Nova Patents
CA2280997C

Dna-based transposon system for the introduction of nucleic acid into dna of a cell

Abstract

This invention relates to a system for introducing nucleic acid into the DNA of a cell. The system includes the use of a member of the SB family of transposases (SB) or nucleic acid encoding the transposase and a nucleic acid fragment that includes a nucleic acid sequence with flanking inverted repeats. The transposase recognizes at least a portion of an inverted repeats and incorporates the nucleic acid sequence into the DNA. Methods for use of this system are discussed.

CA2280997C, drawing sheet 1
Sheet 1 of 15

Term

Term ended

Expired 11 March 2018, 8.5 years ago.

  1. Priority
  2. Filed
  3. Granted
  4. Expired
  5. Today

13 claims: 3 independent, 10 dependent

  1. 1
    CLAIMS:1. A nucleic acid fragment comprising: a nucleic acid sequence positioned between at least two inverted repeats that bind to an SB protein, the inverted repeats comprising at least one direct repeat, wherein each direct repeat comprises a nucleic acid sequence that has at least 80% nucleic acid sequence identity to SEQ ID NO:10 and wherein each direct repeat binds to the SB protein;wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1 and binds to at least one of SEQ ID NO:4 and SEQ ID NO:5, and wherein the amino acid at position 31 is arginine.
  2. 6
    The fragment of any one of claims 1 to 5 wherein the nucleic acid fragment is part of a plasmid.
  3. 7
    The fragment of any one of claims 1 to 5 wherein the fragment is present in a cell. CA 02280997 2012-10-30 76433-16
  4. 11
    15. 28. The gene transfer system of any one of claims 19 to 27 wherein the nucleic acid sequence comprises at least a regulatory region of a gene. 29. The gene transfer system of claim 28 wherein the regulatory region is a transcriptional regulatory region.
  5. 13
    25 32. The gene transfer system of any one of claims 19 to 26 wherein the nucleic acid sequence comprises a promoter operably linked to at least a portion of an open reading frame. CA 02280997 2010-01-15 76433-16 33. The gene transfer system of claim 31 wherein the cell is a vertebrate or an invertebrate cell. 34. The gene transfer system of claim 33 wherein the invertebrate cell is a crustacean or a mollusk cell. 35. The gene transfer system of claim 33 wherein the cell is a fish or a bird cell. 36. The gene transfer system of claim 33 wherein the vertebrate cell is a mammalian cell. 37. The gene transfer system of claim 36 wherein the cell is a rodent, ungulate, sheep, swine, or human cell. 38. The gene transfer system of any one of claims 19 to 37 wherein the DNA in the cell is the cell genome or extrachromosal DNA, wherein the extrachromosomal DNA is an episome or a plasmid. 39. The gene transfer system of any one of claims 19 to 38 wherein at least one of the inverted repeats comprises SEQ ID NO:4 or SEQ ID NO:5. 40. The gene transfer system of any one of claims 19 to 38 wherein at least one of the inverted repeats comprises at least one direct repeat and wherein the at least one direct repeat sequence comprises SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9. 41. The gene transfer system of any one of claims 19 to 40 wherein the nucleic acid sequence is part of a library of recombinant sequences. 42. The gene transfer system of claim 22 wherein the nucleic acid sequence is for introduction into the cell using : CA 02280997 2012-08-21 76433-16 particle bombardment;electroporation;microinj ection;lipid-containing vesicles or DNA condensing reagents;or 5 a viral vector comprising the nucleic acid fragment. 43. A nucleic acid encoding an SB protein, wherein the nucleic acid encodes a protein comprising SEQ ID N0:l or a protein comprising an amino acid sequence with at least 80% identity to SEQ ID N0:l and an arginine at position 31, a gene expression vector. 