SOMATIC MUTATION & AFFINITY MATURATION OF A CHIMERIC IG

体细胞突变

• 批准号: 2074625
• 负责人: ROBERTA R POLLOCK
• 金额: $ 10.33万
• 依托单位: OCCIDENTAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2074625
• 关键词: B lymphocyte; T lymphocyte; antibody specificity; chimeric proteins; gene mutation; gene rearrangement; genetically modified animals; human genetic material tag; hybridomas; immunoglobulin genes; immunoglobulins; introns; laboratory mouse; leukocyte activation /transformation

The generation of antibody diversity is a complex and highly regulated process. Immunoglobulin (Ig) gene rearrangement, to form functional variable (V) region genes (which bind antigen) is the major way this diversity is initially generated, and occurs before antigen is encountered. Additional diversity arises during an immune response by somatic mutation and antigen selection of mutated antibodies with higher antigen affinity. The goal of this proposal is to study, in transgenic mice, how expression of chimeric immunoglobulins composed of rearranged murine variable region genes and human constant (C) region genes, affects the rearrangement of endogenous variable region genes and whether such chimeric genes undergo somatic mutation and affinity maturation. Initial studies will be done on mice expressing only a chimeric Ig heavy chain; later studies will be done on mice expressing chimeric heavy and light chains. Two types of chimeric transgenes will be used. Both the heavy and the light chain murine variable regions are derived from a hybridoma making an anti-dansyl response (dansyl is a hapten, which is a small, simple molecule that contains only one antigenic determinant). The heavy chain constructs consist of a murine heavy chain anti-dansyl V region and a human gamma-1 C region, with the murine V/H promoter, intron enhancer, and plus or minus the 3' alpha enhancer. The light chain construct contains a murine light chain anti-dansyl V region and a human kappa light chain C region, with the V/L promoter, intron enhancer, and 3' enhancer. Since the murine V regions are specific for a known antigen, we can study both somatic mutation and affinity maturation in these mice. In particular, we can ask whether a human gamma-1 constant region can send three different types of signals to murine B lymphocytes; one to shut down endogenous V gene rearrangement, a second to initiate somatic mutation and a third to mediate antigen selection. The specific questions to be asked are: 1) does expression of a human gamma-1 constant region inhibit rearrangement of endogenous heavy chain V genes; 2) do the transgenes undergo somatic mutation in these animals; in other words, can the human gamma-1 gene signal the B lymphocyte that it has encountered antigen, and in the presence of T lymphocyte help turn on somatic mutation, and are the transgenes then mutated; and 3) do the transgenes undergo affinity maturation; are cells expressing mutated transgenes with increased affinity for dansyl selectively stimulated to proliferate, resulting in increased antibody affinity over time after immunization.

抗体多样性的产生是一个复杂且高度调节的 过程。 免疫球蛋白（IG）基因重排以形成功能 变量（v）区域基因（结合抗原）是主要方式 最初是生成多样性的，并且发生在抗原是 遭遇。 通过 较高的突变抗体的体细胞突变和抗原选择 抗原亲和力。 该提议的目的是在转基因小鼠中研究表达方式 由重排鼠可变区组成的嵌合免疫球蛋白 基因和人类常数（c）区域基因影响 内源性可变区域基因以及这种嵌合基因是否经历 体细胞突变和亲和力成熟。 最初的研究将进行 仅表达嵌合IG重链的小鼠；以后的研究将进行 在表达嵌合体重和轻链的小鼠上。 将使用两种类型的嵌合转基因。 重物和 轻链鼠变量区域是从杂交瘤中得出的 抗丹珊基反应（丹珊基是触觉，它是一个小的，简单的 仅包含一个抗原决定因素）的分子。 重链 构造由鼠重链抗丹珊基V区和A组成 人类伽马1 C区，带有鼠V/H启动子，内含子增强子和 加上或减去3'Alpha增强子。 轻链结构包含 鼠轻链抗丹珊基V区和人类Kappa轻链C 区域，带有V/L启动子，内含子增强子和3'增强子。 自从 鼠V区域是针对已知抗原的特异性的，我们可以研究两者 这些小鼠的体细胞突变和亲和力成熟。 特别是我们 可以询问人类伽马1是否可以发送三个不同的区域 鼠B淋巴细胞的信号类型；一个关闭内源V 基因重排，第二次启动躯体突变，三分之一 介导抗原选择。 要问的具体问题是：1）表达人 伽马1恒定区域抑制内源性重链V的重排 基因； 2）使转基因在这些动物中经历体细胞突变；在 其他词语，人γ-1基因是否可以信号淋巴细胞 遇到了抗原，在存在T淋巴细胞的情况下有助于开启 体细胞突变，是转基因，然后突变。 3）做 转基因经历亲和力成熟；是表达突变的细胞 对丹烷的亲和力增加的转基因有选择地刺激 扩散，导致抗体亲和力随着时间的推移而增加 免疫。