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1. 发明申请

US20120276537A1 HOMOLOGOUS RECOMBINATION IN THE OOCYTE 审中-公开
标题翻译：同卵生物的重组
公开(公告)号：US20120276537A1
公开(公告)日：2012-11-01
申请号：US13504587
申请日：2010-10-28
申请人： Ralf Kühn , Wolfgang Wurst , Melanie Meyer
发明人： Ralf Kühn , Wolfgang Wurst , Melanie Meyer
IPC分类号： C12N15/90 , C12Q1/68 , C12N15/89
CPC分类号： A01K67/0278 , A01K2217/072 , A01K2227/105 , A01K2267/01 , C12N9/22 , C12N15/8509 , C12N15/907
摘要： The present invention relates to a method of modifying a target sequence in the genome of a mammalian or avian oocyte by homologous recombination with a donor nucleic acid sequence, the method comprising the steps (a) introducing into the oocyte a zinc finger nuclease or a nucleic acid molecule encoding the zinc finger nuclease in expressible form, wherein the zinc finger nuclease specifically binds within the target sequence and introduces a double strand break within the target sequence; and (b) introducing a nucleic acid molecule into the oocyte, wherein the nucleic acid molecule comprises the donor nucleic acid sequence and regions homologous to the target sequence. The present invention further relates to a method of producing a non-human mammal or an avian carrying a modified target sequence in its genome.
摘要翻译：本发明涉及通过与供体核酸序列同源重组来修饰哺乳动物或禽类卵母细胞基因组中的靶序列的方法，所述方法包括以下步骤：（a）向卵母细胞中引入锌指核酸酶或核酸编码锌指核酸酶的可分解形式的酸分子，其中所述锌指核酸酶在靶序列内特异性结合并在靶序列内引入双链断裂; 和（b）将核酸分子引入所述卵母细胞中，其中所述核酸分子包含供体核酸序列和与靶序列同源的区域。本发明还涉及在其基因组中产生携带修饰的靶序列的非人哺乳动物或禽类的方法。

2. 发明申请

US20130212725A1 FUSION PROTEINS COMPRISING A DNA-BINDING DOMAIN OF A TAL EFFECTOR PROTEIN AND A NON-SPECIFIC CLEAVAGE DOMAIN OF A RESTRICTION NUCLEASE AND THEIR USE 审中-公开
标题翻译：包含T细胞效应蛋白的DNA结合域和限制性核酸的非特异性切割域及其使用的融合蛋白
公开(公告)号：US20130212725A1
公开(公告)日：2013-08-15
申请号：US13702231
申请日：2011-06-07
申请人： Ralf Kühn , Wolfgang Wurst , Melanie Meyer
发明人： Ralf Kühn , Wolfgang Wurst , Melanie Meyer
IPC分类号： C12N15/62
CPC分类号： C12N15/62 , A01K67/0276 , A01K67/0278 , A01K2207/05 , A01K2217/07 , A01K2217/072 , A01K2227/105 , A01K2267/03 , A01K2267/0393 , C07K2319/80 , C12N9/22 , C12N15/907
摘要： The present invention relates to a method of modifying a target sequence in the genome of a eukaryotic cell, the method comprising the step: (a) introducing into the cell a fusion protein comprising a DNA-binding domain of a Tal effector protein and a non-specific cleavage domain of a restriction nuclease or a nucleic acid molecule encoding the fusion protein in expressible form, wherein the fusion protein specifically binds within the target sequence and introduces a double strand break within the target sequence. The present invention further relates to the method of the invention, wherein the modification of the target sequence is by homologous recombination with a donor nucleic acid sequence further comprising the step: (b) introducing a nucleic acid molecule into the cell, wherein the nucleic acid molecule comprises the donor nucleic acid sequence and regions homologous to the target sequence. The present invention also relates to a method of producing a non-human mammal or vertebrate carrying a modified target sequence in its genome. Furthermore, the present invention relates to a fusion protein comprising a DNA-binding domain of a Tal effector protein and a non-specific cleavage domain of a restriction nuclease.
摘要翻译：本发明涉及一种修饰真核细胞基因组中的靶序列的方法，所述方法包括以下步骤：（a）将包含Tal效应蛋白的DNA结合结构域的融合蛋白引入细胞，限制性核酸酶或编码可表达形式的融合蛋白的核酸分子的特异性切割结构域，其中所述融合蛋白在靶序列内特异性结合并在靶序列内引入双链断裂。本发明还涉及本发明的方法，其中靶序列的修饰是通过与供体核酸序列的同源重组而进一步包括步骤：（b）将核酸分子引入细胞，其中核酸分子包含供体核酸序列和与靶序列同源的区域。本发明还涉及在其基因组中产生携带修饰的靶序列的非人哺乳动物或脊椎动物的方法。此外，本发明涉及包含Tal效应蛋白的DNA结合结构域和限制性核酸酶的非特异性切割结构域的融合蛋白。

