Claims
- 1. A nucleic acid fragment comprising a nucleic acid sequence comprising a coding sequence, the nucleic acid sequence positioned between at least two inverted repeats wherein the inverted repeats can bind to an SB protein.
- 2. The fragment of claim 1 wherein the coding sequence is a detectable marker coding sequence that encodes a detectable marker or a selectable marker.
- 3. The fragment of claim 2 wherein the detectable marker is green fluorescent protein.
- 4. The fragment of claim 2 wherein the selectable marker is neomycin.
- 5. The fragment of claim 2 wherein the nucleic acid sequence further comprises a weak promoter operably linked to the detectable marker coding sequence.
- 6. The fragment of claim 5 wherein the promoter comprises a carp β-actin promoter.
- 7. The fragment of claim 2 wherein the nucleic acid sequence further comprises a splice acceptor site and an internal ribosome entry site, each being operably linked to the detectable marker coding sequence.
- 8. The fragment of claim 2 wherein the nucleic acid sequence further comprises (i) an analyte coding sequence located 5′ of the detectable marker coding sequence and (ii) an internal ribosome entry site located therebetween, the internal ribosome entry site being operably linked to the detectable marker coding sequence.
- 9. The fragment of claim 8 wherein the analyte coding sequence is derived from a library of DNA fragments.
- 10. The fragment of claim 8 wherein the analyte coding sequence comprises a preselected coding sequence.
- 11. The fragment of claim 8 wherein the analyte coding sequence is operably linked to a promoter.
- 12. The fragment of claim 11 wherein the promoter is a native promoter.
- 13. A method for identifying an enhancer in a cell comprising:
(a) introducing a nucleic acid fragment and a transposase source into a cell wherein the nucleic acid fragment comprises a nucleic acid sequence comprising a detectable marker coding sequence encoding a detectable marker or a selectable marker, wherein the detectable marker coding sequence is operably linked to a weak promoter, the nucleic acid sequence positioned between at least two inverted repeats, wherein the inverted repeats can bind to an SB protein; (b) detecting the detectable marker or the selectable marker in the cell or its progeny containing the nucleic acid fragment, wherein the expression of the detectable marker or the selectable marker indicates that the nucleic acid fragment has integrated into the DNA of the cell or its progeny within a domain that contains an enhancer.
- 14. The method of claim 13 wherein the transposase is an SB protein having an amino acid sequence identity of least about 80% to SEQ ID NO:1.
- 15. The method of claim 13 wherein the cell is a pluripotent or totipotent cell.
- 16. The method of claim 15 wherein the pluripotent or totipotent cell is selected from the group consisting of an oocyte, a cell of an embryo, an egg and a stem cell.
- 17. The method of claim 13 wherein the detectable marker is green fluorescent protein.
- 18. The method of claim 13 wherein the selectable marker is neomycin.
- 19. The method of claim 13 further comprising (c) determining whether a phenotype of a cell that contains the nucleic acid fragment is altered in comparison to a cell that does not comprise the nucleic acid fragment.
- 20. The method of claim 13 further comprising, prior to (b), growing the cell into an animal.
- 21. The method of claim 20 further comprising (c) determining whether a phenotype of an animal that contains the nucleic acid fragment is altered in comparison to an animal that does not comprise the nucleic acid fragment.
- 22. The method of claim 13 wherein the transposase source is an mRNA encoding the transposase.
- 23. The method of claim 13 wherein the transposase source is a transposase.
- 24. The method of claim 13 further comprising:
(c) cleaving the DNA of the cell with a restriction endonuclease to yield at least one restriction fragment comprising (i) at least a portion of the integrated nucleic acid fragment, which portion comprises at least a portion of an inverted repeat sequence and (ii) genomic DNA of the cell, which genomic DNA is adjacent to the inverted repeat sequence; (d) determining the nucleotide sequence of at least a portion of the genomic DNA; and (e) comparing the nucleotide sequence with sequence information in a computer database.
