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公开(公告)号：US20140154807A1
公开(公告)日：2014-06-05
申请号：US14146858
申请日：2014-01-03
IPC分类号： C12N15/80
CPC分类号： C12N15/80 , C07K14/385 , C12N15/1058 , C12N15/902 , C12N2800/30
摘要： The present invention discloses a method to construct eukaryotic cells having a target sequence in a chromosomal DNA sequence replaced by a replacement sequence of interest comprising: modifying a parent eukaryotic cell with a preference for NHR to provide a eukaryotic cell having an increased HR/NHR ratio as compared to the parent cell, providing two sets of DNA molecules of which the first set comprises DNA molecules each comprising a first non-functional fragment of the replacement sequence of interest flanked at its 5′-side by a DNA sequence substantially homologous to a sequence of the chromosomal DNA flanking the target sequence and the second set comprises DNA molecules each comprising a second non-functional fragment of the DNA replacement sequence of interest overlapping with the first non-functional fragment and flanked at its 3′-side by a DNA sequence substantially homologous to a sequence of the chromosomal DNA flanking the target sequence, wherein the first and second non-functional fragments become functional upon recombination; transforming the modified eukaryotic cells with both sets of DNA molecules; and growing the cells to obtain transformed progeny cells having a DNA molecule from the first set recombined with a DNA molecule from the second set into a DNA molecule comprising a functional replacement sequence and having the target sequence replaced by the functional replacement sequence.
摘要翻译： 本发明公开了一种构建真核细胞的方法，所述真核细胞在染色体DNA序列中被替换的目标序列替代，所述染色体DNA序列被替换的目标序列所替代，包括：以优选NHR修饰亲本真核细胞以提供具有增加的HR / NHR比的真核细胞 与亲本细胞相比，提供两组DNA分子，其中第一组包含DNA分子，每个DNA分子包含在其5'侧两侧的替换序列的第一个非功能性片段，其基本上与 在靶序列侧翼的染色体DNA的序列和第二组包含DNA分子，每个DNA分子包含与第一非功能片段重叠的DNA置换序列的第二个非功能性片段，并在其3'侧通过DNA侧接 基本上与靶序列侧翼的染色体DNA的序列同源的序列，其中第一和第二 非功能性片段在重组时变得有功能; 用两组DNA分子转化修饰的真核细胞; 并生长细胞以获得具有来自第一组的DNA分子的转化后代细胞与来自第二组的DNA分子重组为包含功能性替换序列的DNA分子并且将靶序列替换为功能性替换序列。
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公开(公告)号：US20090170206A1
公开(公告)日：2009-07-02
申请号：US12296506
申请日：2007-03-14
IPC分类号： C12N15/87
CPC分类号： C12N15/815 , C12N15/905 , C12N2800/30 , C12N2800/70
摘要： We disclose a method to construct eukaryotic cells having a target sequence in a chromosomal DNA sequence replaced by a desired replacement sequence, comprising: modifying a parent eukaryotic cell with a preference for NHR to provide a eukaryotic cell having an increased HR/NHR ratio as compared to the parent cell, providing a DNA molecule comprising a first DNA fragment comprising a desired replacement sequence flanked at its 5′ and 3′ sides by DNA sequences substantially homologous to sequences of the chromosomal DNA flanking the target sequence and a second DNA fragment comprising an expression cassette comprising a gene encoding a selection marker operably linked to regulatory sequences functional in the eukaryotic cell, transforming the modified eukaryotic cells with the DNA molecule, selecting transformed progeny cells having the DNA molecule inserted into the chromosome, deselecting transformed progeny cells having the DNA molecule inserted into the chromosome via NHR by expression of the selection marker, and obtaining cells wherein the target sequence in the chromosomal DNA sequence is replaced by the desired replacement sequence.
摘要翻译： 我们公开了一种构建真核细胞的方法，所述真核细胞在染色体DNA序列中被所需的置换序列替代，其包括：以优选NHR修饰亲本真核细胞以提供具有增加的HR / NHR比的真核细胞， 提供DNA分子，其包含第一DNA片段，所述第一DNA片段包含在其5'和3'侧侧翼的所需替换序列，所述DNA序列与靶序列侧翼的染色体DNA的序列基本上同源的DNA序列和包含 表达盒，其包含编码与真核细胞功能性调节序列可操作地连接的选择标记的基因，用DNA分子转化修饰的真核细胞，选择具有插入到染色体中的DNA分子的转化后代细胞，取代具有DNA的转化后代细胞 分子通过NHR通过表达插入到染色体中 选择标记的离子，并获得其中染色体DNA序列中的靶序列被所需替换序列替换的细胞。
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公开(公告)号：US20120282698A1
公开(公告)日：2012-11-08
申请号：US13469762
申请日：2012-05-11
IPC分类号： C12N15/90
CPC分类号： C12N15/815 , C12N15/905 , C12N2800/30 , C12N2800/70
摘要： A method to construct eukaryotic cells having a target sequence in a chromosomal DNA sequence replaced by a desired replacement sequence is disclosed.
摘要翻译： 公开了一种构建真核细胞的方法，所述真核细胞在染色体DNA序列中具有由期望的置换序列替代的靶序列。
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公开(公告)号：US20100120154A1
公开(公告)日：2010-05-13
申请号：US12532205
申请日：2008-03-20
CPC分类号： C12N15/80 , C07K14/385 , C12N15/1058 , C12N15/902 , C12N2800/30
摘要： The present invention discloses a method to construct eukaryotic cells having a target sequence in a chromosomal DNA sequence replaced by a replacement sequence of interest comprising: modifying a parent eukaryotic cell with a preference for NHR to provide a eukaryotic cell having an increased HR/NHR ratio as compared to the parent cell, providing two sets of DNA molecules of which the first set comprises DNA molecules each comprising a first non-functional fragment of the replacement sequence of interest flanked at its 5′-side by a DNA sequence substantially homologous to a sequence of the chromosomal DNA flanking the target sequence and the second set comprises DNA molecules each comprising a second non-functional fragment of the DNA replacement sequence of interest overlapping with the first non-functional fragment and flanked at its 3′-side by a DNA sequence substantially homologous to a sequence of the chromosomal DNA flanking the target sequence, wherein the first and second non-functional fragments become functional upon recombination; transforming the modified eukaryotic cells with both sets of DNA molecules; and growing the cells to obtain transformed progeny cells having a DNA molecule from the first set recombined with a DNA molecule from the second set into a DNA molecule comprising a functional replacement sequence and having the target sequence replaced by the functional replacement sequence.
摘要翻译： 本发明公开了一种构建真核细胞的方法，所述真核细胞在染色体DNA序列中被替换的目标序列替代，所述染色体DNA序列被替换的目标序列所替代，包括：以优选NHR修饰亲本真核细胞以提供具有增加的HR / NHR比的真核细胞 与亲本细胞相比，提供两组DNA分子，其中第一组包含DNA分子，每个DNA分子包含在其5'侧两侧的替换序列的第一个非功能性片段，其基本上与 在靶序列侧翼的染色体DNA的序列和第二组包含DNA分子，每个DNA分子包含与第一非功能片段重叠的DNA置换序列的第二个非功能性片段，并在其3'侧通过DNA侧接 基本上与靶序列侧翼的染色体DNA的序列同源的序列，其中第一和第二 非功能性片段在重组时变得有功能; 用两组DNA分子转化修饰的真核细胞; 并生长细胞以获得具有来自第一组的DNA分子的转化后代细胞与来自第二组的DNA分子重组为包含功能性替换序列的DNA分子并且将靶序列替换为功能性替换序列。
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