3. 发明申请

US20110061118A1 VECTORS AND METHODS FOR GENERATING VECTOR-FREE INDUCED PLURIPOTENT STEM (IPS) CELLS USING SITE-SPECIFIC RECOMBINATION 审中-公开
公开(公告)号：US20110061118A1
公开(公告)日：2011-03-10
申请号：US12933362
申请日：2009-03-17
申请人： Ralf Kühn , Wolfgang Wurst
发明人： Ralf Kühn , Wolfgang Wurst
IPC分类号： A01K67/02 , C07H21/04 , C12N15/63 , C12N5/10 , C12N15/79 , C12Q1/68 , C12Q1/02 , C12N5/071
CPC分类号： C07K14/4702 , C12N5/0696 , C12N15/85 , C12N2501/602 , C12N2501/603 , C12N2501/604 , C12N2501/606 , C12N2510/00 , C12N2800/30 , C12N2830/003 , C12N2840/206
摘要： The present invention relates to a DNA molecule comprising: (a) a first DNA sequence comprising: (aa) a coding sequence giving rise upon transcription to a factor that contributes to the reprogramming of a somatic cell into an induced pluripotent stem (iPS) cell; (ab) a promoter mediating the transcription of said coding sequence; and (ac) two sequence motifs that mediate excision of (aa) and/or (ab) from the DNA molecule, wherein one sequence motif is positioned 5′ and the other sequence motif is positioned 3′ of the sequence to be excised; (b) a second DNA sequence comprising a sequence motif that mediates site-specific integration of (a) into another DNA molecule. Further, the invention relates to DNA molecule comprising: (a) a first DNA sequence comprising: (aa) a coding sequence giving rise upon transcription to a factor that contributes to the reprogramming of a somatic cell into an induced pluripotent stem cell; and (ab) a promoter mediating the transcription of said coding sequence; (b) a second DNA sequence comprising: (ba) a sequence motif that mediates extrachromosomal self-replication of the DNA-molecule; and (bb) two sequence motifs that mediate excision of at least said sequence motif of (ba) from the second DNA sequence (b), wherein one sequence motif is located 5′ of (ba) and the other sequence motif 3′ of (ba). Also, the invention relates to a vector comprising the DNA molecule of the invention, a method for assembly of said vector and a somatic cell comprising said DNA molecule or said vector of the invention. Furthermore, the invention relates to methods to generate an induced pluripotent stem (iPS) cell, an induced pluripotent stem cell obtainable by said methods, to a kit comprising the DNA molecule of the invention, to a cell line or cell culture collection comprising the induced pluripotent stem cell of the invention, to the use of said cell or cell line as a research tool, to a method to generate a transgenic non-human animal and to a non-human animal generated by said method. Finally, the invention relates to a composition for gene therapy, regenerative medicine, cell therapy or drug screening.

