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Last Updated: December 1, 2020

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Claims for Patent: 6,096,717

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Summary for Patent: 6,096,717
Title: Method for producing tagged genes transcripts and proteins
Abstract:The invention described here is a method whereby a molecular tag is put on a gene, transcript and protein in a single recombinational event. The protein tag takes the form of a unique peptide that can be recognized by an antibody or other specific reagent, the transcript tag takes the form of the sequence of nucleotides encoding the peptide that can be recognized by a specific polynucleotide probe, and the gene tag takes the form of a larger sequence of nucleotides that includes the peptide-encoding sequence and other associated nucleotide sequences. The central feature of the invention in its essential form is that the tag-creating DNA has a structure such that when it is inserted into an intron within a gene it creates two hybrid introns separated by a new exon encoding the protein tag. A major virtue of the method is that it allows one to identify new proteins or protein-containing structures, and, having done so, to readily identify and analyze the genes encoding those proteins.
Inventor(s): Jarvik; Jonathan W. (Pittsburg, PA)
Assignee:
Application Number:08/745,404
Patent Claims:1. A method for tagging genes, transcripts and proteins in cells, comprising:

(1) producing a tagged gene by inserting a DNA sequence into an intron of a gene by:

(a) selecting a DNA sequence

(i) having a 5' portion free of any nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT;

(ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, TTCTGAC and TACTAAC, and any nucleotide sequence identical to a known splice branch site in a known gene, followed by;

(iii) a sequence selected from the group consisting of a sequence 14 to 34 nucleotides in length and any nucleotide sequence identical in length to a known spacer region between splice branch and acceptor sites in a known gene, followed by;

(iv) a nucleotide sequence selected from the group consisting of CAGG, TAGG and any sequence identical to a known splice acceptor site in a known gene, followed by;

(v) an open reading frame 3N-1 nucleotides in length, said open reading frame encoding a known peptide tag recognizable by a known reaction characteristic of said known peptide tag, followed by;

(vi) a nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT and any sequence identical to a known splice donor site in a known gene; and

(b) inserting said DNA sequence into said intron within said gene to create a tagged gene; and

(2) incubating said tagged gene within a cell so as to maintain intact or to introduce said tagged gene within the genome of said cell.

2. The method of claim 1 wherein said DNA sequence is introduced into said intron by in vitro recombination methods.

3. The method of claim 1 wherein said DNA sequence is introduced into said intron by in vivo recombination.

4. The method of claim 1 wherein said cell is that of a microorganism.

5. The method of claim 1 wherein said cell belongs to a culture of pleuripotent stem cells derived from a multicellular organism.

6. The method of claim 1 wherein said cell belongs to a somatic cell culture derived from a multicellular organism.

7. The method of claim 1 wherein expression of said gene is promoted by introducing said DNA sequence into said cell by a method chosen from the following group: transformation, electroporation, transduction, transfection, viral infection, bulk loading and liposome fusion.

8. The method of claim 1 wherein said DNA sequence is introduced into said intron by the method of transposon insertion.

9. The method of claim 1 wherein said DNA sequence is part of a recombinant plasmid.

10. The method of claim 1 wherein said DNA sequence is part of a recombinant virus.

11. The method of claim 1 wherein said DNA sequence is part of a recombinant transposon.

12. The method of claim 1 wherein said DNA sequence becomes stably incorporated into the genome of said cell.

13. The method of claim 1 wherein said peptide tag is recognized by specific monoclonal antibodies.

14. The method of claim 1 wherein said peptide tag is recognized by specific polyclonal antibodies.

15. The method of claim 1 wherein said peptide tag is recognized by specific reagents that are not antibodies.

16. The method of claim 1 wherein said gene is contained in a living cell.

17. The method of claim 1 wherein said gene is contained in isolated genomic, viral or organelle DNA.

18. The method according to claim 1 wherein said open reading frame encodes a polypeptide sequence chosen from the group consisting of neomycin phosphotransferase, beta-galactosidase, green fluorescent protein and a substrate for biotin ligase.

