TRANSGENIC ANIMAL MODELS USING YACS

使用 YACS 的转基因动物模型

• 批准号: 3489572
• 负责人: Ted Choi
• 金额: $ 4.99万
• 依托单位: GENPHARM INTERNATIONAL, INC.
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1992-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3489572
• 关键词: artificial chromosomes; biological models; cell type; enzyme linked immunosorbent assay; flow cytometry; gene rearrangement; genetically modified animals; germ cells; hybridomas; immunoglobulin M; immunoglobulin genes; laboratory mouse; method development; microinjections; model design /development; molecular cloning; monoclonal antibody; northern blottings; oligonucleotides; radiotracer; southern blotting

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.

GenPharm项目的目标是开发引入 酵母人工染色体（YACs）进入小鼠的生殖系， 将该技术应用于转基因小鼠的产生， 一种广泛的全人类免疫球蛋白库 这些老鼠会允许 人单克隆抗体的分离与 啮齿动物单克隆抗体。 第一阶段的目标是引入携带V、D、J和Cmu的小型YAC， 将人重链免疫球蛋白基因的多个区域导入 小鼠来产生携带人免疫球蛋白小基因的转基因小鼠。 将测试小鼠中人IgM蛋白的表达。 血清和脾B细胞中的DNA重排。 在第II阶段，第I阶段产生的动物将与人 表达κ轻链的转基因小鼠，以及由此产生的试验小鼠， 测试小鼠表达表面抗原的主要库的能力 并分泌完全的人IgM。 此外，附加常量和变量 将在YAC中克隆区域，以添加或替换I期YAC， 允许类别转换、体细胞突变和IgG产生。 如果 必要的序列修饰，如引入额外的 还将添加增强子元件。 转基因小鼠也将 产生携带未重排的人类kappa基因的大片段 并培育成II期重链小鼠。 最后，这些老鼠将 用缺乏功能性内源性免疫球蛋白基因的小鼠繁殖。 杂交瘤将在项目的各个阶段产生， 以确定转基因的体细胞突变是否可以 有效地发生，导致广泛的高亲和力，充分 产生人IgG的B细胞。