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    • 2. 发明授权
    • Transcriptional activators with graded transactivation potential
    • 具有分级反式激活潜能的转录激活子
    • US06271341B1
    • 2001-08-07
    • US09577027
    • 2000-05-23
    • Udo BaronManfred GossenHermann Bujard
    • Udo BaronManfred GossenHermann Bujard
    • C07K1900
    • C07K14/005A01K2217/05A61K48/00C07K2319/00C12N15/635C12N15/85C12N2710/16622C12N2830/003C12N2830/006
    • Transcriptional activators which differ in their activation potential by more than 3 orders of magnitude are provided. The transactivators are fusions between a DNA binding protein (e.g., a Tet repressor) and minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16). Substitution mutations at amino acid position 442 within the minimal VP16 domain provide transactivators with differing transactivation ability. Moreover, chimeric activation domains comprising both wild type and mutant minimal VP16 domains provide additional variants with differing transactivation ability. Various aspects of the invention pertain to nucleic acid molecules, vectors, host cells, fusion proteins, transgenic and homologous recombinant organisms and methods of regulating gene transcription.
    • 提供了其激活电位不同于3个数量级的转录激活子。 反式激活因子是DNA结合蛋白(例如Tet阻遏物)和衍生自单纯疱疹病毒蛋白16(VP16)的最小转录激活结构域之间的融合物。 在最小VP16结构域内的氨基酸位置442处的取代突变体提供具有不同反式激活能力的反式激活因子。 此外,包含野生型和突变型最小VP16结构域的嵌合活化域提供了具有不同反式激活能力的另外的变体。 本发明的各个方面涉及核酸分子,载体,宿主细胞,融合蛋白,转基因和同源重组生物以及调节基因转录的方法。
    • 3. 发明授权
    • Tetracycline-regulated transcriptional inhibitors
    • 四环素调节的转录抑制剂
    • US5789156A
    • 1998-08-04
    • US383754
    • 1995-02-03
    • Hermann BujardManfred Gossen
    • Hermann BujardManfred Gossen
    • A01K67/027C07K14/035C07K14/245C12N1/21C12N15/63C12N15/67C12N15/85C12Q1/68C07H21/04
    • C12N15/8509C07K14/005C07K14/245C12N15/63C12N15/635C12N15/67C12N15/85A01K2217/05A01K2217/075A01K2217/20A01K2267/01A01K2267/02A01K2267/03A01K2267/0393C07K2319/00C07K2319/09C07K2319/61C07K2319/71C07K2319/80C12N2710/16622C12N2830/003C12N2830/006Y10S435/81
    • Nucleic acid molecules and proteins useful for regulating the expression of genes in eukaryotic cells and organisms in a highly controlled manner are disclosed. In the regulatory system of the invention, transcription of a tet operator-linked nucleotide sequence is inhibited by a transcriptional inhibitor fusion protein composed of two polypeptides, a first polypeptide which binds to tet operator sequences either (i) in the absence but not the presence of tetracycline (or an analogue thereof) or (ii) in the presence but not the absence of tetracycline (or an analogue thereof), and a second polypeptide which directly or indirectly inhibits transcription in eukaryotic cells. In one embodiment, the fusion protein comprises a Tet repressor operatively linked to a transcriptional silencer polypeptide. In another embodiment, the fusion protein comprises a mutated Tet repressor operatively linked to a transcriptional silencer polypeptide. The fusion proteins of the invention are useful for reducing the level of transcription of a tet operator-linked target gene. Moreover, the fusion proteins of the invention can be used in combination with tetracycline-regulated transcriptional activator fusion proteins to allow for precise regulation of the expression of one or multiple target genes. Kits including the components of the regulatory system of the invention are also encompassed by the invention.