- 25. A method for identifying a genomic coding sequence in a cell comprising:
(a) introducing a nucleic acid fragment and a transposase source into a cell wherein the nucleic acid fragment comprises a nucleic acid sequence comprising (i) a detectable marker coding sequence encoding a detectable marker or a selectable marker, (ii) a splice acceptor site and (iii) an internal ribosome entry site; wherein the splice acceptor site and internal ribosome entry site are each operably linked to the detectable marker coding sequence, the nucleic acid sequence positioned between at least two inverted repeats, and wherein the inverted repeats can bind to an SB protein; and (b) detecting the detectable marker or the selectable marker in the cell or its progeny containing the nucleic acid fragment, wherein expression of the detectable marker or the selectable marker indicates that the nucleic acid fragment has integrated within a genomic coding sequence of the cell or its progeny.
- 26. The method of claim 25 wherein the detectable marker or the selectable marker is expressed spatially and temporally in the same way as the genomic coding sequence is expressed when not interrupted.
- 27. The method of claim 25 wherein the transposase is an SB protein having an amino acid sequence identity of least about 80% to SEQ ID NO:1.
- 28. The method of claim 25 wherein the cell is a pluripotent or totipotent cell.
- 29. The method of claim 28 wherein the pluripotent or totipotent cell is selected from the group consisting of an oocyte, a cell of an embryo, an egg and a stem cell.
- 30. The method of claim 25 wherein the detectable marker is green fluorescent protein.
- 31. The method of claim 25 wherein the selectable marker is neomycin.
- 32. The method of claim 25 further comprising (c) determining whether a phenotype of a cell that contains the nucleic acid fragment is altered in comparison to a cell that does not comprise the nucleic acid fragment.
- 33. The method of claim 25 further comprising, prior to (b), growing the cell into an animal.
- 34. The method of claim 33 further comprising step (c) of determining whether a phenotype of an animal that contains the nucleic acid fragment is altered in comparison to an animal that does not comprise the nucleic acid fragment.
- 35. The method of claim 25 wherein the transposase source is an mRNA encoding the transposase.
- 36. The method of claim 25 wherein the transposase source is a transposase.
- 37. The method of claim 25 further comprising:
(c) cleaving the DNA of the cell with a restriction endonuclease to yield at least one restriction fragment comprising (i) at least a portion of the integrated nucleic acid fragment, which portion comprises at least a portion of an inverted repeat sequence and (ii) genomic DNA of the cell, which genomic DNA is adjacent to the inverted repeat sequence; (d) determining the nucleotide sequence of at least a portion of the genomic DNA; and (e) comparing the nucleotide sequence with sequence information in a computer database.
- 38. A method for identifying the function of an analyte coding sequence comprising:
(a) introducing a nucleic acid fragment and a transposase source into a cell wherein the nucleic acid fragment comprises a nucleic acid sequence comprising (i) a detectable marker coding sequence encoding a detectable marker or a selectable marker, (ii) an analyte coding sequence located 5′ of the detectable marker coding sequence and (iii) an internal ribosome entry site located therebetween, the internal ribosome entry site being operably linked to the detectable marker coding sequence, the nucleic acid fragment positioned between at least two inverted repeats, and wherein the inverted repeats can bind to an SB protein; (b) detecting the detectable marker or the selectable marker in the cell or its progeny containing the nucleic acid fragment, wherein the expression of the detectable marker or the selectable marker indicates that the nucleic acid fragment has integrated into the DNA of the cell and that the analyte coding sequence is expressed; and (c) determining whether a phenotype of the cell or its progeny containing the nucleic acid fragment is altered in comparison to a cell that does not comprise the nucleic acid fragment, wherein an altered phenotype indicates that the analyte coding sequence plays a function in the phenotype.
- 39. The method of claim 38 wherein the transposase is an SB protein having an amino acid sequence identity of least about 80% to SEQ ID NO:1.
- 40. The method of claim 38 wherein the cell is a pluripotent or totipotent cell.
- 41. The method of claim 40 wherein the pluripotent or totipotent cell is selected from the group consisting of an oocyte, a cell of an embryo, an egg and a stem cell.
- 42. The method of claim 38 wherein the detectable marker is green fluorescent protein.
- 43. The method of claim 38 wherein the selectable marker is neomycin.
- 44. The method of claim 38 further comprising, prior to (b), growing the cell into an animal.
- 45. The method of claim 44 wherein step (c) comprises determining the phenotype of an animal.
- 46. The method of claim 38 wherein the analyte coding sequence is derived from a library of DNA fragments.
- 47. The method of claim 38 wherein the analyte coding sequence comprises a preselected coding sequence.
- 48. The method of claim 38 wherein the analyte coding sequence is operably linked to a promoter.
