Acquiring Techniques to Study Somatic Mutation in Immunoglobulin Genes

点击翻译按钮获取中文摘要

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.