4. 发明申请

US20140304847A1 RECOMBINATION EFFICIENCY BY INHIBITION OF NHEJ DNA REPAIR 审中-公开
标题翻译：通过抑制NHEJ DNA修复的重组效率
公开(公告)号：US20140304847A1
公开(公告)日：2014-10-09
申请号：US14124106
申请日：2012-06-06
申请人： Ralf Kühn , Wolfgang Wurst
发明人： Ralf Kühn , Wolfgang Wurst
IPC分类号： C12N15/85
CPC分类号： C12N15/85 , C12N15/1024 , C12N15/8509 , C12N15/907
摘要： The present invention relates to a method for modifying a target sequence in the genome of a mammalian cell, the method comprising the step of introducing into a mammalian cell: a. one or more compounds that introduce double-strand breaks in said target sequence; b. one or more DNA molecules comprising a donor DNA sequence to be incorporated by homologous recombination into the genomic DNA of said mammalian cell within said target sequence, wherein said donor DNA sequence is flanked upstream by a first flanking element and downstream by a second flanking element, wherein said first and second flanking element are different and wherein each of said first and second flanking sequence are homologous to a continuous DNA sequence on either side of the double-strand break introduced by said one or more compounds of a. within said target sequence in the genome of said mammalian cell; and c. one or more compounds that decrease the activity of the non-homologous end joining (NHEJ) DNA repair complex in said mammalian cell. Further, the invention relates to a method of producing a non-human mammal carrying a modified target sequence in its genome.
摘要翻译：本发明涉及一种用于修饰哺乳动物细胞基因组中靶序列的方法，所述方法包括引入哺乳动物细胞的步骤：a。一种或多种在所述靶序列中引入双链断裂的化合物; b。一个或多个DNA分子，其包含通过同源重组并入所述靶序列内的所述哺乳动物细胞的基因组DNA中的供体DNA序列，其中所述供体DNA序列在第一侧翼元件的上游侧和第二侧翼元件的下游，其中所述第一和第二侧翼元件是不同的，并且其中所述第一和第二侧翼序列中的每一个与由所述一种或多种a的化合物引入的双链断裂的任一侧上的连续DNA序列同源。在所述哺乳动物细胞的基因组中的所述靶序列内; 和c。一种或多种降低所述哺乳动物细胞中非同源末端连接（NHEJ）DNA修复复合物的活性的化合物。此外，本发明涉及在其基因组中生产携带修饰的靶序列的非人哺乳动物的方法。

5. 发明申请

US20100299771A1 MEANS AND METHODS FOR shRNA MEDIATED CONDITIONAL KNOCKDOWN OF GENES 审中-公开
公开(公告)号：US20100299771A1
公开(公告)日：2010-11-25
申请号：US12678645
申请日：2008-09-17
申请人： Ralf Kühn , Wolfgang Wurst , Patricia Steuber-Buchberger
发明人： Ralf Kühn , Wolfgang Wurst , Patricia Steuber-Buchberger
IPC分类号： A01K67/027 , C12N15/63 , A01K67/00 , C12N5/10 , C12N15/09 , C12Q1/68
CPC分类号： C12N15/111 , C12N2310/111 , C12N2310/14 , C12N2320/12 , C12N2320/50
摘要： The present invention relates to a combination of DNA segments comprising: (a) a first segment comprising in 5′ to 3′ or 3′ to 5′ order: (aa) a promoter; (ab) a first DNA sequence comprising: (i) a DNA sequence giving rise upon transcription to the sense strand of an shRNA molecule; (ii) a transcriptional stop element which is flanked by a first type of recombinase recognition sequences; and (iii) a DNA sequence giving rise upon transcription to the antisense strand of an shRNA molecule; (b) a second segment comprising in 5′ to 3′ or 3′ to 5′ order: (ba) a promoter; (bb) a second DNA sequence comprising: (i) a DNA sequence giving rise upon transcription to the sense strand of an shRNA molecule; (ii) a transcriptional stop element which is flanked by a second type of recombinase recognition sequences; and (iii) a DNA sequence giving rise upon transcription to the antisense strand of an shRNA molecule; wherein (i) said first type of recombinase recognition sequences are recognized and recombined by a recombinase but not recombined with said second type of recombinase recognition sequences; (ii) said second type of recombinase recognition sequences are recognized and recombined by the recombinase of (i) but not recombined with said first type of recombinase recognition sequences; and (iii) said DNA sequence of (ab) and (bb) is expressed under the control of said promoters of (aa) and (ba) upon removal of said transcriptional stop elements of (ab) and (bb) by the activity of a recombinase, resulting in transcription of said shRNA molecule in a cell. Further, the invention relates to a genetically engineered non-human animal and a method to produce said transgenic non-human animal. Also, the invention relates to a cell genetically engineered with the DNA molecule of the invention and a method of simultaneously knocking down two genes in a cell. Furthermore, envisaged is a method of identifying a combination of two target genes as a potential drug target and the use of the DNA molecule of the invention for the preparation of a composition for gene therapy.

