Claims for Patent: 7,351,877

Summary for Patent: 7,351,877

Title:	Lambda integrase mediated recombination in plants
Abstract:	The present disclosure provides methods for obtaining the targeted integration of a DNA molecule into the genome of a host cell using a recombinase. The methods disclosed herein can be used with a variety of host cells, including, for example, dicotyledonous and monocotyledonous plant cells. The present disclosure provides a method for effecting site-specific recombination of DNA within a plant cell, comprising: introducing into the plant cell a target nucleotide sequence comprising a first Int recognition site; introducing into the plant cell a donor nucleotide sequence comprising a second Int recognition site; and introducing into the plant cell an integrase or integrase complex.
Inventor(s):	Suttie; Janet Louise (Research Triangle Park, NC), Chilton; Mary-Dell (Research Triangle Park, NC), Que; Qiudeng (Research Triangle Park, NC)
Assignee:	Syngenta Participations AG (Basel, CH)
Application Number:	10/403,232
Patent Claims:	1. A method for obtaining site-specific recombination of DNA within a plant cell, comprising: introducing into a plant cell a target sequence comprising a first Int recognition site capable of being recognized by a Lambda bacteriophage integrase or integrase complex; introducing into said plant cell a donor sequence comprising a second Int recognition site capable of being recognized by a Lambda bacteriophage integrase or integrase complex; and introducing into said plant cell a Lambda bacteriophage integrase or integrase complex, such that site-specific recombination of said donor sequence is obtained. 2. The method of claim 1, wherein said plant cell is a monocotyledonous plant cell. 3. The method of claim 1, wherein said plant cell is a dicotyledonous plant cell. 4. The method of claim 1, wherein said plant cell is one of a wheat cell, a maize cell, a rice cell, a barley cell, a soybean cell, a cotton plant cell, a tomato cell, and a tobacco cell. 5. The method of claim 1, wherein said target sequence is introduced into said plant cell by one of Agrobacterium-mediated transformation, microprojectile bombardment, electroporation, PEG-mediated transformation, and microinjection. 6. The method of claim 1, wherein said target sequence is stably integrated into a genome of said plant cell. 7. The method of claim 6, wherein said plant cell is a target line cell comprising a single copy of said target sequence. 8. The method of claim 1, wherein said first Int recognition site is a modified Lambda bacteriophage integrase recognition site. 9. The method of claim 1, wherein said first Int recognition site comprises attL (SEQ ID NO:174). 10. The method of claim 1, wherein said target sequence comprises at least one of a sequence of interest, a molecular marker, a selectable marker, a visible marker, a negative selectable marker, a promoter, an expression cassette, an intron, and a portion of any of these. 11. The method of claim 10, wherein said target sequence comprises at least one of a PPO gene, a LUC gene, an NPTII gene, a GUS gene, a PMI gene, a HPT gene, and a portion of any of these. 12. The method of claim 1, wherein said target sequence further comprises a third Int recognition site capable of being recognition by a Lambda bacteriophage integrase or integrase complex. 13. The method of claim 12, wherein said first Int recognition site and said third Int recognition site are identical. 14. The method of claim 12, wherein said first Int recognition site and said third Int recognition site are non-identical. 15. The method of claim 12, wherein said first Int recognition site and said third Int recognition site are incapable of recombining with each other. 16. The method of claim 12, wherein at least one of said first Int recognition site and said third Int recognition site is a modified Lambda bacteriophage integrase recognition site. 17. The method of claim 12, wherein said first Int recognition site and said third Int recognition site are in direct orientation with respect to each other. 18. The method of claim 12, wherein said first Int recognition site and said third Int recognition site are in an inverted orientation with respect to each other. 19. The method of claim 12, wherein said first Int recognition site and said third Int recognition site are positioned within said target sequence such that said first Int recognition site and said third Int recognition site are adjacent to one another. 20. The method of claim 12, wherein said first Int recognition site and said third Int recognition site are positioned within said target sequence such that a first nucleotide sequence is located between said first Int recognition site and said third Int recognition site. 21. The method of claim 20, wherein said first nucleotide sequence comprises at least one of a sequence of interest, a molecular marker, a selectable marker, a visible marker, a negative selectable marker, a promoter, an expression cassette, an intron, and a portion of any of these. 