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公开(公告)号：US06262341B1
公开(公告)日：2001-07-17
申请号：US09193503
申请日：1998-11-17
申请人： Christopher L. Baszczynski , Leszek Alexander Lyznik , William J. Gordon-Kamm , Xueni Guan , Argula Gururaj Rao , Laura A. Tagliani
发明人： Christopher L. Baszczynski , Leszek Alexander Lyznik , William J. Gordon-Kamm , Xueni Guan , Argula Gururaj Rao , Laura A. Tagliani
IPC分类号： C12N504
CPC分类号： C12N15/90 , C07K2319/00 , C12N9/00 , C12N15/8203 , C12N15/8205 , C12N15/8213
摘要： Compositions and methods for introducing a DNA of interest into a genomic target site are provided. In particular, the methods and compositions involve the use of a combination of target sites for two site specific recombinases and expression of a chimeric recombinase with dual target site specificity. Thus, the compositions comprise novel site-specific recombinases with specificities to multiple target sites, and nucleotide sequences and expression cassettes encoding these recombinases or target sites. The methods involve transforming a eukaryotic cell having target sites for the novel recombinase with a DNA of interest that is flanked by corresponding target sites. Expression of the recombinase results in integration of the DNA of interest into the genome of the cell. The compositions and methods of the invention have use in the construction of stably transformed eukaryotic cells, and in particular, plant cells. The methods result in the efficient targeted genomic integration of DNA by site-specific recombination.
摘要翻译： 提供了将目的DNA引入基因组靶位点的组合物和方法。 特别地，所述方法和组合物包括使用两个位点特异性重组酶的靶位点的组合和具有双靶点位点特异性的嵌合重组酶的表达。 因此，组合物包含对多个靶位点具有特异性的新的位点特异性重组酶，以及编码这些重组酶或靶位点的核苷酸序列和表达盒。 所述方法包括用具有相应靶位点侧翼的感兴趣的DNA转化具有新重组酶靶位点的真核细胞。 重组酶的表达导致感兴趣的DNA整合到细胞的基因组中。 本发明的组合物和方法用于构建稳定转化的真核细胞，特别是植物细胞。 该方法通过位点特异性重组导致DNA的有效靶向基因组整合。
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公开(公告)号：US06541231B1
公开(公告)日：2003-04-01
申请号：US09415839
申请日：1999-10-12
申请人： Christopher L. Baszczynski , Leszek Alexander Lyznik , William J. Gordon-Kamm , Xueni Guan , Aragula Gururaj Rao , Laura A. Tagliani
发明人： Christopher L. Baszczynski , Leszek Alexander Lyznik , William J. Gordon-Kamm , Xueni Guan , Aragula Gururaj Rao , Laura A. Tagliani
IPC分类号： C12N900
CPC分类号： C12N15/90 , C07K2319/00 , C12N9/00 , C12N15/8203 , C12N15/8205 , C12N15/8213
摘要： Compositions and methods for introducing a DNA of interest into a genomic target site are provided. In particular, the methods and compositions involve the use of a combination of target sites for two site specific recombinases and expression of a chimeric recombinase with dual target site specificity. Thus, the compositions comprise novel site-specific recombinases with specificities to multiple target sites, and nucleotide sequences and expression cassettes encoding these recombinases or target sites. The methods involve transforming a eukaryotic cell having target sites for the novel recombinase with a DNA of interest that is flanked by corresponding target sites. Expression of the recombinase results in integration of the DNA of interest into the genome of the cell. The compositions and methods of the invention have use in the construction of stably transformed eukaryotic cells, and in particular, plant cells. The methods result in the efficient targeted genomic integration of DNA by site-specific recombination.