    • 公开了用于以高度受控的方式调节真核细胞和生物体中基因表达的核酸分子和蛋白质。 在本发明的调节系统中,tet操纵子连接的核苷酸序列的转录受到由两个多肽组成的转录抑制剂融合蛋白​​的抑制,所述两个多肽是结合tet操纵子序列的第一个多肽,(i)在不存在但不存在的情况下 的四环素(或其类似物)或(ii)在存在但不存在四环素(或其类似物)的情况下,以及在真核细胞中直接或间接抑制转录的第二多肽。 在一个实施方案中,融合蛋白包含可操作地连接到转录沉默子多肽的Tet阻遏物。 在另一个实施方案中,融合蛋白包含与转录沉默子多肽有效连接的突变Tet阻遏物。 本发明的融合蛋白可用于降低与tet操纵子连接的靶基因的转录水平。 此外,本发明的融合蛋白可以与四环素调节的转录激活物融合蛋白组合使用,以允许精确调节一个或多个靶基因的表达。 包括本发明的调节系统的组分的试剂盒也包括在本发明中。
    • 4. 发明授权
    • Tetracycline-inducible transcriptional activator and
tetracycline-regulated transcription units
    • 四环素诱导型转录激活因子和四环素调节转录单位
    • US5654168A
    • 1997-08-05
    • US275876
    • 1994-07-15
    • Hermann BujardManfred Gossen
    • Hermann BujardManfred Gossen
    • A01K67/027C07K14/245C12N15/63C12N15/67C12N15/85C12P21/00C07H21/04C12N15/00
    • C12N15/8509C07K14/245C12N15/63C12N15/635C12N15/67C12N15/85A01K2217/05A01K2217/20A01K2267/01A01K2267/03C07K2319/00C07K2319/09C07K2319/61C07K2319/71C07K2319/80C12N2830/003C12N2830/006
    • Nucleic acid molecules and proteins useful for regulating the expression of genes in eukaryotic cells and organisms in an inducible manner are disclosed. In the regulatory system of the invention, transcription of a tet operator-linked nucleotide sequence is stimulated by a transcriptional activator fusion protein composed of two polypeptides, a first polypeptide which binds to tet operator sequences in the presence of tetracycline or a tetracycline analogue and a second polypeptide which directly or indirectly activates transcription in eukaryotic cells. In one embodiment, the fusion protein comprises a mutated Tet repressor operatively linked to a transcriptional activation polypeptide, such as a portion of herpes simplex virus virion protein 16. In the absence of an inducing agent (tetracycline or a tetracycline analogue), transcription of the tet operator-linked nucleotide sequence remains uninduced. In the presence of the inducing agent, transcription of the tet operator-linked nucleotide sequence is stimulated by the transactivator fusion protein of the invention. Novel transcription units which allow for coordinate or independent tetracycline-regulated expression of two or more nucleotide sequences by the transactivator fusion protein of the invention are also disclosed. Kits including the components of the regulatory system of the invention are also encompassed by the invention.
    • 公开了用于以可诱导的方式调节真核细胞和生物体中基因表达的核酸分子和蛋白质。 在本发明的调节系统中,tet操纵子连接的核苷酸序列的转录受到由两个多肽组成的转录激活物融合蛋白的刺激,该多肽是在四环素或四环素类似物存在下与tet操纵子序列结合的第一多肽 在真核细胞中直接或间接激活转录的第二多肽。 在一个实施方案中,融合蛋白包含可操作地连接到转录激活多肽(例如一部分单纯疱疹病毒颗粒蛋白16)的突变的Tet阻遏物。在不存在诱导剂(四环素或四环素类似物)的情况下, tet操纵子连接的核苷酸序列仍未引起。 在诱导剂存在下,通过本发明的反式激活因子融合蛋白刺激tet操纵子连接的核苷酸序列的转录。 还公开了通过本发明的反式激活物融合蛋白允许两个或更多个核苷酸序列的协调或独立的四环素调节的表达的新型转录单位。 包括本发明的调节系统的组分的试剂盒也包括在本发明中。
    • 5. 发明授权
    • Tetracycline inducible transcription control sequence
    • 四环素诱导型转录调控序列
    • US09181556B2
    • 2015-11-10
    • US13121673
    • 2009-08-19
    • Hermann BujardRainer Loew
    • Hermann BujardRainer Loew
    • C07H21/04C12N15/63A61K31/70
    • C12N15/635C12N2770/40043C12N2830/003
    • The present invention relates to inducible transcription control sequences for the regulation of gene expression. Specifically, it relates to an transcription control sequence comprising at least two tet operator sequence motifs allowing the binding of tetracycline-dependent transcriptional regulators, wherein each of the tetracycline-dependent transcriptional regulators binds with respect to its neighbor to an opposite face of the DNA helix, and a minimal promoter comprising a TATA box which is linked at its 5′ end to a general transcription factor binding site. Further, the present invention relates to a vector, a host cell, a plant or a non-human transgenic animal comprising the transcription control sequence. Also contemplated is a method for regulating the expression of a nucleic acid sequence being operatively linked to the transcription control sequence of the present invention in a host cell, a plant or a non-human transgenic animal.