22. The method of claim 21, wherein said first nucleotide sequence comprises at least one of a PPO gene, a LUC gene, an NPTII gene, a GUS gene, a PMI gene, a HPT gene, and a portion of any of these. 23. The method of claim 12, wherein each of said first Int recognition site comprises attL (SEQ ID NO:174) and said third Int recognition site comprises attL1 (SEQ ID NO:180). 24. The method of claim 1, wherein said donor sequence is introduced into said plant cell by one of Agrobacterium-mediated transformation, microprojectile bombardment, electroporation, PEG-mediated transformation, and microinjection. 25. The method of claim 1, wherein said donor sequence is introduced into said plant cell on a viral replicon. 26. The method of claim 25, wherein said viral replicon is capable of autonomous replication within said plant cell. 27. The method of claim 25, wherein said viral replicon is derived from a geminivirus. 28. The method of claim 27, wherein said geminivirus is one of a maize streak virus, a wheat dwarf virus, a tobacco golden mosaic virus, and a beet curly top virus. 29. The method of claim 25, wherein said viral replicon is introduced into said plant cell by one of Agrobacterium-mediated transformation, microprojectile bombardment, electroporation, PEG-mediated transformation, and microinjection. 30. The method of claim 1, wherein said second Int recognition site is a modified Lambda bacteriophage integrase recognition site. 31. The method of claim 1, wherein said second Int recognition site comprises attB (SEQ ID NO:175). 32. The method of claim 1, wherein said donor sequence comprises at least one of a sequence of interest, a molecular marker, a selectable marker, a visible marker, a negative selectable marker, a promoter, an expression cassette, an intron, and a portion of any of these. 33. The method of claim 32, wherein said donor sequence comprises at least one of a PPO gene, a LUC gene, an NPTII gene, a GUS gene, a PMI gene, a HPT gene, and a portion of any of these. 34. The method of claim 12, wherein said donor sequence further comprises a fourth Int recognition site capable of being recognized by a Lambda bacteriophage integrase or integrase complex. 35. The method of claim 34, wherein said second Int recognition site and said fourth Int recognition site are identical. 36. The method of claim 35, wherein said second Int recognition site and said fourth Int recognition site are in inverted orientation with respect to each other. 37. The method of claim 34, wherein said second Int recognition site and said fourth Int recognition site are non-identical. 38. The method of claim 37, wherein said second Int recognition site and said fourth Int recognition site are in direct orientation with respect to each other. 39. The method of claim 37, wherein said second Int recognition site and said fourth Int recognition site are in inverted orientation with respect to each other. 40. The method of claim 34, wherein said second Int recognition site and said fourth Int recognition site are incapable of recombining with each other. 41. The method of claim 34, wherein at least one of said second Int recognition site and said fourth Int recognition site is a modified Lambda bacteriophage integrase recognition site. 42. The method of claim 34, wherein said second Int recognition site and said fourth Int recognition site are positioned within said donor sequence such that said second Int recognition site and said fourth Int recognition site are adjacent to one another. 43. The method of claim 34, wherein said second Int recognition site and said fourth Int recognition site are positioned within said donor sequence such that a pre-selected nucleotide sequence is located between said second Int recognition site and said fourth Int recognition site. 44. The method of claim 43, wherein said pre-selected nucleotide sequence comprises at least one of a sequence of interest, a molecular marker, a selectable marker, a visible marker, a negative selectable marker, a promoter, an expression cassette, an intron, and a portion of any of these. 45. The method of claim 44, wherein said pre-selected nucleotide sequence comprises at least one of a PPO gene, a LUC gene, an NPTII gene, a GUS gene, a PMI gene, a HPT gene, and a portion of any of these. 46. The method of claim 34, wherein said second Int recognition site is capable of recombining with said first Int recognition site and said fourth Int recognition site is capable of recombining with said third Int recognition site. 47. The method of claim 46, wherein each of said second Int recognition site and said fourth Int recognition site comprises attR1 (SEQ ID NO:181). 