the amplification cell type is prokaryotic.

21. The expression vector of claim 19, wherein the selectable marker facilitates selection in an expression cell type.

22. The expression vector of claim 19, wherein the selectable marker facilitates selection in an amplification cell type.

23. The expression vector of claim 18, wherein the origins of replication are derived from SV40 and pBR322.

24. The expression vector of claim 10, further comprising a promoter 5′

to the cloning site.

25. The expression vector of claim 24, wherein the promoter is a constitutive promoter.

26. The expression vector of claim 24, wherein the promoter is an inducible promoter.

27. The expression vector of claim 24, wherein the promoter is a tissue-specific promoter.

28. The expression vector of claim 10, further comprising a terminator immediately 3′

to the region of (b).

29. The expression vector of claim 10, wherein the vector is a viral vector selected from the group consisting of retroviral vector, adeno-associate vial vector, and adenoviral vector.

30. The expression vector of claim 10, wherein the vector is a non-viral vector.

31. The expression vector of claim 10, wherein the cell transformation is characterized by a phenotypic change selected from the group consisting of formation of cell foci, reduced requirement of serum for cell growth in vitro, and loss of anchorage dependence.

32. The expression vector of claim 31, wherein the loss of anchorage dependence is further characterized by cell growth in soft agar.

33. The expression vector of claim 10, wherein the subcellular localization sequence encodes a signal peptide.

34. The expression vector of claim 10, wherein the subcellular localization sequence encodes a transmembrane domain.

35. The expression vector of claim 10, wherein the subcellular localization sequence encodes a nuclear localization sequence.

36. The expression vector of claim 10, wherein the defective oncogene is a defective v-sis that lacks a functional subcellular localization sequence.

37. The expression vector of claim 10, wherein the defective oncogene is selected from the group consisting of a defective ras, src, v-fos, hedgehog, Wnt1, FGF-8, FGF-9, Mob-5, WISP-1, Int2, and matrix metalloproteinase genes.

38. The expression vector of claim 10, wherein the gene fragment encodes a polypeptide selected from the group consisting of a membrane bound protein, a secreted protein, and a nuclear protein.

39. The expression vector of claim 10, wherein the gene fragment encodes an animal protein or a plant protein.

40. A selectable library comprising a plurality of expression vectors, at least one being a vector of claim 10.

41. A selectable library comprising a plurality of expression vectors at least one being a vector of claim 11.

45. A host cell comprising the expression vector of claim 10 or 11.

46. A population of host cells transfected with a selectable library of claim 41 or 43.

49. A method for conferring a transformation phenotype on a eukaryotic cell, comprising the step of introducing into the cell an expression vector according to claim 11.

50. A method of isolating a gene fragment comprising a functional subcellular localization sequence, the method comprising:

(a) transfecting a population of non-transformed cells a selectable library of expression vectors of claim 41 or 43;

(b) culturing the transfected cells;

(c) identifying transformed cells; and

(d) isolating the gene fragment comprising the functional subcellular localization sequence from the cells exhibiting a transformation phenotype.

51. A method of isolating a gene fragment comprising a functional subcellular localization sequence, the method comprising:

(a) providing a selectable library of expression vectors of claim 41 or 43;

(b) transfecting a population of non-transformed cells with the library of expression vectors;

(c) culturing the transfected cells under conditions and for a time sufficient for expression of the oncogene, and sufficient for cells to exhibit a transformation phenotype; and

(d) isolating the gene fragment comprising the functional subcellular localization sequence from the cells exhibiting a transformation phenotype.

52. The method of claim 51, wherein the gene fragment encodes a polypeptide with a restricted subcellular expression pattern.

53. The method of claim 51, wherein the gene fragment encodes an animal protein or a plant protein.

54. The method of claim 51, wherein the gene fragment comprises a functional signal sequence and encodes a secreted polypeptide.