    • 本发明涉及用于调节基因表达的诱导型转录调控序列。 具体地说,本发明涉及包含至少两个允许四环素依赖性转录调节因子结合的tet操纵子序列基序的转录控制序列,其中四环素依赖性转录调节因子中的每一个相对于其邻近的DNA螺旋结构 ,以及包含在其5'末端与一般转录因子结合位点连接的TATA盒的最小启动子。 此外,本发明涉及包含转录调控序列的载体,宿主细胞,植物或非人转基因动物。 还考虑了在宿主细胞,植物或非人转基因动物中调节与本发明的转录调控序列有效连接的核酸序列的表达的方法。
    • 9. 发明授权
    • Tetracycline-inducible transcriptional inhibitor fusion proteins
    • 四环素诱导型转录抑制剂融合蛋白
    • US06271348B1
    • 2001-08-07
    • US09489777
    • 2000-01-24
    • Hermann BujardManfred Gossen
    • Hermann BujardManfred Gossen
    • C07K1900
    • C07K14/005A01K67/0275A01K2217/05A01K2217/075A01K2217/20A01K2227/105A01K2267/0393A61K48/00C07K14/245C07K2319/00C07K2319/09C07K2319/61C07K2319/71C07K2319/80C12N15/62C12N15/63C12N15/635C12N15/67C12N15/85C12N15/8509C12N2710/16622C12N2830/003C12N2830/006C12N2830/32
    • Methods of regulating gene expression in subjects using tetracycline-responsive fusion proteins are disclosed. In one embodiment, the method involves introducing into a cell the subject a nucleic acid molecule encoding a fusion protein which inhibits transcription, the fusion protein comprising a first polypeptide which binds to a tet operator sequence, operatively linked to a heterologous second polypeptide which inhibits transcription in eukaryotic cells; and modulating the concentration of a tetracycline, or analogue thereof, in the subject. The first polypeptide can binds to a tet operator sequence in the absence, but not the presence, of tetracycline. Alternatively, the first polypeptide can binds to a tet operator sequence in the presence, but not the absence, of tetracycline. In another embodiment, the method of the invention involves obtaining a cell from a subject, introducing into the cell a first nucleic acid molecule which operatively links a gene to at least one tet operator sequence, introducing into the cell a second nucleic acid molecule encoding an inhibitory fusion protein of the invention to form a modified cell, administering the modified cell to the subject and modulating the concentration of a tetracycline, or analogue thereof, in the subject. The first and second nucleic acid molecules can be linked or can be separate molecules.
    • 公开了使用四环素响应融合蛋白调节受试者基因表达的方法。 在一个实施方案中,该方法包括向受试者引入编码抑制转录的融合蛋白的核酸分子,该融合蛋白包含与tet操纵子序列结合的第一多肽,该第一多肽与抑制转录的异源第二多肽有效连接 在真核细胞中 并调节受试者中四环素或其类似物的浓度。 在不存在但不存在四环素的情况下,第一种多肽可以结合tet操纵子序列。 或者,第一多肽可以在四环素存在下但不存在时与tet操纵子序列结合。 在另一个实施方案中,本发明的方法涉及从受试者获得细胞,将在第一核酸分子中引入至少一个tet操纵子序列的第一核酸分子,将编码 本发明的抑制性融合蛋白以形成修饰的细胞,向受试者施用修饰的细胞并调节受试者中四环素或其类似物的浓度。 第一和第二核酸分子可以连接或可以是分开的分子。