摘要翻译： 提供了将目的DNA引入基因组靶位点的组合物和方法。 特别地，所述方法和组合物包括使用两个位点特异性重组酶的靶位点的组合和具有双靶点位点特异性的嵌合重组酶的表达。 因此，组合物包含对多个靶位点具有特异性的新的位点特异性重组酶，以及编码这些重组酶或靶位点的核苷酸序列和表达盒。 所述方法包括用具有相应靶位点侧翼的感兴趣的DNA转化具有新重组酶靶位点的真核细胞。 重组酶的表达导致感兴趣的DNA整合到细胞的基因组中。 本发明的组合物和方法用于构建稳定转化的真核细胞，特别是植物细胞。 该方法通过位点特异性重组导致DNA的有效靶向基因组整合。
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3.发明授权Mobilization of viral genomes from T-DNA using site-specific recombination systems 有权使用位点特异性重组系统动员来自T-DNA的病毒基因组公开(公告)号：US06300545B1
公开(公告)日：2001-10-09
申请号：US09193475
申请日：1998-11-17
IPC分类号： C12N504
CPC分类号： C12N15/8213 , C07K2319/00 , C12N9/00 , C12N15/8203 , C12N15/8205 , C12N15/90
摘要： The invention relates to methods and compositions for site-specific recombinase-mediated mobilization of viral replicons from T-DNA. The methods comprise Agrobacterium-mediated transfer of T-DNA to a plant cell, wherein the T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon. The DNA of interest may also contain a non-identical target site for the recombinase. An expression cassette for the site-specific recombinase is present on the T-DNA or the plant genome, or is transiently introduced into the plant cell. Expression of the site-specific recombinase in the plant cell results in excision of the viral replicon and the associated DNA of interest. The viral replicon and DNA of interest are then replicated to high copy number in the host plant cell. The compositions of the invention comprise nucleic acids, such as T-DNAs containing a viral DNA flanked by directly repeated target sites for a site-specific recombinase. The nucleic acids of the invention may additionally contain expression cassettes encoding the cognate site-specific recombinase for the target sites flanking the viral genome. The compositions of the invention further comprise Agrobacterium containing the nucleic acids of the invention. The compositions and methods of the invention have use in increasing the efficiency of agroinfection, providing high copy numbers of a DNA of interest for transient expression or for integration into a plant chromosome, and in simplifying the construction and stable maintenance of vectors for agroinfection and transformation.
摘要翻译： 本发明涉及用于来自T-DNA的病毒复制子的位点特异性重组酶介导的动员的方法和组合物。 所述方法包括农杆菌介导的T-DNA向植物细胞的转移，其中所述T-DNA含有侧翼于病毒复制子的病毒复制子，所述病毒复制子侧接于位点特异性重组酶和任选的与所述病毒复制子连接的感兴趣的DNA。 感兴趣的DNA还可以含有重组酶的不相同的靶位点。 用于位点特异性重组酶的表达盒存在于T-DNA或植物基因组上，或瞬时引入植物细胞。 位点特异性重组酶在植物细胞中的表达导致病毒复制子和感兴趣的相关DNA的切除。 然后将病毒复制子和感兴趣的DNA复制到宿主植物细胞中的高拷贝数。 本发明的组合物包含核酸，例如含有侧翼为用于位点特异性重组酶的直接重复靶位点的病毒DNA的T-DNA。 本发明的核酸可以另外含有编码针对病毒基因组侧翼的靶位点的同源位点特异性重组酶的表达盒。 本发明的组合物还包含含有本发明核酸的农杆菌。 本发明的组合物和方法用于增加农业感染的效率，为临时表达提供高拷贝数的目的DNA，或用于整合到植物染色体中，以及简化用于农业感染和转化的载体的构建和稳定维持 。
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4.发明授权Mobilization of viral genomes from T-DNA using site-specific recombination systems 失效使用位点特异性重组系统动员来自T-DNA的病毒基因组公开(公告)号：US07364902B2
公开(公告)日：2008-04-29
申请号：US11400293
申请日：2006-04-07
CPC分类号： C12N15/8213 , C07K2319/00 , C12N9/00 , C12N15/8203 , C12N15/8205 , C12N15/90
摘要： The invention relates to methods and compositions for site-specific recombinase-mediated mobilization of viral replicons and associated DNAs of interest from T-DNA. The methods of the invention comprise Agrobacterium-mediated transfer of T-DNA to a plant cell, wherein the T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon. The DNA of interest may also contain a non-identical target site for the recombinase. An expression cassette for the site-specific recombinase is present on the T-DNA or the plant genome, or is transiently introduced into the plant cell. Expression of the site-specific recombinase in the plant cell results in excision of the viral replicon and the associated DNA of interest. The viral replicon and DNA of interest are then replicated to high copy number in the host plant cell. The compositions of the invention comprise nucleic acids, such as T-DNAs containing a viral DNA flanked by directly repeated target sites for a site-specific recombinase. The nucleic acids of the invention may additionally contain expression cassettes encoding the cognate site-specific recombinase for the target sites flanking the viral genome. The compositions of the invention further comprise Agrobacterium containing the nucleic acids of the invention.
