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    • 2. 发明申请
    • NOVEL METHODS
    • 新方法
    • WO2008079943A3
    • 2008-11-06
    • PCT/US2007088310
    • 2007-12-20
    • SMITHKLINE BEECHAM CORPTAYLOR ALEXANDER HKRISHNA DELFI
    • TAYLOR ALEXANDER HKRISHNA DELFI
    • C12N15/09C12N15/00C12N15/63
    • C12N15/1082C12N15/1086
    • The present invention provides methods for site-specifically integrating at least one first nucleic acid into a genome of at least one cell, comprising: transforming said genome with a reporter nucleic acid construct that is linked to a first att site; selecting at least one first transformed cell having at least one integrated reporter nucleic acid construct; introducing said at least one first nucleic acid and a homologous recombination mediating enzyme into said cell wherein said at least one first nucleic acid comprises at least one second att site that is complementary to said first att site; and maintaining said cell under conditions sufficient for said at least one first nucleic acid to integrate into said first att site producing at least one stably intergrated cell.
    • 本发明提供了用于将至少一种第一核酸位点特异性整合到至少一种细胞的基因组中的方法,包括:用与第一att位点连接的报道核酸构建体转化所述基因组; 选择具有至少一个整合的报道子核酸构建体的至少一个第一转化细胞; 将所述至少一种第一核酸和同源重组介导酶导入所述细胞中,其中所述至少一种第一核酸包含与所述第一att位点互补的至少一个第二att位点; 并且将所述细胞保持在足以使所述至少一种第一核酸整合到所述第一att位点的条件下,从而产生至少一个稳定整合的细胞。
    • 3. 发明申请
    • NOVEL METHODS
    • 新方法
    • WO2008079943A2
    • 2008-07-03
    • PCT/US2007/088310
    • 2007-12-20
    • SMITHKLINE BEECHAM CORPORATIONTAYLOR, Alexander, H.KRISHNA, Delfi
    • TAYLOR, Alexander, H.KRISHNA, Delfi
    • C12N15/74
    • C12N15/1082C12N15/1086
    • The present invention provides methods for site-specifically integrating at least one first nucleic acid into a genome of at least one cell, comprising: transforming said genome with a reporter nucleic acid construct that is linked to a first att site; selecting at least one first transformed cell having at least one integrated reporter nucleic acid construct; introducing said at least one first nucleic acid and a homologous recombination mediating enzyme into said cell wherein said at least one first nucleic acid comprises at least one second att site that is complementary to said first att site; and maintaining said cell under conditions sufficient for said at least one first nucleic acid to integrate into said first att site producing at least one stably intergrated cell.
    • 本发明提供了将至少一种第一核酸位点特异性整合至至少一种细胞的基因组的方法,所述方法包括:用连接到第一att位点的报道核酸构建体转化所述基因组; 选择至少一个具有至少一个整合的报告核酸构建体的第一转化细胞; 将所述至少一种第一核酸和同源重组介导酶引入所述细胞中,其中所述至少一种第一核酸包含与所述第一att位点互补的至少一个第二att位点; 以及在足以使所述至少一种第一核酸整合入所述第一att位点的条件下维持所述细胞,产生至少一个稳定整合的细胞。
    • 5. 发明申请
    • HOST CELLS AND METHODS OF USE
    • 宿主细胞和使用方法
    • WO2010099195A1
    • 2010-09-02
    • PCT/US2010/025223
    • 2010-02-24
    • GLAXOSMITHKLINE LLC.JIN, Yong, HwanJOWETT, James, D.TAYLOR, Alexander, H.ZHU, Yuan
    • JIN, Yong, HwanJOWETT, James, D.TAYLOR, Alexander, H.ZHU, Yuan
    • C12N1/00C07H21/04
    • C12N15/815C07K14/605C07K2319/31C12N9/1051C12N9/60
    • The present invention provides genetically modified Pichia strains wherein at least one nucleic acid sequence encoding a functional gene product and/or at least one nucleic acid sequence necessary for expression of at least one functional gene product in said Pichia strain is genetically modified, wherein said gene product is responsible for proteolysis and/or glycosylation in said genetically modified Pichia strain. In particular, Pichia strains are provided wherein nucleic acid sequence encoding a functional gene product or expression of said gene product are genetically modified: PEP 4 , PRBl, YPSl , YPS2 , YMPl , YMP2 , YMP 3 and PMT4 . Also provided herein are genetically modified host cells wherein wild type parent of said genetically modified host cell comprises a gene encoding a polypeptide having at least 60% sequence identity to amino acids 1-865 of SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO: 8 wherein said gene is genetically modified in the genome of said host cell such that the gene product is reduced or eliminated in said genetically modified host cell compared with said wild type host cell.
    • 本发明提供了遗传修饰的毕赤酵母菌株,其中至少一个编码功能性基因产物的核酸序列和/或在所述毕赤酵母属菌株中表达至少一种功能性基因产物所必需的至少一种核酸序列被遗传修饰,其中所述基因 产物负责所述遗传修饰的毕赤酵母菌株中的蛋白水解和/或糖基化。 特别地,提供了毕赤酵母菌株,其中编码功能性基因产物的核酸序列或所述基因产物的表达被遗传修饰:PEP4,PRB1,YPS1,YPS2,YMP1,YMP2,YMP3和PMT4。 本文还提供遗传修饰的宿主细胞,其中所述经遗传修饰的宿主细胞的野生型亲本包含编码与SEQ ID NO:4,SEQ ID NO:6的氨基酸1-865至少60%序列同一性的多肽的基因和 SEQ ID NO:8,其中所述基因在所述宿主细胞的基因组中被遗传修饰,使得与所述野生型宿主细胞相比,在所述遗传修饰的宿主细胞中基因产物被还原或消除。