6. 发明申请

US20100242127A1 METHODS TO IDENTIFY MODULATORS OF B-RAF PROTEIN KINASE AND THEIR USE FOR THE TREATMENT OF ANXIETY AND DEPRESSION 审中-公开
标题翻译：鉴定B-RAF蛋白激酶调节剂的方法及其用于治疗放射性疾病和抑郁症的用途
公开(公告)号：US20100242127A1
公开(公告)日：2010-09-23
申请号：US12602753
申请日：2008-06-03
申请人： Christiane Hitz , Sabine Hölter , Ralf Kühn , Wolfgang Wurst , Benedikt Wefers
发明人： Christiane Hitz , Sabine Hölter , Ralf Kühn , Wolfgang Wurst , Benedikt Wefers
IPC分类号： A01K67/00 , C12Q1/68 , C12Q1/48 , G01N33/53 , A61K31/44 , A61P25/00 , C07D213/62
CPC分类号： C12Q1/485 , C12Q1/6883 , C12Q2600/136 , C12Q2600/158 , G01N33/6893 , G01N2500/02 , G01N2500/10 , G01N2800/301 , G01N2800/304
摘要： The present invention relates to a method for identifying a compound capable of modulating an anxiety or depression disorder comprising the steps of: (a) contacting a composition comprising a B-Raf protein or a B-Raf gene in expressible form or a transcript thereof with a compound under conditions that allow for an interaction of the B-Raf protein or the B-Raf gene or a transcript thereof and the compound; and (b) measuring whether said interaction, if any, results in (i) a change of B-Raf kinase activity compared to B-Raf kinase activity in the absence of said compound; (ii) a modulation of the expression of the B-Raf gene compared to B-Raf gene expression in the absence of said compound; or (iii) the formation of a complex between the compound and the B-Raf protein, wherein such a change in activity, modulation of expression or the formation of a complex is indicative of the compound being a modulator of an anxiety or depression disorder. Further, the invention relates to a method for treating an anxiety or depression disorder in an individual comprising administering to the individual an effective amount of a compound inhibiting B-Raf kinase activity or gene expression and to a use of a compound that inhibits B-Raf kinase activity or gene expression in the manufacture of a pharmaceutical composition for treating an anxiety or depression disorder. Moreover, the invention relates to a method of diagnosing a B-Raf associated anxiety or depression disorder and to a genetically engineered mouse. Finally, the invention also relates to a method of identifying another gene contributing to the pathophysiology of an anxiety or depression disorder apart from B-Raf.
摘要翻译：本发明涉及一种鉴定能够调节焦虑或抑郁症的化合物的方法，包括以下步骤：（a）将包含可表达形式的B-Raf蛋白或B-Raf基因或其转录物的组合物与在允许B-Raf蛋白或B-Raf基因或其转录物与所述化合物相互作用的条件下的化合物; 和（b）测量所述相互作用（如果有的话）导致（i）与不存在所述化合物的B-Raf激酶活性相比，B-Raf激酶活性的变化; （ii）与不存在所述化合物的B-Raf基因表达相比，B-Raf基因表达的调节; 或（iii）化合物与B-Raf蛋白质之间的复合物的形成，其中这种活性变化，表达调节或复合物的形成指示该化合物为焦虑或抑郁症的调节剂。此外，本发明涉及一种治疗个体中的焦虑或抑郁症的方法，包括向个体施用有效量的抑制B-Raf激酶活性或基因表达的化合物以及使用抑制B-Raf的化合物在制备用于治疗焦虑或抑郁症的药物组合物中的激酶活性或基因表达。此外，本发明涉及一种诊断B-Raf相关焦虑症或抑郁症病症和遗传工程化小鼠的方法。最后，本发明还涉及一种识别除了B-Raf之外有助于焦虑或抑郁症病理生理学的另一种基因的方法。