摘要翻译： 本发明涉及用于位点特异性重组酶介导的来自T-DNA的感兴趣的病毒复制子和相关DNA的动员的方法和组合物。 本发明的方法包括农杆菌介导的T-DNA向植物细胞的转移，其中T-DNA含有病毒复制子，其侧翼是用于位点特异性重组酶的直接重复的靶位点，以及任选的与病毒连接的感兴趣的DNA 复制品。 感兴趣的DNA还可以含有重组酶的不相同的靶位点。 用于位点特异性重组酶的表达盒存在于T-DNA或植物基因组上，或瞬时引入植物细胞。 位点特异性重组酶在植物细胞中的表达导致病毒复制子和感兴趣的相关DNA的切除。 然后将病毒复制子和感兴趣的DNA复制到宿主植物细胞中的高拷贝数。 本发明的组合物包含核酸，例如含有侧翼为用于位点特异性重组酶的直接重复靶位点的病毒DNA的T-DNA。 本发明的核酸可以另外含有编码针对病毒基因组侧翼的靶位点的同源位点特异性重组酶的表达盒。 本发明的组合物还包含含有本发明核酸的农杆菌。
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公开(公告)号：US07223601B2
公开(公告)日：2007-05-29
申请号：US10353445
申请日：2003-01-29
申请人： Christopher L. Baszczynski , Leszek Alexander Lyznik , William J. Gordon-Kamm , Xueni Guan , Aragula Gururaj Rao , Laura A. Tagliani
发明人： Christopher L. Baszczynski , Leszek Alexander Lyznik , William J. Gordon-Kamm , Xueni Guan , Aragula Gururaj Rao , Laura A. Tagliani
IPC分类号： C12N15/87
CPC分类号： C12N15/90 , C07K2319/00 , C12N9/00 , C12N15/8203 , C12N15/8205 , C12N15/8213
摘要： Compositions and methods for introducing a DNA of interest into a genomic target site are provided. In particular, the methods and compositions involve the use of a combination of target sites for two site specific recombinases and expression of a chimeric recombinase with dual target site specificity. Thus, the compositions comprise novel site-specific recombinases with specificities to multiple target sites, and nucleotide sequences and expression cassettes encoding these recombinases or target sites. The methods involve transforming a eukaryotic cell having target sites for the novel recombinase with a DNA of interest that is flanked by corresponding target sites. Expression of the recombinase results in integration of the DNA of interest into the genome of the cell. The compositions and methods of the invention have use in the construction of stably transformed eukaryotic cells, and in particular, plant cells. The methods result in the efficient targeted genomic integration of DNA by site-specific recombination.
摘要翻译： 提供了将目的DNA引入基因组靶位点的组合物和方法。 特别地，所述方法和组合物涉及使用两个位点特异性重组酶的靶位点的组合和具有双重靶位点特异性的嵌合重组酶的表达。 因此，组合物包含对多个靶位点具有特异性的新的位点特异性重组酶，以及编码这些重组酶或靶位点的核苷酸序列和表达盒。 所述方法包括用具有相应靶位点侧翼的感兴趣的DNA转化具有新重组酶靶位点的真核细胞。 重组酶的表达导致感兴趣的DNA整合到细胞的基因组中。 本发明的组合物和方法用于构建稳定转化的真核细胞，特别是植物细胞。 该方法通过位点特异性重组导致DNA的有效靶向基因组整合。
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6.发明授权Mobilization of viral genomes from T-DNA using site-specific recombination systems 有权使用位点特异性重组系统动员来自T-DNA的病毒基因组公开(公告)号：US07179599B2
公开(公告)日：2007-02-20
申请号：US10444664
申请日：2003-05-23
CPC分类号： C12N15/8213 , C07K2319/00 , C12N9/00 , C12N15/8203 , C12N15/8205 , C12N15/90
摘要： The invention relates to methods and compositions for site-specific recombinase-mediated mobilization of viral replicons and associated DNAs of interest from T-DNA. The methods of the invention comprise Agrobacterium-mediated transfer of T-DNA to a plant cell, wherein the T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon. The DNA of interest may also contain a non-identical target site for the recombinase. An expression cassette for the site-specific recombinase is present on the T-DNA or the plant genome, or is transiently introduced into the plant cell. Expression of the site-specific recombinase in the plant cell results in excision of the viral replicon and the associated DNA of interest. The viral replicon and DNA of interest are then replicated to high copy number in the host plant cell. The compositions of the invention comprise nucleic acids, such as T-DNAs containing a viral DNA flanked by directly repeated target sites for a site-specific recombinase. The nucleic acids of the invention may additionally contain expression cassettes encoding the cognate site-specific recombinase for the target sites flanking the viral genome. The compositions of the invention further comprise Agrobacterium containing the nucleic acids of the invention. The compositions and methods of the invention have use in increasing the efficiency of agroinfection, providing high copy numbers of a DNA of interest for transient expression or for integration into a plant chromosome, and in simplifying the construction and stable maintenance of vectors for agroinfection and transformation.
