TRANSGENIC ANIMAL MODELS USING YACS

Genpharm计划的目标是开发引入 将酵母人工染色体(YAC)导入小鼠的生殖系，并 将该技术应用于转基因小鼠的制备 一个广泛的，完全的人类免疫球蛋白曲目。这些老鼠会允许 以类似于以下方法分离人的单抗 啮齿动物的单抗。 第一阶段的目标是引入携带V、D、J和CMU的小型YAC 将人类重链免疫球蛋白基因的某些区域导入猪的种系 小鼠创造携带人类免疫球蛋白微型基因的转基因小鼠。 将测试小鼠体内人IgM蛋白的表达情况 血清和脾B细胞DNA重排。 在第二阶段，第一阶段产生的动物将与人类繁殖 表达Kappa轻链的转基因小鼠及其产物 小鼠测试其表达表面的主要曲目的能力 并分泌出完全人类的免疫球蛋白。另外，附加的常量和变量 将在YAC中克隆区域，以添加或替换第一阶段YAC，以 允许类别转换、体细胞突变和产生免疫球蛋白。如果 必要的序列修改，如引入额外的 还将添加增强器元素。转基因小鼠也将被 产生携带未重排的人类kappa基因的大片段 并培育成二期重链小鼠。最后，这些老鼠将会是 用缺乏功能性内源性免疫球蛋白基因的小鼠饲养。 杂交瘤将在项目的不同阶段从他们的 以确定转基因的体细胞突变是否可以 高效地发生，导致广泛的高亲和力的曲目，完全 产生人免疫球蛋白的B细胞。

The goal of GenPharm's program is to develop methods for the introduction of yeast artificial chromosomes (YACs) into the germline of mice, and to apply the technology toward the generation of transgenic mice expressing a broad, fully human immunoglobulin repertoire. These mice would permit the isolation of human monoclonal antibodies in an analogous manner to rodent monoclonal antibodies. The goal of Phase I to introduce small YACs carrying V,D,J and Cmu regions of the human heavy chain immunoglobulin gene into the germline of mice to create transgenic mice carrying a human immunoglobulin minigene. The mice will be tested for expression of human IgM proteins in the serum, and for DNA rearrangement in spleen B-cells. In Phase II, the animals generated in Phase I will be bred with Human kappa light chain expressing transgenic mice, and the resultant pilot mouse tested for its capacity to express a primary repertoire of surface and secreted fully human IgMs. Also, additional constant and variable regions will be cloned in YACs to add to or replace the Phase I YACs, to permit class switching, somatic mutation and IgG production. If necessary, sequence modifications such as the introduction of additional enhancer elements will also be added. Transgenic mice will also be generated carrying large fragments of the unrearranged human kappa gene and bred to the Phase II heavy chain mice. Finally, these mice will be bred with mice lacking functional endogenous immunoglobulin genes. Hybridomas will be generated at various stages of the project from their offspring to determine if somatic mutation of the transgene can efficiently occur, leading to a broad repertoire of high affinity, fully human IgG producing B cells.