Harnessing Somatic Hypermutation for Drug Addiction Research

利用体细胞超突变进行毒瘾研究

• 批准号: 7512188
• 负责人: Howard H Gu
• 金额: $ 15万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-10 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7512188
• 关键词: Amphetamines; Antibodies; Antibody Diversity; B-Cell Lymphomas; B-Lymphocytes; Cell Line; Cell division; Cells; Cocaine; Cultured Cells; DNA; Drug Addiction; Drug Binding Site; Future; Gene Expression; Genes; Genetic Recombination; Genome; Hour; Immunoglobulin Somatic Hypermutation; Immunoglobulin Variable Region; In Vitro; Interphase Cell; Lymphoma; Methamphetamine; Methods; Mutagenesis; Mutate; Mutation; Pharmaceutical Preparations; Procedures; Property; Proteins; Prozac; Rate; Regulation; Reporter Genes; Research; Retroviral Vector; Ritalin; Screening procedure; Site; Structure; System; Therapeutic; Time; Work; cell type; desire; ecstasy; homologous recombination; interest; monoamine; mutant; novel; promoter; protein structure function; recombinase; tool

DESCRIPTION (provided by applicant): Site-directed and random mutagenesis is an indispensable tool in studying the structures and functions of proteins including those that are critical in drug addiction. However, the in vitro mutagenesis procedures are very labor intensive and of low throughput. Somatic hypermutation (SHM), one of the main mechanisms that generate the great diversity of antibodies in B lymphocytes, can be used to randomly mutate exogenous Genes Of Interest (GOI) that encode non-antibody proteins. Since mutations occur independently in each cell, millions of random mutants are easily generated and can be screened quickly when a proper selection method is set up. The SHM rate is much higher (up to 106 fold) at certain loci (such as loci for immunoglobulin V regions) than the rest of the cell genome. However, there is no convenient way to insert the GOI at loci of high SHM. Targeted homologous recombination can not be performed because the sequences at these loci are heterogeneous and different from cell to cell. Therefore, we propose to generate novel B-lymphoma cell lines that allow easy insertion of a GOI to loci of high SHM. Millions of mutants can be generated and expressed in the cell lines. Certain properties of the GOI mutants can be screened and selected for further studies. The mutagenesis procedure can be repeated multiple times to evolve the GOI to desired properties. We will generate the cell lines by integrating a DNA construct into the cell genome randomly that contains an insertion guide marker and a mutation reporter gene. We will perform a prescreening and two rounds of screening to isolate cells with the reporter gene mutated in a single cell division. Such cells should have the reporter gene integrated at loci of very high SHM rates. The insertion guide marker will allow easy and specific insertion of a GOI at the high SHM loci. In addition, the GOI will be controlled by an inducible promoter and thus SHM can be turned up and down because SHM rates are proportional to the levels of gene expression. In future studies, we plan to use this system to screen for mutants of the monoamine transporters that are insensitive to addictive drugs such as cocaine, amphetamine, methamphetamine, MDMA (ecstacy) or therapeutic drugs, such as Ritalin and Prozac. The cell lines can be used to study many other proteins on their structures, functions, drug binding sites, regulations, etc.

描述（由申请人提供）：位于现场定向和随机诱变是研究蛋白质的结构和功能的必不可少的工具，包括在药物成瘾中至关重要的蛋白质。但是，体外诱变程序是劳动密集型的，吞吐量很低。体细胞超突变（SHM）是在B淋巴细胞中产生巨大抗体多样性的主要机制之一，可用于随机突变的外源性基因（GOI），该基因编码非抗体蛋白。由于突变在每个单元格中独立发生，因此很容易生成数百万个随机突变体，并在设置适当的选择方法时可以快速筛选。在某些基因座（例如免疫球蛋白V区域）的SHM速率比其他细胞基因组更高（例如，用于免疫球蛋白V区域）。但是，没有方便的方法可以在High SHM的位置插入GOI。无法进行靶向同源重组，因为这些基因座的序列是异质的，并且在细胞之间不同。因此，我们建议生成新型的B淋巴瘤细胞系，这些细胞系可以轻松地将GOI插入高SHM的基因座。可以在细胞系中生成并表达数百万个突变体。可以筛选并选择GOI突变体的某些特性进行进一步研究。可以多次重复诱变过程，以将GOI演变为所需的特性。我们将通过随机地将DNA构建体整合到细胞基因组中，该基因组随机地包含插入指南和突变报告基因。我们将进行预筛选和两轮筛选，以分离单个细胞分裂中突变的报告基因。此类细胞应将报告基因集成在非常高的SHM速率的基因座。插入指南标记将允许在高SHM基因座的GOI轻松插入。此外，GOI将由诱导启动子控制，因此可以向上和降低SHM，因为SHM速率与基因表达水平成正比。在未来的研究中，我们计划使用该系统来筛选单胺转运蛋白的突变体，这些突变体对可卡因，苯丙胺，甲基苯丙胺，MDMA（狂喜）或治疗药物（例如Ritalin and Prozac）不敏感。细胞系可用于研究许多其他蛋白质的结构，功能，药物结合位点，法规等。