48. The method of claim 47, wherein each of said first Int recognition site comprises (SEQ ID NO:174) and said third Int recognition site comprises attL1 (SEQ ID NO:181). 49. The method of claim 1, wherein said integrase or integrase complex comprises one of a wild-type Lambda bacteriophage integrase and a modified Lambda bacteriophage integrase. 50. The method of claim 49, wherein said modified Lambda integrase comprises one of Int-h and Int-h/218. 51. The method of claim 50, wherein said integrase or integrase complex further comprises an integration host factor. 52. The method of claim 51, wherein said integrase or integrase complex further comprises an excisionase. 53. The method of claim 1, wherein said integrase or integrase complex is introduced into said plant cell as one or more nucleotide sequences comprising a coding region for each constituent protein of said integrase or integrase complex. 54. The method of claim 53, wherein said one or more nucleotide sequences comprises SEQ ID NO:21. 55. The method of claim 54, wherein SEQ ID NO:21 is modified such that base pair 520 is changed from a "G" to an "A." 56. The method of claim 55, wherein SEQ ID NO:21 is further modified such that base pair 652 is changed from a "G" to an "A." 57. The method of claim 53, wherein said coding region for each constituent protein is operably linked to a plant expressible promoter. 58. The method of claim 57, wherein said plant expressible promoter is one of a constitutive promoter, an inducible promoter, a tissue-specific promoter, a tissue-preferred promoter, a developmentally-regulated promoter, a cell-specific promoter, and an organellar-specific promoter. 59. The method of claim 53, wherein said integrase or integrase complex is stably integrated into a genome of said plant cell. 60. The method of claim 53, wherein said integrase or integrase complex is transiently expressed within said plant cell. 61. The method of claim 60, wherein said integrase or integrase complex is introduced into said plant cell on a viral replicon. 62. The method of claim 61, wherein said viral replicon is capable of autonomous replication within said plant cell. 63. The method of claim 61, wherein said viral replicon is derived from a geminivirus. 64. The method of claim 63, wherein said geminivirus is one of a maize streak virus, a wheat dwarf virus, a tobacco golden mosaic virus, and a beet curly top virus. 65. The method of claim 61, wherein said viral replicon is introduced into said plant cell by one of Agrobacterium-mediated transformation, microprojectile bombardment, electroporation, PEG-mediated transformation, and microinjection. 66. The method of claim 60, wherein said integrase or integrase complex is introduced into said plant cell as one or more RNA molecules. 67. The method of claim 53, wherein said coding region for each constituent protein is optimized for expression in said plant cell. 68. The method of claim 1, wherein said integrase or integrase complex is introduced into said plant cell as one or more proteins. 69. The method of claim 68, wherein said one or more proteins is introduced into said plant cell by one of electroporation and microinjection. 70. The method of claim 68, wherein said one or more proteins is introduced into said plant cell through Agrobacterium comprising a VirE or a VirF fusion protein. 71. The method of claim 1, wherein introducing said donor sequence and introducing said integrase or integrase complex are performed simultaneously. 72. The method of claim 1, wherein said target sequence is introduced into said plant cell by a first method, said donor sequence is introduced by a second method, and said integrase or integrase complex is introduced by a third method. 73. The method of claim 72, wherein each of said first method, said second method, and said third method is independently selected from the group consisting of Agrobacterium-mediated transformation, microprojectile bombardment, electroporation, PEG-mediated transformation, microinjection, and sexual reproduction, and wherein at least one of said first method, said second method, and said third method is other than sexual reproduction. 74. The method of claim 73, wherein said donor sequence and said integrase or integrase complex are introduced into said plant cell simultaneously. 75. The method of claim 73, wherein said target sequence is introduced into said plant cell prior to an introduction of said donor sequence and said integrase or integrase complex. 