48. A cell comprising the nucleic acid of any one of 20 claims 43 to 47, with the proviso that said cell is not totipotent. 49. The cell of claim 48 wherein the cell is an animal cell. CA 02280997 2010-01-15 76433-16 50. The cell of claim 49 wherein the cell is a vertebrate or an invertebrate cell. 51. The cell of claim 50 wherein the vertebrate cell is a fish cell. 52. The cell of claim 50 wherein the vertebrate cell is a mammalian cell. 53. The nucleic acid of any one of claims 43 to 47 integrated in DNA of a cell. 54. An SB protein comprising the amino acid sequence of SEQ ID N0:l. 55. A method for producing a transgenic animal comprising the steps of: introducing a nucleic acid fragment and a transposase into a pluripotent or totipotent cell wherein the nucleic acid fragment comprises a nucleic acid sequence positioned between at least two inverted repeats that bind to an SB protein, the inverted repeats comprising at least one direct repeat, wherein each direct repeat comprises a nucleic acid sequence that has at least 80% nucleic acid sequence identity to SEQ ID NO:10 and wherein each direct repeat binds to the SB protein;wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID N0:l and binds to at least one of SEQ ID NO:4 and SEQ ID NO:5, wherein the amino acid at position 31 is arginine, and wherein the transposase is an SB protein having an amino acid sequence identity of at least 80% to SEQ ID NO:1 and the amino acid at position 31 is arginine;and CA 02280997 2010-01-15 76433-16 growing the cell into an animal. 56. The method of claim 55 wherein the pluripotent or totipotent cell is an oocyte, a cell of an embryo, an egg, or a stem cell. 5· 57. The method of claim 55 or 56 wherein the introducing step comprises: microinjection;electroporation;combining the nucleic acid fragment with cationic 10 lipid vesicles or DNA condensing reagents;or incorporating the nucleic acid fragment into a viral vector and contacting the viral vector with the cell. 58. The method of claim 57 wherein the viral vector is a retroviral vector, an adenovirus vector, an 15 adeno-associated viral vector, or a herpes virus. 59. The method of any one of claims 55 to 58 wherein the animal is a mouse, a fish, an ungulate, a bird, or a sheep. 60. An in vitro or ex vivo method for introducing 20 nucleic acid into DNA in a cell comprising the step of: introducing into the cell a nucleic acid fragment comprising a riucleic acid sequence positioned between at least two inverted repeats that bind to an SB protein, the inverted repeats comprising at least one direct repeat, 25 wherein each direct repeat comprises a nucleic acid sequence that has at least 80% nucleic acid sequence identity to CA 02280997 2010-01-15 76433-16 SEQ ID NO:10 and wherein each direct repeat binds to the SB protein, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID N0:l and binds to at least one of SEQ ID NO:4 and SEQ ID NO:5, and wherein the amino acid at position 31 is arginine. 61. The method of claim 60 wherein the method further comprises introducing the SB protein into the cell, wherein the introduced SB protein has an amino acid sequence comprising at least 80% identity to SEQ ID NO:1. 62. The method of claim 61 wherein the introduced SB protein is introduced into the cell as RNA. 63. The method of claim 60 wherein the cell comprises nucleic acid encoding the SB protein. 64. The method of claim 63 wherein the nucleic acid encoding the SB protein is integrated into the DNA in the cell. 65. The method of claim 63 wherein the SB protein is stably expressed in the cell. 66. The method of claim 63 wherein expression of the SB protein is under the control of an inducible promoter. 