Acquiring Techniques to Study Somatic Mutation in Immunoglobulin Genes

本研究旨在探讨体细胞突变在基因突变中的作用。 抗体开发中免疫球蛋白可变（V）基因 多样性一种含有重排的V基因与 将构建lacZ基因5 ′的终止密码子。lacZ基因 可以通过荧光或组织化学容易地检测到。融合 蛋白质将用于制造转基因小鼠和细胞系。的 终止密码子阻止lacZ“外显子”的翻译，除非体细胞突变。 发生突变，导致终止密码子和表达的逆转 lacZ的。将使用荧光激活细胞分选（FACS）来 分离表达lacZ的细胞;并且假定的突变将被 通过PCR后测序确认。的表达和定位 个体发育期间的体细胞突变将通过以下方式确定： 分离细胞的组织化学和FACS分析。这应该 有助于确定体细胞突变的机制 %%% 抗体是一种蛋白质， 各种化合物，除其他外，是一个主要的来源， 防御致病微生物。 抗体与每 化合物与其他化合物的作用不相同。的 获得这种特异性的机制仍然不清楚， 完全理解一种研究体细胞作用的方法 产生抗体多样性的突变涉及融合 一种含有重排的V基因片段的构建体， 一种叫做lacZ的报告蛋白。重排的V基因含有 阻止lacZ蛋白合成的“停止”信号。 lacZ基因的产物可以容易地检测。仅当a 突变发生在“停止”信号可以这个产品的形式。小鼠 含有融合构建体的细胞将用于几项研究， 看看何时何地可以检测到lacZ这可能有助于 增加我们对抗体多样性的理解。

This proposal is to study the role of somatic mutation of immunoglobulin variable (V) genes in development of antibody diversity. A fusion protein containing a rearranged V gene with a stop codon 5' to the lacZ gene will be constructed. The lacZ gene can be easily detected by fluoresence or histochemistry. The fusion protein will be used to make transgenic mice and in cell lines. The stop codon prevents translation of the lacZ "exon" unless a somatic mutation occurs, causing reversion of the stop codon and expression of lacZ. Fluorescence activated cell sorting (FACS) will be used to isolate cells expressing lacZ; and putative mutations will be confirmed by sequencing after PCR. The expression and location of somatic mutation during ontogeny will be determined by histochemistry and FACS analysis of isolated cells. This should help determine the mechanism of somatic mutation. %%% Antibodies are proteins which react with millions of different kinds of compounds, and among other things are a major source of defense from disease-causing organisms. Antibodies to each compound are not identical to those against other compounds. The mechanism by which this specificity is obtained is still not completely understood. A method to study the role of somatic mutation in producing antibody diversity involves making a fusion construct which contains a piece of rearranged V gene and the gene for a reporter protein called lacZ. The rearranged V gene contains a "stop" signal which will prevent synthesis of the lacZ protein. The product of the lacZ gene can be easily detected. Only if a mutation occurs in the "stop" signal can this product form. Mice containing the fusion construct will be used for several studies, to see where and when the lacZ can be detected. This may help increase our understanding of antibody diversity.