76. The method of claim 73, wherein said integrase or integrase complex is introduced into said plant cell after said target sequence and said donor sequence have been introduced into said plant cell. 77. The method of claim 73, wherein said donor sequence is stably integrated into a genome of a second plant cell prior to being introduced into said plant cell. 78. The method of claim 34, wherein said target sequence comprises an incomplete nucleotide sequence and said donor sequence comprises a completion sequence, such that recombination between said target sequence and said donor sequence produces a complete nucleotide sequence. 79. The method of claim 78, wherein said incomplete nucleotide sequence comprises at least one of a sequence of interest, a gene, an intron, a promoter, an expression cassette, a selectable marker, a visible marker, and a negative selectable marker. 80. The method of claim 1, wherein said donor sequence comprises a third Int recognition site that does not recombine with said first Int recognition site of said target sequence and is capable of being used in a subsequent recombination of DNA within said plant cell. 81. The method of claim 80, wherein said first Int recognition site, attL (SEQ ID NO:174), said second Int recognition site comprises attR (SEQ ID NO:175), and said third Int recognition site comprises attL1 (SEQ ID NO:180). 82. The method of claim 34, wherein said donor sequence comprises a fifth Int recognition site that does not recombine with either of said first Int recognition site or said third Int recognition site of said target sequence and is capable of being used in a subsequent recombination of DNA within said plant cell. 83. The method of claim 82, wherein said first Int recognition site, comprises attL (SEQ ID NO:174), said second Int recognition site comprises attR (SEQ ID NO:175), said third Int recognition site attL1 (SEQ ID NO:180), said fourth Int recognition site comprises attR1 (SEQ ID NO:181), and said fifth Int recognition site comprises attP2 (SEQ ID NO:179). 84. The method of claim 1, further comprising: identifying a recombination product obtained through sequence exchange between said target sequence and said donor sequence. 85. The method of claim 84, wherein said recombination product comprises at least one newly created Int recognition site, and wherein said newly created Int recognition site is flanked on a first side by sequence obtained from said target sequence and flanked on a second side by sequence obtained from said donor sequence. 86. The method of claim 34, further comprising: identifying a recombination product obtained through sequence exchange between said target sequence and said donor sequence. 87. The method of claim 86, wherein said recombination product comprises at least one newly created Int recognition site, and wherein said newly created Int recognition site is flanked on a first side by sequence obtained from said target sequence and flanked on a second side by sequence obtained from said donor sequence. 88. A method for manipulating a target sequence within a plant cell, comprising: introducing into a plant cell a target sequence comprising (a) a first Int recognition site capable of being recognized by a Lambda bacteriophage integrase or integrase complex and a second Int recognition site capable of being recognized by a Lambda bacteriophage integrase or integrase complex that are capable of recombining with each other and (b) a first nucleotide sequence that is situated between said first Int recognition site and said second Int recognition site; introducing into said plant cell a Lambda bacteriophage integrase or integrase complex; and identifying a recombination product comprising an altered target sequence. 89. The method of claim 88, wherein said first Int recognition site and said second Int recognition site are in direct orientation, and said altered target sequence does not comprise said first nucleotide sequence. 90. The method of claim 88, wherein said first Int recognition site and said second Int recognition site are in inverted orientation, and said altered target sequence comprises said first nucleotide sequence in an inverted orientation relative to an original orientation of said first nucleotide sequence. 91. The method of claim 88, wherein each of said first Int recognition site comprises attB (SEQ ID NO:172) and said second Int recognition site comprises attP (SEQ ID NO:173). 92. The method of claim 88, wherein said first nucleotide sequence comprises at least one of a sequence of interest, a molecular marker, a selectable marker, a visible marker, a negative selectable marker, a promoter, an expression cassette, an intron, and a portion of any of these. 93. The method of claim 88, wherein said target sequence further comprises a second nucleotide sequence that is not positioned between said first Int recognition site and said second Int recognition site. 94. The method of claim 93, wherein said second nucleotide sequence comprises at least one of a sequence of interest, a molecular marker, a selectable marker, a visible marker, a negative selectable marker, a promoter, an expression cassette, an intron, and a portion of any of these. 