67. The method of claim 60 wherein the introducing step comprises: microinjection;electroporation;combining the nucleic acid fragment with cationic lipid vesicles or DNA condensing reagents;or CA 02280997 2012-08-21 76433-16 incorporating the nucleic acid fragment into a viral vector and contacting the viral vector with the cell. 68. The method of claim 67 wherein the viral vector is a retroviral vector, an adenovirus vector, an adeno-associated viral vector, or herpes virus. 69. The method of claim 60 wherein the method further comprises introducing an SB protein or RNA encoding an SB protein into the cell. 70. The method of any one of claims 60 to 69 wherein the cell is a pluripotent or a totipotent cell. 71. A transgenic animal cell produced by the method of claim 70, wherein the transgenic animal cell comprises a nucleic acid fragment comprising a nucleic acid sequence flanked by inverted repeats comprising at least one direct repeat, wherein each direct repeat comprises a nucleic acid sequence that has at least 80% identity to SEQ ID NO:10 and wherein each direct repeat binds to the SB protein, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID N0:l and binds to at least one of SEQ ID NO:4 and SEQ ID NO:5, and wherein the nucleic acid sequence flanked by inverted repeats encodes a recombinant protein, with the proviso that the cell is not totipotent. 72. The transgenic animal cell of claim 71 wherein the recombinant protein is a marker protein. 73. A cell comprising a nucleic acid fragment comprising a nucleic acid sequence flanked by inverted repeats comprising at least one direct repeat, wherein each direct repeat comprises a nucleic acid sequence that has at least 80% nucleic acid sequence CA 02280997 2012-08-21 76433-16 identity to SEQ ID NO:10 and wherein each direct repeat binds to the SB protein, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1 and an arginine at position 31, and wherein the nucleic acid sequence flanked by inverted repeats encodes a recombinant protein, with the proviso that the cell is not totipotent. 74. A transgenic animal cell comprising a nucleic acid fragment comprising a nucleic acid sequence flanked by inverted repeats comprising at least one direct repeat, wherein each direct repeat comprises a nucleic acid sequence that has at least 80% nucleic acid sequence identity to SEQ ID NO:10 and wherein each direct repeat binds to the SB protein, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID N0:l and an arginine at position 31, and wherein the nucleic acid sequence flanked by inverted repeats encodes a recombinant protein, with the proviso that the cell is not totipotent. 75. A protein comprising the following characteristics: an ability to catalyze the integration of nucleic acid into DNA of a cell, wherein the nucleic acid sequence is positioned between at least two inverted repeats, the inverted repeats comprising at least one direct repeat, wherein each direct repeat comprises a nucleic acid sequence that has at least 80% nucleic acid sequence identity to SEQ ID NO:10;capable of binding to the inverted repeat sequence of SEQ ID NOS :4 or 5;and at least 80% amino acid sequence identity to SEQ ID NO:1 and an arginine at position 31. 76. A protein comprising the following characteristics: CA 02280997 2010-01-15 76433-16 at least 80% amino acid sequence identity to SEQ ID NO:1 and an arginine at position 31;transposase activity;a molecular weight range of about 35 kD to about 40 kD on about a 10% SDS-polyacrylamide gel;and a nuclear localization domain sequence, a DNA binding domain and a catalytic domain wherein the protein has at least about five-fold improvement in the rate for introducing a nucleic acid fragment into the nucleic acid of a cell as compared to the level obtained by nonhomologous recombination. 