摘要翻译： 本发明涉及用于位点特异性重组酶介导的来自T-DNA的感兴趣的病毒复制子和相关DNA的动员的方法和组合物。 本发明的方法包括农杆菌介导的T-DNA向植物细胞的转移，其中T-DNA含有病毒复制子，其侧翼是用于位点特异性重组酶的直接重复的靶位点，以及任选的与病毒连接的感兴趣的DNA 复制品。 感兴趣的DNA还可以含有重组酶的不相同的靶位点。 用于位点特异性重组酶的表达盒存在于T-DNA或植物基因组上，或瞬时引入植物细胞。 位点特异性重组酶在植物细胞中的表达导致病毒复制子和感兴趣的相关DNA的切除。 然后将病毒复制子和感兴趣的DNA复制到宿主植物细胞中的高拷贝数。 本发明的组合物包含核酸，例如含有侧翼为用于位点特异性重组酶的直接重复靶位点的病毒DNA的T-DNA。 本发明的核酸可以另外含有编码针对病毒基因组侧翼的靶位点的同源位点特异性重组酶的表达盒。 本发明的组合物还包含含有本发明核酸的农杆菌。 本发明的组合物和方法用于增加农业感染的效率，为临时表达提供高拷贝数的目的DNA，或用于整合到植物染色体中，以及简化用于农业感染和转化的载体的构建和稳定维持 。
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7.发明授权Mobilization of viral genomes from T-DNA using site-specific recombination systems 失效使用位点特异性重组系统动员来自T-DNA的病毒基因组公开(公告)号：US06664108B1
公开(公告)日：2003-12-16
申请号：US09411826
申请日：1999-10-01
IPC分类号： C12N1582
CPC分类号： C12N15/8213 , C07K2319/00 , C12N9/00 , C12N15/8203 , C12N15/8205 , C12N15/90
摘要： The invention relates to methods and compositions for site-specific recombinase-mediated mobilization of viral replicons and associated DNAs of interest from T-DNA. The methods of the invention comprise Agrobacterium-mediated transfer of T-DNA to a plant cell, wherein the T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon. The DNA of interest may also contain a non-identical target site for the recombinase. An expression cassette for the site-specific recombinase is present on the T-DNA or the plant genome, or is transiently introduced into the plant cell. Expression of the site-specific recombinase in the plant cell results in excision of the viral replicon and the associated DNA of interest. The viral replicon and DNA of interest are then replicated to high copy number in the host plant cell. The compositions of the invention comprise nucleic acids, such as T-DNAs containing a viral DNA flanked by directly repeated target sites for a site-specific recombinase. The nucleic acids of the invention may additionally contain expression cassettes encoding the cognate site-specific recombinase for the target sites flanking the viral genome. The compositions of the invention further comprise Agrobacterium containing the nucleic acids of the invention. The compositions and methods of the invention have use in increasing the efficiency of agroinfection, providing high copy numbers of a DNA of interest for transient expression or for integration into a plant chromosome, and in simplifying the construction and stable maintenance of vectors for agroinfection and transformation.
摘要翻译： 本发明涉及用于位点特异性重组酶介导的来自T-DNA的感兴趣的病毒复制子和相关DNA的动员的方法和组合物。 本发明的方法包括农杆菌介导的T-DNA向植物细胞的转移，其中T-DNA含有病毒复制子，其侧翼是用于位点特异性重组酶的直接重复的靶位点，以及任选的与病毒连接的感兴趣的DNA 复制品。 感兴趣的DNA还可以含有重组酶的不相同的靶位点。 用于位点特异性重组酶的表达盒存在于T-DNA或植物基因组上，或瞬时引入植物细胞。 位点特异性重组酶在植物细胞中的表达导致病毒复制子和感兴趣的相关DNA的切除。 然后将病毒复制子和感兴趣的DNA复制到宿主植物细胞中的高拷贝数。 本发明的组合物包含核酸，例如含有侧翼为用于位点特异性重组酶的直接重复靶位点的病毒DNA的T-DNA。 本发明的核酸可以另外含有编码针对病毒基因组侧翼的靶位点的同源位点特异性重组酶的表达盒。 本发明的组合物还包含含有本发明核酸的农杆菌。 本发明的组合物和方法用于增加农业感染的效率，为临时表达提供高拷贝数的目的DNA，或用于整合到植物染色体中，以及简化用于农业感染和转化的载体的构建和稳定维持 。
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公开(公告)号：US06331661B1
公开(公告)日：2001-12-18
申请号：US09193484
申请日：1998-11-17
IPC分类号： C12N1590
CPC分类号： C12N15/8216 , C07K2319/00 , C12N9/00 , C12N15/102 , C12N15/1137 , C12N15/8203 , C12N15/8205 , C12N15/8213 , C12N15/8274 , C12N15/90 , C12N2310/321 , C12N2310/53 , C12N2310/3521
摘要： The present invention is drawn to compositions and methods for introducing nucleotide sequences at preferred genomic target sites in a eukaryotic genome. The compositions comprise transfer cassettes which are flanked by nonhomologous recombination sites. The method involves transforming eukaryotic cells containing target sites utilizing non-integrating transformation methods. The method results in efficient integration of nucleotides into predetermined genetic locations and eliminates random DNA integration.