7. 发明申请

US20090113561A1 GENE TRAP CASSETTES FOR RANDOM AND TARGETED CONDITIONAL GENE INACTIVATION 审中-公开
标题翻译：用于随机和有针对性的条件基因失活的基因捕获系统
公开(公告)号：US20090113561A1
公开(公告)日：2009-04-30
申请号：US11720231
申请日：2005-11-28
申请人： Harald Von Melchner , Frank Schnutgen , Wolfgang Wurst , Particia Ruiz , Silke De-Zolt , Thomas Floss , Jens Hansen
发明人： Harald Von Melchner , Frank Schnutgen , Wolfgang Wurst , Particia Ruiz , Silke De-Zolt , Thomas Floss , Jens Hansen
IPC分类号： A01K67/027 , C12N15/11 , C12N15/87 , C12Q1/68 , C12N5/06
CPC分类号： C12N15/1051 , C12N15/907 , C12N2799/027 , C12N2800/30 , C12N2800/60
摘要： A new type of gene trap cassette, which can induce conditional mutations, relies on directional site-specific recombination systems, which can repair and re-induce gene trap mutations when activated in succession. After the gene trap cassettes are inserted into the genome of the target organism, mutations can be activated at a particular time and place in somatic cells. The gene trap cassettes also create multipurpose alleles amendable to a wide range of post-insertional modifications. Such gene trap cassettes can be used to mutationally inactivate all cellular genes temporally and/or spatially. Cells which contain the inventive gene trap cassette can be used for identification and/or isolation of genes and for the creation of transgenic organisms to study gene function at various developmental stages, including the adult, as well as for the creation of animal models of human disease useful for in vivo drug target validation.
摘要翻译：可以诱导条件突变的新型基因陷阱盒依赖于定向位点特异性重组系统，其可以在连续激活时修复和再诱导基因陷阱突变。将基因陷阱盒插入目标生物体的基因组后，可以在特定时间激活突变并置于体细胞中。基因陷阱盒还创建了可以进行广泛的插入后修饰的多用途等位基因。这种基因捕获盒可用于在时间和/或空间上突变灭活所有细胞基因。含有本发明基因捕获盒的细胞可用于鉴定和/或分离基因，并用于产生转基因生物以研究各种发育阶段的基因功能，包括成人，以及用于创建人的动物模型用于体内药物靶标验证的疾病。

8. 发明授权

US5806168A Tool for the contemporary crimping of a plurality of insulated wires in an electrical connector 失效
标题翻译：用于电连接器中多个绝缘电线的当代压接的工具
公开(公告)号：US5806168A
公开(公告)日：1998-09-15
申请号：US850289
申请日：1997-05-05
申请人： Wolfgang Wurst , Eckhard Wolter
发明人： Wolfgang Wurst , Eckhard Wolter
IPC分类号： H01R43/01
CPC分类号： H01R43/015 , Y10T29/5151 , Y10T29/53226 , Y10T29/53243
摘要： A tool (10) for the termination of insulated wires in electrical connectors having different heights, which tool has a mechanism affording adjustment of the spacing between a pressing body (50), accommodated by a splice head, and a retaining body for the connector. The adjustment in height between the bodies is afforded by step adjustment and by an intermediate member (56, 58) affording smaller pitches, and the tool has an attachment mechanism for releasably attaching the tool to the retaining body.
摘要翻译：一种用于在具有不同高度的电连接器中终止绝缘电线的工具（10），该工具具有一种机构，用于调节由接头头容纳的按压体（50）和用于连接器的保持体之间的间隔。主体之间的高度调节是通过阶梯调节和提供较小间距的中间构件（56,58）提供的，并且该工具具有用于将工具可释放地附接到保持体的附接机构。