55. The method of claim 51, wherein the gene fragment comprises a functional membrane anchorage domain and encodes a membrane protein.

56. The method of claim 51, wherein the membrane anchorage domain is a transmembrane domain of an integral membrane protein.

57. The method of claim 51, wherein the gene fragment comprises a functional nuclear localization sequence, and encodes a nuclear protein.

58. The method of claim 51, where the non-transformed cells are eukaryotic cells.

59. The method of claim 51, where the non-transformed cells are mammalian cells.

60. The method of claim 51, where the non-transformed cells have a species origin being selected from the group consisting of human, mouse, rat, fruit fly, Chinese hamster, and worm.

61. The method of claim 51, wherein the gene fragment is fused in-frame from 5′

to 3′

with the oncogene.

62. The method of claim 51, wherein the gene fragment is fused in-frame from 3′

to 5′

with the oncogene.

63. The method of claim 51, wherein the vector further comprises at least two origins of replication, wherein at least one first origin facilitates replication in an expression cell type, and at least one second origin facilitates replication in an amplification cell type.

64. The method of claim 51, wherein the vector further comprises at least one gene encoding a selectable marker.

65. The method of claim 63, wherein the expression cell type is eukaryotic and the amplification cell type is prokaryotic.

66. The method of claim 64, wherein the at least one selectable marker facilitates selection in an expression cell type.

67. The method of claim 64, wherein the at least one selectable marker facilitates selection in an amplification cell type.

68. The method of claim 63, wherein the origins of replication are derived from SV40 and pBR322.

69. The method of claim 51, wherein the cell transforming is characterized by a phenotypic change selected from the group consisting of formation of cell foci, reduced requirement of serum for cell growth in vitro, and loss of anchorage dependence.

70. The method of claim 51, wherein the gene fragment comprises genomic DNA.

71. The method of claim 51, wherein the gene fragment comprises cDNA.

72. The method of claim 51, wherein the defective oncogene is a defective v-sis.

73. The method of claim 51, wherein the defective oncogene is selected from the group consisting of a defective ras, src, v-fos, hedgehog, Wnt1, FGF-8, FGF-9, Mob-5, WISP-1, Int2, and matrix metalloproteinase genes.

74. The method of claim 51, wherein the non-constitutively active oncogene is c-raf

76. A kit comprising an expression vector of claim 10 in suitable packaging.

77. A kit comprising a selectable library of expression vectors of any one of claims 40, 41, 42, and 43 in suitable packaging.

42. A selectable library comprising a plurality of expression vectors, wherein at least one vector comprises:

(a) a cloning site;

(b) a region encoding a non-constitutively active oncogene, wherein upon inserting in the cloning site a gene fragment comprising a subcellular localization sequence, in-frame with the non-constitutively active oncogene, the expression thereof results in constitutive activation of the oncogene and cell transformation.

43. The selectable library of claim 42, wherein the gene fragment is inserted in-frame with the non-constitutively active oncogene.

44. The selectable library of claim 42, wherein the non-constitutively active oncogene is c-raf.

47. The population of host cells of claim 46, where the cells are eukaryotic cells.

48. The population of eukaryotic host cells of claim 47, where the cells have a species origin selected from the group consisting of human, mouse, rat, fruit fly, Chinese hamster, and worm.

75. A method of determining subcellular location of a polypeptide, comprising:

(a) providing an expression vector having a polynucleotide encoding the polypeptide, wherein the polynucleotide is fused in-frame with a defective oncogene or a non-constitutively active oncogene, and wherein the subcellular location at which the oncoprotein encoded by the oncogene acts to transform a cell is known;

(b) transfecting a population of non-transformed cells with the expression vector; and

(c) culturing the transfected cells under conditions and for a time sufficient for expression of the oncogene and sufficient for cells to exhibit a transformation phenotype, wherein an observation of cell transformation indicates that the polypeptide is located in the subcellular location where the oncoprotein acts to transform the cell.