77. An in vitro or ex vivo method for mobilizing a nucleic acid sequence in a cell comprising: introducing the protein of claim 75 or 76 into the cell wherein the cell comprises DNA containing the nucleic acid fragment according to claim 1, wherein the protein mobilizes the nucleic acid fragment from a first position within the DNA to a second position within the DNA. 78. The method of claim 77 wherein the DNA of the cell. 79. The method of claim 77 wherein the within the DNA is extrachromosomal DNA. 80. The method of claim 77 wherein the within the DNA is extrachromosomal DNA. DNA is genomic first position second position 81. The method of any one of claims 77 to 80 wherein the protein is introduced into the cell as nucleic acid. CA 02280997 2010-01-15 76433-16 82. An in vitro or ex vivo method for identifying a gene in a genome of a cell comprising the steps of : introducing a nucleic acid fragment and an SB protein into a cell, wherein the nucleic acid fragment comprises a nucleic acid sequence positioned between at least two inverted repeats, the inverted repeats comprising at least one direct repeat, wherein each direct repeat comprises a nucleic acid sequence that has at least 80% nucleic acid sequence identity to SEQ ID NO:10 and wherein each direct repeat binds to the SB protein, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1 and an arginine at position 31, and binds to at least one of SEQ ID NO:4 and SEQ ID NO:5, and wherein the nucleic acid fragment is capable of integrating into DNA in a cell in the presence of the SB protein;digesting the DNA of the cell with a restriction endonuclease capable of cleaving the nucleic acid sequence;identifying the inverted repeat sequences,· sequencing the DNA of the cell close to the inverted repeat sequences to obtain DNA sequence from an open reading frame;comparing the DNA sequence with sequence information in a computer database;and identifying a gene comprising the DNA sequence. 83. The method of claim 82 wherein the restriction endonuclease recognizes a 6-base recognition sequence. CA 02280997 2010-01-15 76433-16 84. The method of claim 83 wherein the digesting step further comprises cloning the digested fragments or PCR amplifying the digested fragments. 85. A stable transgenic vertebrate cell line comprising a gene operably linked to a promoter, wherein the gene and promoter are flanked by inverted repeats, the inverted repeats comprising at least one direct repeat, wherein each direct repeat comprises a nucleic acid sequence that has at least 80% nucleic acid sequence identity to SEQ ID NO:10 and wherein each direct repeat binds to the SB protein, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID N0:l and an arginine at position 31, and binds to at least one of SEQ ID NO:4 and SEQ ID NO:5, wherein the cell line is not a totipotent stem cell line. 86. The stable transgenic vertebrate cell line of claim 85 wherein the vertebrate cell is a fish cell. 87. The stable transgenic vertebrate cell line of claim 86 wherein the vertebrate cell is a zebrafish cell. 88. The stable transgenic vertebrate cell line of claim 86 wherein the vertebrate cell is a mouse cell. 89. A protein with transposase activity that can bind to one or more of the following sequences: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, wherein the protein comprises an amino acid sequence having at least 80% identity to SEQ ID N0:l, and wherein the amino acid at position 31 is arginine. CA 02280997 2010-01-15 76433-16 90. The nucleic acid of claim 53 wherein the DNA is the cell genome or extrachromosomal DNA, wherein the extrachromosomal DNA is an episome or a plasmid. 5. 91. The method of claim 64 wherein the DNA is the cell genome or extrachromosomal DNA, wherein the extrachromosomal DNA is an episome or a plasmid. 