- 49. A gene transfer system to introduce a nucleic acid sequence into the DNA of a cell comprising:
(a) a nucleic acid fragment comprising a nucleic acid sequence comprising a coding sequence, the nucleic acid sequence positioned between at least two inverted repeats wherein the inverted repeats can bind to an SB protein; and (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
- 50. The gene transfer system of 49 wherein the SB protein has an amino acid sequence identity of least about 80% to SEQ ID NO:1.
- 51. The gene transfer system of claim 49 wherein the cell is a pluripotent or totipotent cell.
- 52. The gene transfer system of claim 51 wherein the pluripotent or totipotent cell is selected from the group consisting of an oocyte, a cell of an embryo, an egg and a stem cell.
- 53. The gene transfer system of claim 49 wherein the coding sequence is a detectable marker coding sequence that encodes a detectable marker or a selectable marker.
- 54. The gene transfer system of claim 53 wherein the detectable marker is green fluorescent protein.
- 55. The gene transfer system of claim 53 wherein the selectable marker is neomycin.
- 56. The gene transfer system of claim 53 wherein the nucleic acid sequence further comprises a weak promoter operably linked to the detectable marker coding sequence.
- 57. The gene transfer system of claim 56 wherein the promoter comprises a carp β-actin promoter.
- 58. The gene transfer system of claim 53 wherein the nucleic acid sequence further comprises a splice acceptor site and an internal ribosome entry site, each being operably linked to the detectable marker coding sequence.
- 59. The gene transfer system of claim 53 wherein the nucleic acid sequence further comprises (i) an analyte coding sequence located 5′ of the detectable marker coding sequence and (ii) an internal ribosome entry site located therebetween, the internal ribosome entry site being operably linked to the detectable marker coding sequence.
- 60. The gene transfer system of claim 59 wherein the analyte coding sequence is derived from a library of DNA fragments.
- 61. The gene transfer system of claim 59 wherein the analyte coding sequence comprises a preselected coding sequence.
- 62. The gene transfer system of claim 59 wherein the analyte coding sequence is operably linked to a promoter.
- 63. The gene transfer system of claim 49 wherein the transposase source is an mRNA.
- 64. The gene transfer system of claim 49 wherein the transposase source is a transposase.
- 65. The gene transfer system of claim 49 wherein the nucleic acid encoding the transposase is integrated into the genome of the cell.
- 66. The gene transfer system of claim 49 wherein the nucleic acid fragment is part of a plasmid or a recombinant viral vector.
- 67. The gene transfer system of claim 49 wherein the cell is an animal cell.
- 68. The gene transfer system of claim 67 wherein the cell is a vertebrate or an invertebrate cell.
- 69. The gene transfer system of claim 68 wherein the invertebrate is a crustacean or a mollusk.
- 70. The gene transfer system of claim 68 wherein the cell is a fish cell or a bird cell.
- 71. The gene transfer system of claim 68 wherein the vertebrate is a mammal.
- 72. The gene transfer system of claim 71 wherein the cell is selected from the group consisting of a rodent cell, an ungulate cell, a sheep cell, a swine cell and a human cell.
- 73. A method for producing a transgenic animal comprising:
(a) introducing a nucleic acid fragment and a transposase source into a cell wherein the nucleic acid fragment comprises a nucleic acid sequence comprising a heterologous coding sequence, the nucleic acid sequence positioned between at least two inverted repeats, wherein the inverted repeats can bind to an SB protein to yield a transgenic cell; and (b) growing the transgenic cell into a transgenic animal.
- 74. The method of 73 wherein the SB protein has an amino acid sequence identity of least about 80% to SEQ ID NO:1.
- 75. The method of claim 73 wherein the cell is a pluripotent or totipotent cell.
- 76. The method of claim 75 wherein the pluripotent or totipotent cell is selected from the group consisting of an oocyte, a cell of an embryo, an egg and a stem cell.
- 77. The method of claim 73 wherein the animal is a mouse, a fish, an ungulate, a bird, or a sheep.
- 78. The transgenic animal of claim 73 and its progeny.
- 79. A gene transfer system to introduce a nucleic acid sequence into the DNA of a fish comprising a nucleic acid fragment comprising a nucleic acid sequence comprising an internal ribosome entry site, wherein the nucleic acid fragment is capable of integrating into the genomic DNA of a fish.
