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    • 6. 发明申请
    • COMPOSITION AND METHOD FOR DIVERSIFICATION OF TARGET SEQUENCES
    • 用于目标序列多样化的组合物和方法
    • WO2013138518A1
    • 2013-09-19
    • PCT/US2013/031036
    • 2013-03-13
    • OMEROS CORPORATIONUNIVERSITY OF WASHINGTON THROUGH ITS CENTER FOR COMMERCIALIZATION
    • CUMMINGS, W., JasonTJOELKER, Larry, W.WOOD, Christi, L.YABUKI, MunehisaALLISON, Daniel, S.LEPPARD, John, B.MAIZELS, Nancy
    • C12N15/63C12N15/13C12N15/70C12N15/79
    • C12N15/1082C07K16/00C07K16/2863C07K16/30C07K2317/24C07K2317/92
    • The disclosure relates generally to the targeting of genes to, and their integration into, an innnnunoglobulin (antibody) heavy chain locus. In particular, the methods described herein contemplate replacing the single rearranged heavy chain V, D, and J genes of a B cell lymphoma such as DT40 with independently rearranged V H -D-J H genes of chicken, in a system for generating immunoglobulin diversity. Also contemplated is replacement of the chicken V H -D-J H with rearranged V H -D-J H genes of other vertebrates including human in a system for generating immunoglobulin diversity, with the exception of any substitution disclosed and claimed in PCT application WO 2009/029315 A2. Regions upstream of the rearranged V H gene, and downstream of the rearranged J H genes, are identified and incorporated into the immunoglobulin heavy chain gene-targeting vectors for efficient gene replacement. Also described is construction of a diverse chicken immunoglobulin heavy chain VDJ library in DT40 by homologous gene replacement of the single endogenous rearranged VDJ gene with a chicken VDJ repertoire using the described targeting vectors. The methods are useful for generating a diverse library of immunoglobulins that can be screened for binding to desired target antigens. Compositions include the unique targeting vectors, the diverse chicken heavy chain VDJ library in the targeting vectors, and the diverse chicken VDJ DT40 library that is created by homologous gene replacement of the single endogenous rearranged VDJ gene with a chicken VDJ repertoire, using the targeting vectors.
    • 本公开一般涉及将基因靶向于其和无融合蛋白(抗体)重链基因座的融合体。 特别地,本文描述的方法考虑在用于产生免疫球蛋白多样性的系统中将具有独立重排的鸡的VH-D-JH基因的B细胞淋巴瘤(例如DT40)的单重排列重链V,D和J基因替换为鸡的VH-D-JH基因。 还考虑到,除了在PCT申请WO 2009/029315A2中公开和要求保护的任何替代物之外,在用于产生免疫球蛋白多样性的系统中替换包括人在内的其它脊椎动物的重排的VH-D-JH基因的鸡VH-D-JH 。 鉴定重新排列的VH基因上游区域和重新排列的JH基因的下游区域,并将其并入免疫球蛋白重链基因靶向载体中以进行有效的基因替换。 还描述了使用所述靶向载体通过用单独的内源重排的VDJ基因与鸡VDJ库的同源基因替换来构建DT40中的多种鸡免疫球蛋白重链VDJ文库。 该方法可用于产生可筛选以结合所需靶抗原的免疫球蛋白的多样化文库。 组合物包括独特的靶向载体,靶向载体中不同的鸡重链VDJ文库,以及通过使用靶向载体的单个内源重排的VDJ基因的同源基因替换产生的多样化的鸡VDJ DT40文库 。