19. A method for tagging genes, transcripts and proteins in cells, comprising:

(1) producing a tagged gene by inserting a DNA sequence into an intron of a gene by:

(a) selecting a first DNA sequence

(i) having a 5' portion free of any nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT;

(ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, TTCTGAC and TACTAAC, and any nucleotide sequence identical to a known splice branch site in a known gene, followed by;

(iii) a sequence selected from the group consisting of a sequence 14 to 34 nucleotides in length and any nucleotide sequence identical in length to a known spacer region between splice branch and acceptor sites in a known gene, followed by;

(iv) a nucleotide sequence selected from the group consisting of CAGG, TAGG and any sequence identical to a known splice acceptor site in a known gene, followed by;

(v) an open reading frame 3N nucleotides in length, said open reading frame encoding a known peptide tag recognizable by a known reaction characteristic of said known peptide tag, followed by;

(vi) a nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT and any sequence identical to a known splice donor site in a known gene;

(b) selecting a second DNA sequence

(i) having a 5' portion free of any nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT;

(ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, TTCTGAC and TACTAAC, and any nucleotide sequence identical to a known splice branch site in a known gene, followed by;

(iii) a sequence selected from the group consisting of a sequence 14 to 34 nucleotides in length and any nucleotide sequence identical in length to a known spacer region between splice branch and acceptor sites in a known gene, followed by;

(iv) a nucleotide sequence selected from the group consisting of CAGG, TAGG and any sequence identical to a known splice acceptor site in a known gene, followed by;

(v) an open reading frame 3N+1 nucleotides in length, said open reading frame encoding a known peptide tag recognizable by a known reaction characteristic of said known peptide tag, followed by;

(vi) a nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT and any sequence identical to a known splice donor site in a known gene; and

(c) inserting said first and second DNA sequences in tandem into said intron within said gene to create a tagged gene; and

(2) incubating said tagged gene within a cell so as to maintain intact or to introduce said tagged gene within the genome of said cell.

20. The method of claim 19 wherein said DNA sequence is introduced into said intron by in vitro recombination methods.

21. The method of claim 19 wherein said DNA sequence is introduced into said intron by in vivo recombination.

22. The method of claim 19 wherein said cell is that of a microorganism.

23. The method of claim 19 wherein said cell belongs to a culture of pleuripotent stem cells derived from a multicellular organism.

24. The method of claim 19 wherein said cell belongs to a somatic cell culture derived from a multicellular organism.

25. The method of claim 19 wherein expression of said gene is promoted by introducing said DNA sequence into said cell by a method chosen from the following group: transformation, electroporation, transduction, transfection, viral infection, bulk loading and liposome fusion.

26. The method of claim 19 wherein said DNA sequence is introduced into said intron by the method of transposon insertion.

27. The method of claim 19 wherein said DNA sequence is part of a recombinant plasmid.

28. The method of claim 19 wherein said DNA sequence is part of a recombinant virus.

29. The method of claim 19 wherein said DNA sequence is part of a recombinant transposon.

30. The method of claim 19 wherein said DNA sequence becomes stably incorporated into the genome of said cell.

31. The method of claim 19 wherein said peptide tag is recognized by specific monoclonal antibodies.

32. The method of claim 19 wherein said peptide tag is recognized by specific polyclonal antibodies.

33. The method of claim 19 wherein said peptide tag is recognized by specific reagents that are not antibodies.

34. The method of claim 19 wherein said gene is contained in a living cell.

35. The method of claim 19 wherein said gene is contained in isolated genomic, viral or organelle DNA.

36. The method according to claim 19 wherein said first sequence is 5' to said second sequence.

37. The method according to claim 19 wherein said first sequence is 3' to said second sequence.

38. The method according to claim 19 wherein said first or second sequence is flanked by nucleotide sequences known to be recombinogenic.

39. The method according to claim 38 wherein the recombinogenic sequences are lox sites.

40. The method according to claim 38 wherein the recombinogenic sequences are FRT sites.

41. A method for creating a frameshift mutation in a gene in a cell by tagging said gene by the method of claim 38 followed by the activation of the recombinogenic sequences so as to delete said sequence flanked by the recombinogenic sequences.

42. The method according to claim 41 wherein the activation of the recombinogenic sequences is catalyzed by cre recombinase.

43. The method according to claim 41 wherein the activation of said recombinogenic sequences is catalyzed by FLP recombinase.

44. The method according to claim 19 wherein one or both of said open reading frames encode a polypeptide sequence chosen from the group consisting of neomycin phosphotransferase, beta-galactosidase, green fluorescent protein and a substrate for biotin ligase.

45. A method for tagging genes, transcripts and proteins in cells, comprising:

(1) producing a tagged gene by inserting a DNA sequence into an intron of a gene by:

(a) selecting a first DNA sequence

(i) having a 5' portion free of any nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT;

(ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, TTCTGAC and TACTAAC, and any nucleotide sequence identical to a known splice branch site in a known gene, followed by;

(iii) a sequence selected from the group consisting of a sequence 14 to 34 nucleotides in length and any nucleotide sequence identical in length to a known spacer region between splice branch and acceptor sites in a known gene, followed by;

(iv) a nucleotide sequence selected from the group consisting of CAGG, TAGG and any sequence identical to a known splice acceptor site in a known gene, followed by;