- 80. The gene transfer system of claim 79 wherein the nucleic acid sequence further comprises a coding sequence located 3′ to and operably linked to the internal ribosome entry site.
- 81. The gene transfer system of claim 80 wherein the coding sequence is a first coding sequence, the nucleic acid sequence further comprising a second coding sequence located 5′ to both the first coding sequence and the internal ribosome entry site.
- 82. A transgenic fish or fish cell comprising a heterologous internal ribosome entry site.
- 83. A nucleic acid fragment comprising a nucleic acid sequence comprising a weak promoter operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein.
- 84. A nucleic acid fragment comprising a nucleic acid sequence comprising a splice acceptor site and an internal ribosome entry site, each operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein.
- 85. A nucleic acid fragment comprising a nucleic acid sequence comprising (i) a detectable marker coding sequence that encodes a detectable marker or a selectable marker, (ii) an analyte coding sequence located 5′ of the detectable marker coding sequence and (iii) an internal ribosome entry site located therebetween and operably linked to the detectable marker coding sequence, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein.
- 86. The fragment of claim 85 wherein the analyte coding sequence is derived from a library of DNA fragments.
- 87. The fragment of claim 85 wherein the analyte coding sequence comprises a preselected coding sequence.
- 88. The fragment of claim 85 wherein the analyte coding sequence is operably linked to a promoter.
- 89. The gene transfer system of claims 83, 84, or 85, wherein the SB protein has an amino acid sequence identity of least about 80% to SEQ ID NO:1.
- 90. An organism comprising the nucleic acid fragment of claims 83, 84, or 85.
- 91. A gene transfer system to introduce a nucleic acid sequence into the DNA of a cell comprising:
(a) a nucleic acid fragment comprising a nucleic acid sequence comprising a weak promoter operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein; and (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
- 92. A gene transfer system to introduce a nucleic acid sequence into the DNA of a cell comprising:
(a) a nucleic acid fragment comprising a nucleic acid sequence comprising a splice acceptor site and an internal ribosome entry site, each operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein; and (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
- 93. A gene transfer system to introduce a nucleic acid sequence into the DNA of a cell comprising:
(a) a nucleic acid fragment comprising a nucleic acid sequence comprising (i) a detectable marker coding sequence that encodes a detectable marker or a selectable marker, (ii) an analyte coding sequence located 5′ of the detectable marker coding sequence and (iii) an internal ribosome entry site located therebetween and operably linked to the detectable marker coding sequence, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein.; and (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
- 94. The gene transfer system of claim 93 wherein the analyte coding sequence is derived from a library of DNA fragments.
- 95. The gene transfer system of claim 93 wherein the analyte coding sequence comprises a preselected coding sequence.
- 96. The gene transfer system of claim 93 wherein the analyte coding sequence is operably linked to a promoter.
- 97. The gene transfer system of claims 91, 92 or 93, wherein the SB protein has an amino acid sequence identity of least about 80% to SEQ ID NO:1.
- 98. The gene transfer system of claims 91, 92 or 93, wherein the cell is a pluripotent or totipotent cell.
- 99. The gene transfer system of claim 98 wherein the pluripotent or totipotent cell is selected from the group consisting of an oocyte, a cell of an embryo, an egg and a stem cell.
- 100. The gene transfer system of claims 91, 92 or 93, wherein the transposase source is an mRNA.
- 101. The gene transfer system of claims 91, 92 or 93, wherein the transposase source is a transposase.
- 102. The gene transfer system of claims 91, 92 or 93, wherein the transposase source is a nucleic acid encoding the transposase that is integrated into the genome of the cell.
- 103. The gene transfer system of claims 91, 92 or 93, wherein the nucleic acid fragment is part of a plasmid or a recombinant viral vector.
- 104. The gene transfer system of claims 91, 92 or 93, wherein the cell is an animal cell.
- 105. The gene transfer system of claim 104 wherein the cell is a vertebrate or an invertebrate cell.
- 106. The gene transfer system of claim 105 wherein the cell is a fish cell or a bird cell.
- 107. The gene transfer system of claim 105 wherein the vertebrate is a mammal.
- 108. The gene transfer system of claim 107 wherein the cell is selected from the group consisting of a rodent cell, an ungulate cell, a sheep cell, a swine cell and a human cell.