9. 发明授权

US09085767B2 Enhancer-containing gene trap vectors for random and targeted gene trapping 有权
标题翻译：用于随机和靶向基因捕获的含增强子基因捕获载体
公开(公告)号：US09085767B2
公开(公告)日：2015-07-21
申请号：US11720227
申请日：2005-11-28
申请人： Harald Von Melchner , Frank Schnütgen , Wolfgang Wurst , Patricia Ruiz
发明人： Harald Von Melchner , Frank Schnütgen , Wolfgang Wurst , Patricia Ruiz
IPC分类号： C12N15/86 , C12N15/867 , C12N15/65 , C12N15/90 , C12N15/10
CPC分类号： C12N15/1051 , C12N15/63 , C12N2740/13043 , C12N2800/30 , C12N2800/60
摘要： The present invention relates to a novel class of gene trap vector (enhanced gene trap vectors, eGTV) for efficiently identifying silent or weakly expressed target genes in mammalian genomes, methods of their production and methods for identifying and mutating target genes by using the enhanced gene trap vectors. The gene trap vectors of the present invention can also be used for inducing the expression of silent genes and enhancing the expression of weakly expressed genes. The use of the enhanced gene trap vectors for creating transgenic organisms to identify gene function and to validate pharmaceutical compounds prior to clinical applications is a further aspect of the present invention.
摘要翻译：本发明涉及用于有效识别哺乳动物基因组中的沉默或弱表达的靶基因的新型基因捕获载体（增强型基因捕获载体，eGTV），其生产方法以及通过使用增强型基因鉴定和突变靶基因的方法陷阱向量。本发明的基因捕获载体也可用于诱导沉默基因的表达和增强弱表达基因的表达。使用增强型基因捕获载体产生转基因生物以识别基因功能并在临床应用之前验证药物化合物是本发明的另一方面。

10. 发明授权

US08530168B2 Fusion protein comprising a Caspase domain and a nuclear hormone receptor binding domain and methods and uses thereof 有权
标题翻译：包含胱天蛋白酶结构域和核激素受体结合结构域的融合蛋白及其方法和用途
公开(公告)号：US08530168B2
公开(公告)日：2013-09-10
申请号：US12665680
申请日：2008-06-20
申请人： Yuanyuan Chu , Ralf Kuehn , Wolfgang Wurst
发明人： Yuanyuan Chu , Ralf Kuehn , Wolfgang Wurst
IPC分类号： G01N33/53 , C07H21/04 , C12P21/06 , C12N15/00 , C12N5/00 , C07K1/00
CPC分类号： C12N9/6475 , C07K14/72 , C07K14/721 , C07K2319/00
摘要： The present invention relates to a fusion protein comprising a Caspase domain or a functionally active variant thereof and a ligand binding domain of a nuclear hormone receptor, a nucleic acid coding for the fusion protein, a vector or cell comprising the nucleic acid, a method of producing the fusion protein, a non-human transgenic animal containing the nucleic acid, the use of the fusion protein for ligand-mediated induction of apoptosis of a cell, or for studying the function of a cell, tissue and/or organ or the use of a transgenic organism for studying the function of a cell at various developmental stages or as a disease model, a method for inducing apoptosis of a cell expressing a fusion protein or for identifying a ligand, or a medicament comprising a fusion protein, the nucleic acid, the vector or the cell, particularly for the treatment of cancer or for or after transplantation, particularly as safety mechanism.
摘要翻译：本发明涉及包含胱天蛋白酶结构域或其功能活性变体和核激素受体的配体结合结构域，编码融合蛋白的核酸，包含核酸的载体或细胞的融合蛋白，产生融合蛋白，含有核酸的非人转基因动物，融合蛋白用于配体介导的诱导细胞凋亡的用途，或用于研究细胞，组织和/或器官的功能或用途用于研究各种发育阶段的细胞功能或作为疾病模型的转基因生物体，诱导表达融合蛋白或鉴定配体的细胞凋亡的方法或包含融合蛋白的药物，所述核酸，载体或细胞，特别是用于治疗癌症或移植后或移植后，特别是作为安全机制。

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