(v) an open reading frame 3N-1 nucleotides in length, said open reading frame encoding a known peptide tag recognizable by a known reaction characteristic of said known peptide tag, followed by;

(vi) a nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT and any sequence identical to a known splice donor site in a known gene;

(b) selecting a second DNA sequence

(i) having a 5' portion free of any nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT;

(ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, TTCTGAC and TACTAAC, and any nucleotide sequence identical to a known splice branch site in a known gene, followed by;

(iii) a sequence selected from the group consisting of a sequence 14 to 34 nucleotides in length and any nucleotide sequence identical in length to a known spacer region between splice branch and acceptor sites in a known gene, followed by;

(iv) a nucleotide sequence selected from the group consisting of CAGG, TAGG and any sequence identical to a known splice acceptor site in a known gene, followed by;

(v) an open reading frame 3N or 3N+1 nucleotides in length, said open reading frame encoding a known peptide tag recognizable by a known reaction characteristic of said known peptide tag, followed by;

(vi) a nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT and any sequence identical to a known splice donor site in a known gene; and

(c) inserting said first and second DNA sequences in tandem into said intron within said gene to create a tagged gene; and

(2) incubating said tagged gene within a cell so as to maintain intact or to introduce said tagged gene within the genome of said cell.

46. The method of claim 45 wherein said DNA sequence is introduced into said intron by in vitro recombination methods.

47. The method of claim 45 wherein said DNA sequence is introduced into said intron by in vivo recombination.

48. The method of claim 45 wherein said cell is that of a microorganism.

49. The method of claim 45 wherein said cell belongs to a culture of pleuripotent stem cells derived from a multicellular organism.

50. The method of claim 45 wherein said cell belongs to a somatic cell culture derived from a multicellular organism.

51. The method of claim 45 wherein expression of said gene is promoted by introducing said DNA sequence into said cell by a method chosen from the following group: transformation, electroporation, transduction, transfection, viral infection, bulk loading and liposome fusion.

52. The method of claim 45 wherein said DNA sequence is introduced into said intron by the method of transposon insertion.

53. The method of claim 45 wherein said DNA sequence is part of a recombinant plasmid.

54. The method of claim 45 wherein said DNA sequence is part of a recombinant virus.

55. The method of claim 45 wherein said DNA sequence is part of a recombinant transposon.

56. The method of claim 45 wherein said DNA sequence becomes stably incorporated into the genome of said cell.

57. The method of claim 45 wherein said peptide tag is recognized by specific monoclonal antibodies.

58. The method of claim 45 wherein said peptide tag is recognized by specific polyclonal antibodies.

59. The method of claim 45 wherein said peptide tag is recognized by specific reagents that are not antibodies.

60. The method of claim 45 wherein said gene is contained in a living cell.

61. The method of claim 45 wherein said gene is contained in isolated genomic, viral or organelle DNA.

62. The method according to claim 45 wherein said first sequence is 5' to said second sequence.

63. The method according to claim 45 wherein said first sequence is 3' to said second sequence.

64. The method according to claim 45 wherein said first or second sequence is flanked by nucleotide sequences known to be recombinogenic.

65. The method according to claim 64 wherein the recombinogenic sequences are lox sites.

66. The method according to claim 64 wherein the recombinogenic sequences are FRT sites.

67. A method for creating a frameshift mutation in a gene in a cell by tagging said gene by the method of claim 64 followed by the activation of the recombinogenic sequences so as to delete said sequence flanked by the recombinogenic sequences.

68. The method according to claim 64 wherein the activation of the recombinogenic sequences is catalyzed by cre recombinase.

69. The method according to claim 64 wherein the activation of the recombinogenic sequences is catalyzed by FLP recombinase.

70. The method according to claim 45 wherein one or both of said open reading frames encode a polypeptide sequence chosen from the group consisting of neomycin phosphotransferase, beta-galactosidase, green fluorescent protein and a substrate for biotin ligase.

71. A method for tagging genes, transcripts and proteins in cells, comprising:

(1) producing a tagged gene by inserting a DNA sequence into an intron of a gene by:

(a) selecting a first DNA sequence

(i) having a 5' portion free of any nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT;

(ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, TTCTGAC and TACTAAC, and any nucleotide sequence identical to a known splice branch site in a known gene, followed by;

(iii) a sequence selected from the group consisting of a sequence 14 to 34 nucleotides in length and any nucleotide sequence identical in length to a known spacer region between splice branch and acceptor sites in a known gene, followed by;

(iv) a nucleotide sequence selected from the group consisting of CAGG, TAGG and any sequence identical to a known splice acceptor site in a known gene, followed by;

(v) an open reading frame 3N-1 nucleotides in length, said open reading frame encoding a known peptide tag recognizable by a known reaction characteristic of said known peptide tag, followed by;