- 109. The method of claim 13, 25 or 38 wherein the vertebrate cell is selected from the group consisting of a fish cell, a human cell and a mouse cell.
- 110. A method for identifying an enhancer in an isolated or cultured vertebrate cell comprising:
(a) introducing into an isolated or cultured vertebrate cell
(i) a nucleic acid fragment comprising a nucleic acid sequence comprising a detectable marker coding sequence encoding a detectable marker or a selectable marker, wherein the detectable marker coding sequence is operably linked to a weak promoter, the nucleic acid sequence positioned between at least two inverted repeats that can bind to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, and wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell; and (ii) a transposase source selected from the group consisting of a transposase and a nucleic acid encoding a transposase, wherein the transposase is an SB protein; (b) detecting the detectable marker or the selectable marker in the cell or its progeny containing the nucleic acid fragment, wherein the expression of the detectable marker or the selectable marker indicates that the nucleic acid fragment has integrated into the DNA of the cell or its progeny within a domain that contains an enhancer.
- 111. The method of claim 110 wherein the vertebrate cell is a pluripotent or totipotent vertebrate cell.
- 112. The method of claim 111 wherein the pluripotent or totipotent vertebrate cell is selected from the group consisting of an oocyte, a cell isolated from an embryo, an egg cell and a stem cell.
- 113. The method of claim 110 wherein the detectable marker is green fluorescent protein.
- 114. The method of claim 110 wherein the selectable marker is neomycin.
- 115. The method of claim 110 further comprising (c) determining whether a phenotype of a cell that contains the nucleic acid fragment is altered in comparison to a cell that does not comprise the nucleic acid fragment.
- 116. The method of claim 110 further comprising, prior to (b), growing the cell into an animal.
- 117. The method of claim 116 further comprising (c) determining whether a phenotype of an animal that contains the nucleic acid fragment is altered in comparison to an animal that does not comprise the nucleic acid fragment.
- 118. The method of claim 110 wherein the transposase source is an mRNA encoding the transposase.
- 119. The method of claim 110 wherein the transposase source is a transposase.
- 120. The method of claim 110 further comprising:
(c) cleaving the DNA of the cell with a restriction endonuclease to yield at least one restriction fragment comprising (i) at least a portion of the integrated nucleic acid fragment, which portion comprises at least a portion of an inverted repeat sequence and (ii) genomic DNA of the cell, which genomic DNA is adjacent to the inverted repeat sequence; (d) determining the nucleotide sequence of at least a portion of the genomic DNA; and (e) comparing the nucleotide sequence with sequence information in a computer database.
- 121. A method for identifying a genomic coding sequence in an isolated or cultured vertebrate cell comprising:
(a) introducing into an isolated or cultured vertebrate cell
(i) a nucleic acid fragment comprising a nucleic acid sequence comprising (i) a detectable marker coding sequence encoding a detectable marker or a selectable marker, (ii) a splice acceptor site and (iii) an internal ribosome entry site; wherein the splice acceptor site and internal ribosome entry site are each operably linked to the detectable marker coding sequence, the nucleic acid sequence positioned between at least two inverted repeats that can bind to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, and wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell; and (ii) a transposase source selected from the group consisting of a transposase and a nucleic acid encoding a transposase, wherein the transposase is an SB protein; (b) detecting the detectable marker or the selectable marker in the cell or its progeny containing the nucleic acid fragment, wherein expression of the detectable marker or the selectable marker indicates that the nucleic acid fragment has integrated within a genomic coding sequence of the cell or its progeny.
- 122. The method of claim 121 wherein the detectable marker or the selectable marker is expressed spatially and temporally in the same way as the genomic coding sequence is expressed when not interrupted.
- 123. The method of claim 121 wherein the vertebrate cell is a pluripotent or totipotent vertebrate cell.
- 124. The method of claim 123 wherein the pluripotent or totipotent vertebrate cell is selected from the group consisting of an oocyte, a cell of an embryo, an egg and a stem cell.
- 125. The method of claim 121 wherein the detectable marker is green fluorescent protein.
- 126. The method of claim 121 wherein the selectable marker is neomycin.
- 127. The method of claim 121 further comprising (c) determining whether a phenotype of a cell that contains the nucleic acid fragment is altered in comparison to a cell that does not comprise the nucleic acid fragment.
- 128. The method of claim 121 further comprising, prior to (b), growing the cell into an animal.