92. Use, for introducing nucleic acid into DNA on a cell of: a nucleic acid fragment which is adapted for 10 introduction into the cell, said fragment comprising a nucleic acid sequence positioned between at least two inverted repeats that bind to an SB protein, the inverted repeats comprising at least one direct repeat, wherein each direct repeat comprises a nucleic acid sequence that has at 15 least 80% nucleic acid sequence identity to SEQ ID NO:10 and wherein each direct repeat binds to the SB protein, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1 and an arginine at position 31, and binds to at least one of 20 SEQ ID NO:4 and SEQ ID NO:5. 93. The use of claim 92, further comprising use of the SB protein, wherein the SB protein has an amino acid sequence comprising at least 80% identity to SEQ ID NO:1. 94. The use of claim 93 wherein the SB protein is 25 expressed from an RNA which was introduced into the cell. 95. The use of claim 92 wherein the cell comprises nucleic acid encoding the SB protein. CA 02280997 2010-01-15 76433-16 96. The use of claim 95 wherein the nucleic acid encoding the SB protein is integrated into the DNA in the cell. 97. The use of claim 95 wherein the SB protein is stably expressed in the cell. 98. The use of claim 95 wherein expression of the SB protein is under the control of an inducible promoter. 99. The use of claim 92 wherein the nucleic acid fragment is adapted for introduction into the cell by: microinj ection;electroporation;combination of the nucleic acid fragment with cationic lipid vesicles or DNA condensing reagents;or incorporation of the nucleic acid fragment into a viral vector which is adapted to contact the cell. 100. The use of claim 99 wherein the viral vector is a retroviral vector, an adenovirus vector, an adeno-associated viral vector, or herpes virus. 101. The use of claim 92 further comprising use of the SB protein or RNA encoding the SB protein which are adapted for introduction into the cell. 102. The use of any one of claims 92 to 101 wherein the cell is a pluripotent or a totipotent cell. 103. Use/ for mobilizing a nucleic acid sequence in a cell, of: CA 02280997 2010-01-15 76433-16 the protein of claim 75 or 76 in the cell, wherein the cell comprises DNA containing the nucleic acid fragment according to claim 1, wherein the protein mobilizes the nucleic acid fragment from a first position within the DNA to a second position within the DNA. 104. The use of claim 103 wherein the DNA is genomic DNA of the cell. 105. The use of claim 103 wherein the first position within the DNA is extrachromosomal DNA. 106. The use of claim 103 wherein the second position within the DNA is extrachromosomal DNA. 107. The use of any one of claims 103 to 106 wherein the protein is expressed in the cell from a nucleic acid which was introduced in the cell. 108. Use, for identifying a gene in a genome of a cell of : a nucleic acid fragment and an SB protein which are both adapted for introduction into the cell, wherein the nucleic acid fragment comprises a nucleic acid sequence positioned between at least two inverted repeats, the inverted repeats comprising at least one direct repeat, wherein each direct repeat comprises a nucleic acid sequence that has at least 80% nucleic acid sequence identity to SEQ ID NO:10 and wherein each direct repeat binds to the SB protein, wherein the SB protein comprises an amino acid sequence having at least 80% identity to SEQ ID NO:1 and an arginine at position 31, and binds to at least one of SEQ ID NO:4 and SEQ ID NO:5, and wherein the nucleic acid CA 02280997 2012-08-21 76433-16 fragment is capable of integrating into the gene in the genome of the cell in the presence of the SB protein. 109. The use of claim 108, further comprising use of a restriction endonuclease capable of cleaving the nucleic acid 5 sequence. 110. The use of claim 109 wherein the restriction endonuclease recognizes a 6-base recognition sequence. 