(vi) a nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT and any sequence identical to a known splice donor site in a known gene;

(b) selecting a second DNA sequence

(i) having a 5' portion free of any nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT;

(ii) a nucleotide sequence selected from the group consisting of TACTAAC, TGCTAAC, TCCTAAC, TTCTAAC, TACTGAC, TGCTGAC, TCCTGAC, TTCTGAC and TACTAAC, and any nucleotide sequence identical to a known splice branch site in a known gene, followed by;

(iii) a sequence selected from the group consisting of a sequence 14 to 34 nucleotides in length and any nucleotide sequence identical in length to a known spacer region between splice branch and acceptor sites in a known gene, followed by;

(iv) a nucleotide sequence selected from the group consisting of CAGG, TAGG and any sequence identical to a known splice acceptor site in a known gene, followed by;

(v) a sequence of nucleotides containing one or more stop codons in one or more reading frames;

(vi) a nucleotide sequence selected from the group consisting of CAGGTAAGT, CAGGTGAGT, AAGGTAAGT, AAGGTGAGT and any sequence identical to a known splice donor site in a known gene; and

(c) inserting said first and second DNA sequences in tandem into said intron within said gene to create a tagged gene; and

(2) incubating said tagged gene within a cell so as to maintain intact or to introduce said tagged gene within the genome of said cell.

72. The method of claim 71 wherein said DNA sequence is introduced into said intron by in vitro recombination methods.

73. The method of claim 71 wherein said DNA sequence is introduced into said intron by in vivo recombination.

74. The method of claim 71 wherein said cell is that of a microorganism.

75. The method of claim 71 wherein said cell belongs to a culture of pleuripotent stem cells derived from a multicellular organism.

76. The method of claim 71 wherein said cell belongs to a somatic cell culture derived from a multicellular organism.

77. The method of claim 71 wherein expression of said gene is promoted by introducing said DNA sequence into said cell by a method chosen from the following group: transformation, electroporation, transfection, bulk loading and liposome fusion.

78. The method of claim 71 wherein said DNA sequence is introduced into said intron by the method of transposon insertion.

79. The method of claim 71 wherein said DNA sequence is part of a recombinant plasmid.

80. The method of claim 71 wherein said DNA sequence is part of a recombinant virus.

81. The method of claim 71 wherein said DNA sequence is part of a recombinant transposon.

82. The method of claim 71 wherein said DNA sequence becomes stably incorporated into the genome of said cell.

83. The method of claim 71 wherein said peptide tag is recognized by specific monoclonal antibodies.

84. The method of claim 71 wherein said peptide tag is recognized by specific polyclonal antibodies.

85. The method of claim 71 wherein said peptide tag is recognized by specific reagents that are not antibodies.

86. The method of claim 71 wherein said gene is contained in a living cell.

87. The method of claim 71 wherein said gene is contained in isolated genomic, viral or organelle DNA.

88. The method according to claim 71 wherein said first sequence is 5' to said second sequence.

89. The method according to claim 71 wherein said first sequence is 3' to said second sequence.

90. The method according to claim 71 wherein said first or second sequence is flanked by nucleotide sequences known to be recombinogenic.

91. The method according to claim 90 wherein the recombinogenic sequences are lox sites.

92. The method according to claim 90 wherein the recombinogenic sequences are FRT sites.

93. A method for eliminating a nonsense mutation in a gene by tagging said gene by the method of claim 90 followed by the activation of the recombinogenic sequences so as to delete said sequence flanked by the recombinogenic sequences.

94. The method according to claim 93 wherein the activation of the recombinogenic sequences is catalyzed by cre recombinase.

95. The method according to claim 93 wherein the activation of the recombinogenic sequences is catalyzed by FLP recombinase.

96. The method according to claim 71 wherein said open reading frame or said sequence of nucleotides containing one or more stop codons encode a polypeptide sequence chosen from the group consisting of neomycin phosphotransferase, beta-galactosidase, green fluorescent protein and a substrate for biotin ligase.

97. A eukaryotic cell containing at least one gene tagged in accordance with the method of claim 1.

98. A eukaryotic cell containing at least one gene tagged in accordance with the method of claim 19.

99. A eukaryotic cell containing at least one gene tagged in accordance with the method of claim 45.

100. A eukaryotic cell containing at least one gene tagged in accordance with the method of claim 71.

Details for Patent 6,096,717

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Schering INTRON A interferon alfa-2b VIAL 103132 001 1986-06-04   Start Trial 2039-02-26 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 002 1986-06-04   Start Trial 2039-02-26 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 003 1986-06-04   Start Trial 2039-02-26 RX search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

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