- 129. The method of claim 128 further comprising step (c) of determining whether a phenotype of an animal that contains the nucleic acid fragment is altered in comparison to an animal that does not comprise the nucleic acid fragment.
- 130. The method of claim 121 wherein the transposase source is an mRNA encoding the transposase.
- 131. The method of claim 121 wherein the transposase source is a transposase.
- 132. The method of claim 121 further comprising:
(c) cleaving the DNA of the cell with a restriction endonuclease to yield at least one restriction fragment comprising (i) at least a portion of the integrated nucleic acid fragment, which portion comprises at least a portion of an inverted repeat sequence and (ii) genomic DNA of the cell, which genomic DNA is adjacent to the inverted repeat sequence; (d) determining the nucleotide sequence of at least a portion of the genomic DNA; and (e) comparing the nucleotide sequence with sequence information in a computer database.
- 133. A method for identifying the function of an analyte coding sequence comprising:
(a) introducing into an isolated or cultured vertebrate cell
(i) a nucleic acid fragment comprising a nucleic acid sequence comprising (i) a detectable marker coding sequence encoding a detectable marker or a selectable marker, (ii) an analyte coding sequence located 5′ of the detectable marker coding sequence and (iii) an internal ribosome entry site located therebetween, the internal ribosome entry site being operably linked to the detectable marker coding sequence, the nucleic acid fragment positioned between at least two inverted repeats that can bind to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, and wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell; and (ii) a transposase source selected from the group consisting of a transposase and a nucleic acid encoding a transposase, wherein the transposase is an SB protein; (b) detecting the detectable marker or the selectable marker in the cell or its progeny containing the nucleic acid fragment, wherein the expression of the detectable marker or the selectable marker indicates that the nucleic acid fragment has integrated into the DNA of the cell and that the analyte coding sequence is expressed; and (c) determining whether a phenotype of the cell or its progeny containing the nucleic acid fragment is altered in comparison to a cell that does not comprise the nucleic acid fragment, wherein an altered phenotype indicates that the analyte coding sequence plays a function in the phenotype.
- 134. The method of claim 133 wherein the vertebrate cell is a pluripotent or totipotent vertebrate cell.
- 135. The method of claim 134 wherein the pluripotent or totipotent vertebrate cell is selected from the group consisting of an oocyte, a cell isolated from an embryo, an egg cell and a stem cell.
- 136. The method of claim 133 wherein the detectable marker is green fluorescent protein.
- 137. The method of claim 133 wherein the selectable marker is neomycin.
- 138. The method of claim 133 further comprising, prior to (b), growing the cell into an animal.
- 139. The method of claim 138 wherein step (c) comprises determining the phenotype of an animal.
- 140. The method of claim 133 wherein the analyte coding sequence is derived from a library of DNA fragments.
- 141. The method of claim 133 wherein the analyte coding sequence comprises a preselected coding sequence.
- 142. The method of claim 133 wherein the analyte coding sequence is operably linked to a promoter.
- 143. The method of claim 110, 121 or 133 wherein the vertebrate cell is selected from the group consisting of a fish cell, a human cell and a mouse cell.
- 144. A nucleic acid fragment comprising a nucleic acid sequence comprising a weak promoter operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell, and wherein the weak promoter directs higher expression of the detectable or selectable marker when the nucleic acid fragment integrates into the DNA of the cell near an enhancer than when it integrates elsewhere into the DNA of the cell.
- 145. A nucleic acid fragment comprising a nucleic acid sequence comprising a splice acceptor site and an internal ribosome entry site, each operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell.
- 146. A nucleic acid fragment comprising a nucleic acid sequence comprising (i) a detectable marker coding sequence that encodes a detectable marker or a selectable marker, (ii) an analyte coding sequence located 5′ of the detectable marker coding sequence and (iii) an internal ribosome entry site located therebetween and operably linked to the detectable marker coding sequence, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell.
- 147. The fragment of claim 146 wherein the analyte coding sequence is derived from a library of DNA fragments.
- 148. The fragment of claim 146 wherein the analyte coding sequence comprises a preselected coding sequence.
- 149. The fragment of claim 146 wherein the analyte coding sequence is operably linked to a promoter.