摘要翻译： 本发明涉及在真核基因组中优选的基因组靶位点处引入核苷酸序列的组合物和方法。 组合物包含侧翼为非同源重组位点的转移盒。 该方法涉及使用非整合转化方法转化含有靶位点的真核细胞。 该方法导致核苷酸有效整合到预定的遗传位置，并消除随机DNA整合。
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公开(公告)号：US06175058B1
公开(公告)日：2001-01-16
申请号：US09263128
申请日：1999-03-05
申请人： Christopher L. Baszczynski , Benjamin A. Bowen , Bruce J. Drummond , William J. Gordon-Kamm , David J. Peterson , Gary A. Sandahl , Laura A. Tagliani , Zuo-Yu Zhao , Grace marie St. Clair
发明人： Christopher L. Baszczynski , Benjamin A. Bowen , Bruce J. Drummond , William J. Gordon-Kamm , David J. Peterson , Gary A. Sandahl , Laura A. Tagliani , Zuo-Yu Zhao , Grace marie St. Clair
IPC分类号： A01H100
CPC分类号： C12N9/00 , C12N15/8213 , C12N15/8289
摘要： A nucleic acid sequence effectively expressing FLP recombinase in monocot plants, particularly in maize. Stable, transformed maize plants harboring a gene encoding FLP or harboring FRT nucleic acid sequences enable efficient site-directed recombination of nucleic acid sequences in a monocot's genome.
摘要翻译： 在单子叶植物中，特别是在玉米中有效表达FLP重组酶的核酸序列。 含有编码FLP或携带FRT核酸序列的基因的稳定的转化的玉米植物能够在单子叶植物的基因组中有效地进行位点定向重组核酸序列。
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10.发明授权Mobilization of viral genomes from T-DNA using site-specific recombination systems 有权使用位点特异性重组系统动员来自T-DNA的病毒基因组公开(公告)号：US07807868B2
公开(公告)日：2010-10-05
申请号：US12061315
申请日：2008-04-02
CPC分类号： C12N15/8213 , C07K2319/00 , C12N9/00 , C12N15/8203 , C12N15/8205 , C12N15/90
摘要： The invention relates to methods and compositions for site-specific recombinase-mediated mobilization of viral replicons and associated DNAs of interest from T-DNA. The methods of the invention comprise Agrobacterium-mediated transfer of T-DNA to a plant cell, wherein the T-DNA contains a viral replicon flanked by directly repeated target sites for a site-specific recombinase and optionally a DNA of interest linked to the viral replicon. The DNA of interest may also contain a non-identical target site for the recombinase. An expression cassette for the site-specific recombinase is present on the T-DNA or the plant genome, or is transiently introduced into the plant cell. Expression of the site-specific recombinase in the plant cell results in excision of the viral replicon and the associated DNA of interest. The viral replicon and DNA of interest are then replicated to high copy number in the host plant cell.
摘要翻译： 本发明涉及用于位点特异性重组酶介导的来自T-DNA的感兴趣的病毒复制子和相关DNA的动员的方法和组合物。 本发明的方法包括农杆菌介导的T-DNA向植物细胞的转移，其中T-DNA含有病毒复制子，其侧翼是用于位点特异性重组酶的直接重复的靶位点，以及任选的与病毒连接的感兴趣的DNA 复制品。 感兴趣的DNA还可以含有重组酶的不相同的靶位点。 用于位点特异性重组酶的表达盒存在于T-DNA或植物基因组上，或瞬时引入植物细胞。 位点特异性重组酶在植物细胞中的表达导致切除病毒复制子和感兴趣的相关DNA。 然后将病毒复制子和感兴趣的DNA复制到宿主植物细胞中的高拷贝数。
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