111. The use of claim 96 wherein the DNA is the cell genome or extrachromosomal DNA, wherein the extrachromosomal 10 DNA is an episome or a plasmid. CA 02280997 2013-01-22 Application number / Numéro de demande: t — O It ' F i gures :_ Pages: Unscannable items received with this application (Request original documents in File Prep. Section on the 10 th floor) Documents reçu avec cette demande ne pouvant être balayés (Commander les documents originaux dans la section de la préparation des dossiers au lOième étage) CA 02280997 2013-01-22 //Z? . m m CQ W W H Q fl •o ” s g* 2 =6 w c S 5 t Oi ü «S' ·-* ·2“ ϊ 1 O Ç k 7Î «Λ ffl ffl W W et oo en ffl m ffl U) o m ffl SUBSTITUTE cucct/diii oe\ CA 02280997 2013-01-22 (SEQ ID NO-·3) ATGGGAAAA TCAAAAGAAA TCAGCCAAGA CCTCAGAAAA TACCCTTTT AGTTTTCTTT AGTCGGTTCT GGAGTCTTTT 701 CAGGAAGTTA AAGCTTGGTC GCAAATGGGT CTTCCAAATG GACAATGACC GTCCTTCAAT TTCGAACCAG CGTTTACCCA GAAGGTTTAC CTGTTACTGG 751 CCAAGCATAC TTCCAAAGTT GTGGCAAAAT GGCTTAAGGA CAACAAAGTC GGTTCGTATG AAGGTTTCAA CACCGTTTTA CCGAATTCCT GTTGTTTCAG 801 AAGGTATTGG AGTGGCCATC ACAAAGCCCT GACCTCAATC CTATAGAAAA TTCCATAACC TCACCGGTAG TGTTTCGGGA CTGGAGTTAG GATATCTTTT 851 TTTGTGGGCA GAACTGAAAA AGCGTGTGCG AGCAAGGAGG CCTACAAACC AAACACCCGT CTTGACTTTT TCGCACACGC TCGTTCCTCC GGATGTTTGG 901 TGACTCAGTT ACACCAGCTC TGTCAGGAGG AATGGGCCAA AATTCACCCA ACTGAGTCAA TGTGGTCGAG ACAGTCCTCC TTACCCGGTT TTAAGTGGGT 951 ACTTATTGTG GGAAGCTTGT GGAAGGCTAC CCGAAACGTT TGACCCAAGT TGAATAACAC CCTTCGAACA CCTTCCGATG GGCTTTGCAA ACTGGGTTCA 1001 TAAACAATTT AAAGGCAATG CTACCAAATA CTAG. ATTTGTTAAA TTTCCGTTAC GATGGTTTAT GATC SUBSTITUTE SUEET/DIlt c oe\ CA 02280997 2013-01-22 Paired-like domain with Leucine-zipper A W w a Eh A A A H Oi A a A Q a co H co A s A g A A A A A A Q Eh Λ A xj a o A ·* Q i 0 .g 3 *3. ε-ι 0 A A A pci « A A A o A A A A ω A i*i w a Pci A A A A Q A A A ω H A A A Q ω τ—I LT) A A A A A A CO A A A A O A A H Q A A A A A S H Q H A A A A t—I ί—I lO O CM CQ o CM SUBSTITIITC CUCCT /BI II C OCX CA 02280997 2013-01-22 é/f3 SUBSTITIITP PUPPT ZDIII p oc\ CA 02280997 2013-01-22 7/fS - + SUBSTITUTE CUBCT /Dili e «ISA CA 02280997 2013-01-22 8/f3 100 200 300 400 number of transformants per petri dish fjiq. 6 SUBSTITUTE SUEET/BIII E ntt\ CA 02280997 2013-01-22 /0//3 transposon pUCtf vector pUC/9 vector tgdattcgaqctcgqtâcccTA CAGT .. ..Acre TAgqqqatcctctagagtcgac·· (SEQ ID NO-43) + (SEQ ID N0.44) • · /** · · o · * 'ST 0Ί |§ I Ça kJ J o, TO TO 3 TO TO yj en en (0 TO) 4-J 4-J -+j 4-» 4-J υ υ υ kJ (0 to œ to en Oa TO TO TO TO 4- 4-J 5C Ça Γ 4 Vq Ό σ» Cn en en 4—· 4-J ro ro kJ CJ 4-) 4- 4-J 4-J 4-» 4- 4-» 4-J en en 4-J ro 4-) 5C TO = ii kj s s TO TO U kJ (D TO 4-J 4-» en en 4-J 4-J en en 4- 4-J (0 TO 0 ro m +-* 4-» V 4J 4-1 q TO TO a 4-» -+J Σ' 4-J 4- S-H «+j a 2 TO UJ A3 (Q L3 m TO v£ 'T $ $ e A kû f\) . kD \) I—' e iS s 4-J 4-J U J m to TO TO e tj 4-J 4-J 4-1 4-J en en TO ro 4-J 4-j en en TO TO O kJ TO 03 fO 03 TO (TJ 4- 4-J TO Π3 TO 03 en en CX V s «j to to 5? *+-J 4_j u e) en en 4—1 4-J e j kJ O en en 4-J 4_l œ m to ni en 4-» 4-J 4-J -4-J to m 4-» 4-i 4-J 4—· TO TO 4-J 4-J kJ 4-J TO TO 4-J 4-J e e 4-J 4-J TO kJ rü TO en en 4-J -μ TO TO en en -h +- nj TO TO TO 4-J 4-J TO TO e Q 4-J 4-J 4-J 4-J TO TO 4-J 4-J kj e TO TO kJ kJ en en en en ej kj en en 4-J 4-J kJ 4-j ro to 4-J 4-J 4-J 4-J 4-J 4-» en en 4-J 4-J hhe @3 Lx t5 to AS O A UJ O A Q kJ ej ej 4-J 4-J 4-J 4-J TO TO kJ kJ 4-J 4-J ei e TO TO en en 4-J 4-J υ ei TO TO Ό kJ 4-J 4-J 4-J 4-J TO TO 4-J 4-J 4-J 4-J TO TO 4-J TO kJ TO 4-J 4-1 4-J kJ kJ TO TO TO TO 4-J TO kJ TO 5 e -S' S' 3 3 TO 03 kJ kJ -m en 4-1 4-J TO TO kJ kJ TO TO kJ kJ TO TO en en TO (D TO TO TO TO 4-J 4-J TO TO 4-J 4-J Vq '9 ex c*S 'n kq Ko kD SUBSTITUTE succt roui c * e\ CA 02280997 2013-01-22 zz/z? 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Independent claims5