95. The method of claim 93, wherein said altered target sequence comprises said first nucleotide sequence in an inverted orientation relative to an original orientation of said first nucleotide sequence and said second nucleotide sequence in an original orientation. 96. The method of claim 88, wherein said integrase or integrase complex comprises one of a wild-type Lambda bacteriophage integrase and a modified Lambda bacteriophage integrase. 97. The method of claim 96, wherein said modified Lambda integrase comprises one of Int-h and Int-h/218. 98. The method of claim 97, wherein said integrase or integrase complex further comprises an integration host factor. 99. The method of claim 98, wherein said integrase or integrase complex further comprises an excisionase. 100. A method for obtaining site-specific recombination of DNA within a plant cell, comprising: introducing into a genome of a plant cell a target sequence comprising a first Int recognition site that comprises attL (SEQ ID NO:174); introducing into said plant cell a donor sequence comprising a second Int recognition site that comprises attR (SEQ ID NO:175); introducing into said plant cell a Lambda bacteriophage integrase or integrase complex; and identifying a recombination product within said genome of said plant cell obtained through sequence exchange between said target sequence and said donor sequence. 101. The method of claim 100, wherein said integrase or integrase complex comprises one of a wild-type Lambda bacteriophage integrase and a modified Lambda bacteriophage integrase. 102. The method of claim 101, wherein said modified Lambda integrase comprises one of Int-h and Int-h/218. 103. The method of claim 102, wherein said integrase or integrase complex further comprises an integration host factor. 104. The method of claim 103, wherein said integrase or integrase complex further comprises an excisionase. 105. A method for obtaining site-specific recombination of DNA within a plant cell, comprising: introducing into a genome of a plant cell a target sequence comprising attL (SEQ ID NO:174) as a first Int recognition site and comprising attL1 (SEQ ID NO:180) as a third Int recognition site; introducing into said plant cell a donor sequence comprising attR (SEQ ID NO:175) as a second Int recognition site and comprising attR1 (SEQ ID NO:181) as a fourth recognition site; introducing into said plant cell a Lambda bacteriophage integrase or integrase complex; and identifying a recombination product within said genome of said plant cell obtained through sequence exchange between said target sequence and said donor sequence. 106. The method of claim 105, wherein said integrase or integrase complex comprises one of a wild-type Lambda bacteriophage integrase and a modified Lambda bacteriophage integrase. 107. The method of claim 106, wherein said modified Lambda integrase comprises one of Int-h and Int-h/218. 108. The method of claim 107, wherein said integrase or integrase complex further comprises an integration host factor. 109. The method of claim 108, wherein said integrase or integrase complex further comprises an excisionase. 110. The method of claim 1, further comprising identifying a transgenic plant cell comprising a recombination product within a genome of said transgenic plant cell obtained through sequence exchange between said target sequence and said donor sequence. 111. A transgenic plant cell obtained by the method of claim 110, said transgenic plant cell comprising at least one Int recognition site. 112. The transgenic plant cell of claim 111, wherein said Int recognition site comprises attB (SEQ ID NO:172). 113. A transgenic plant comprising the transgenic plant cell of claim 111. 114. A transgenic plant comprising the transgenic plant cell of claim 112. 115. The method of claim 34, further comprising identifying a transgenic plant cell comprising a recombination product within a genome of said transgenic plant cell obtained through sequence exchange between said target sequence and said donor sequence. 116. A transgenic plant cell obtained by the method of claim 115, said transgenic plant cell comprising at least one Int recognition site. 117. The transgenic plant cell of claim 116, wherein said Int recognition site comprises attP (SEQ ID NO:173). 118. A transgenic plant comprising the transgenic plant cell of claim 116. 119. A transgenic plant comprising the transgenic plant cell of claim 117.

Details for Patent 7,351,877

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2022-03-29
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2022-03-29
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2022-03-29