- 150. A gene transfer system to introduce a nucleic acid sequence into the DNA of an isolated or cultured vertebrate cell comprising:
(a) a nucleic acid fragment comprising a nucleic acid sequence comprising a weak promoter operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell, and wherein the weak promoter directs higher expression of the detectable or selectable marker when the nucleic acid fragment integrates into the DNA of the cell near an enhancer than when it integrates elsewhere into the DNA of the cell.; and (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
- 151. A gene transfer system to introduce a nucleic acid sequence into the DNA of an isolated or cultured vertebrate cell comprising:
(a) a nucleic acid fragment comprising a nucleic acid sequence comprising a splice acceptor site and an internal ribosome entry site, each operably linked to a detectable marker coding sequence encoding a detectable marker or a selectable marker, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell; and (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
- 152. A gene transfer system to introduce a nucleic acid sequence into the DNA of an isolated or cultured vertebrate cell comprising:
(a) a nucleic acid fragment comprising a nucleic acid sequence comprising (i) a detectable marker coding sequence that encodes a detectable marker or a selectable marker, (ii) an analyte coding sequence located 5′ of the detectable marker coding sequence and (iii) an internal ribosome entry site located therebetween and operably linked to the detectable marker coding sequence, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, the inverted repeats comprising at least one direct repeat comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1, is capable of binding to the inverted repeat sequence of at least one of SEQ ID NO:4 and SEQ ID NO:5, and is capable of catalyzing the integration of the nucleic acid fragment into DNA in an isolated or cultured vertebrate cell; and (b) a transposase source selected from the group consisting of a transposase and nucleic acid encoding a transposase, wherein the transposase is an SB protein.
- 153. The gene transfer system of claim 152 wherein the analyte coding sequence is derived from a library of DNA fragments.
- 154. The gene transfer system of claim 152 wherein the analyte coding sequence comprises a preselected coding sequence.
- 155. The gene transfer system of claim 152 wherein the analyte coding sequence is operably linked to a promoter.
- 156. The gene transfer system of claims 150, 151 or 152, wherein the vertebrate cell is a pluripotent or totipotent vertebrate cell.
- 157. The gene transfer system of claim 156 wherein the pluripotent or totipotent vertebrate cell is selected from the group consisting of an oocyte, a cell isolated from an embryo, an egg cell and a stem cell.
- 158. The gene transfer system of claims 150, 151 or 152, wherein the transposase source is an mRNA.
- 159. The gene transfer system of claims 150, 151 or 152, wherein the transposase source is an SB transposase.
- 160. The gene transfer system of claims 151, 152 or 153, wherein the transposase source is a nucleic acid encoding the transposase that is integrated into the genome of the vertebrate cell.
- 161. The gene transfer system of claims 151, 152 or 153, wherein the nucleic acid fragment is part of a plasmid or a recombinant viral vector.
- 162. The gene transfer system of claims 150, 151 or 152, wherein the vertebrate cell is a selected from the group consisting of a fish cell, a human cell and a mouse cell.
- 163. A gene transfer system to introduce a nucleic acid sequence into the DNA of a fish comprising a nucleic acid fragment comprising a nucleic acid sequence comprising an internal ribosome entry site, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein, wherein the nucleic acid fragment is capable of integrating into the genomic DNA of a fish.
- 164. The gene transfer system of claim 163 wherein the nucleic acid sequence further comprises a coding sequence located 3′ to and operably linked to the internal ribosome entry site.
- 165. The gene transfer system of claim 164 wherein the coding sequence is a first coding sequence, the nucleic acid sequence further comprising a second coding sequence located 5′ to both the first coding sequence and the internal ribosome entry site.
- 166. A transgenic fish or fish cell comprising a nucleic acid sequence comprising a heterologous internal ribosome entry site, the nucleic acid sequence positioned between at least two inverted repeats capable of binding to an SB protein.
CONTINUING APPLICATION DATA
[0001] This application is a continuation application of U.S. Ser. No. 09/191,572, which is incorporated by reference in its entirety.
STATEMENT OF GOVERNMENT RIGHTS
[0002] The present invention was made with government support under Grant No. 2ROI-RR06625-05, awarded by the National Institutes of Health/National Institutes of Research Resources. The Government may have certain rights in this invention.
Continuations (1)
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Number |
Date |
Country |
Parent |
09191572 |
Nov 1998 |
US |
Child |
10191698 |
Jul 2002 |
US |