NOVEL ASSAY FOR SOMATIC MUTATION IN IG V GENES IN VIVO

体内 IG V 基因体细胞突变的新颖检测

• 批准号: 3455591
• 负责人: ROBERTA R POLLOCK
• 金额: $ 8.42万
• 依托单位: OCCIDENTAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3455591
• 关键词: T lymphocyte; chimeric proteins; flow cytometry; fluorescence microscopy; gene expression; gene mutation; gene rearrangement; genetic manipulation; genetic translation; genetically modified animals; histochemistry /cytochemistry; immunoglobulin genes; laboratory mouse; leukocyte activation /transformation; nucleic acid sequence; reporter genes; tissue /cell culture; transfection

Somatic mutation of immuoglobulin (Ig) variable (V) genes plays an important role in the generation of antibody diversity. The goal of this project is to develop an assay to detect and isolate cells undergoing somatic mutation in vivo and in vitro, by making transgenic mice in which a somatic mutation in an Ig transgene results in expression of a reporter gene, lacZ, that is easily detected. The transgene to be used encodes a fusion protein that pairs a rearranged VDJ, containing a stop codon, with a marker gene, lacZ. The VDJ is 5' of the lacZ; its stop codon therefore prevents translation of the lacZ "exon". If the stop codon is reverted by somatic mutation, the entire fusion protein will be translated, and lacZ expressed. LacZ expression can be readily detected by histochemical or fluorescence assays. Cells that have undergone somatic mutation will turn blue when the chromogenic substrate (X-gal) is used, or fluorescence if the fluorogenic substrate FDG is added. Somatic mutation can be studied in animals by sectioning organs, or even entire embryos, and staining with X-gal; sites of mutation will turn blue. The amount of mutation can be quantitated with FDG and analysis on the Fluorescence Activated Cell Sorter (FACS). Positive cells can be isolated using the FACS and further characterized as to phenotypic and functional properties. The initial step is to make this construct and an appropriate control construct (with a translatable VDJ), transfect them into cell lines to ensure that they are expressed, and then to make transgenic mice. These mice can then be used to directly test many of the current hypotheses regarding somatic mutation, including: 1) somatic mutation occurs in a burst of activity after immunization; 2) somatically mutating B cells are found in germinal centers; 3) such cells are, or are destined to become, memory cells; 4) Lyl+ B cells do not somatically mutate; 5) T cells do not somatically mutate; and 6) somatic mutation is not triggered in responses to thymus independent antigens. The transgene can also be bred onto mice with various immune dysfunctions, such as autoimmune strain MRL/lpr, the autoimmune and immunodeficient motheaten strain, and the immunodeficient scid mouse strain. Somatic mutation in these mice can be analyzed and compared to the normal situation.

免疫球蛋白（Ig）变量（V）基因的体细胞突变播放 在抗体多样性的产生中的重要作用。 目标的目标 项目是开发一种检测和分离经过的细胞的测定 通过使转基因小鼠在体内和体外体外突变 Ig转基因中的体细胞突变导致报告基因的表达 很容易检测到Gene，Lacz。 用于使用的转基因编码A 融合蛋白，配对一个重排的VDJ，包含终止密码子，与 标记基因，lacz。 VDJ是lacz的5'；因此，它的停止密码子 防止LACZ“外显子”的翻译。 如果终止密码子被恢复 体细胞突变，整个融合蛋白将被翻译，lacz 表达。 LACZ表达可以通过组织化学或 荧光测定。 发生体突变的细胞会转动 当使用发色底物（x-gal）或荧光时，蓝色 添加了荧光底物FDG。 可以研究体细胞突变 在动物中，通过切片器官，甚至整个胚胎，并用染色 X-GAL;突变部位会变成蓝色。 突变量可以是 用FDG定量和对荧光激活细胞进行分析 分类器（FACS）。 可以使用FACS和进一步分离阳性细胞 具有表型和功能特性的特征。 最初的步骤是使该结构和适当的控制 构造（使用可翻译的VDJ），将它们转染为单元线 确保表达它们，然后制造转基因小鼠。 这些 然后，可以使用小鼠直接检验许多当前的假设 关于体细胞突变，包括：1）在A中发生体细胞突变 免疫后的活动爆发； 2）体面突变的B细胞是 在生发中心发现； 3）此类细胞是或注定要成为 记忆细胞； 4）LYL+ B细胞不会在体外突变； 5）T细胞不 体积突变； 6）响应中未触发体细胞突变 到胸腺独立抗原。 转基因也可以繁殖到小鼠上 具有各种免疫功能障碍，例如自身免疫性菌株MRL/LPR， 自身免疫性和免疫缺陷的运动菌株以及免疫缺陷型 SCID小鼠应变。 可以分析这些小鼠的体细胞突变，并 与正常情况相比。