Harnessing Somatic Hypermutation for Drug Addiction Research

描述（由申请人提供）：定点和随机诱变是研究蛋白质（包括药物成瘾中的关键蛋白质）结构和功能的不可或缺的工具。然而，体外诱变程序是非常劳动密集型的并且具有低通量。体细胞超突变（SHM）是B淋巴细胞中产生大量抗体多样性的主要机制之一，可用于随机突变编码非抗体蛋白的外源性目的基因（GOI）。由于突变在每个细胞中独立发生，因此很容易产生数百万个随机突变体，并且当建立适当的选择方法时可以快速筛选。SHM率在某些基因座（如免疫球蛋白V区的基因座）比细胞基因组的其余部分高得多（高达106倍）。然而，没有方便的方法将GOI插入高SHM的基因座。不能进行靶向同源重组，因为这些基因座上的序列是异质的，并且在细胞与细胞之间不同。因此，我们建议产生新的B淋巴瘤细胞系，允许容易地插入GOI到高SHM的基因座。可以在细胞系中产生和表达数百万个突变体。可以筛选和选择GOI突变体的某些性质用于进一步研究。诱变程序可重复多次以使GOI进化至所需性质。我们将通过将DNA构建体随机整合到含有插入引导标记和突变报告基因的细胞基因组中来产生细胞系。我们将进行一次预筛选和两轮筛选，以分离在单次细胞分裂中报告基因突变的细胞。这样的细胞应该具有整合在非常高SHM率的基因座处的报告基因。插入引导标记将允许在高SHM基因座处容易且特异性地插入GOI。此外，GOI将由诱导型启动子控制，因此SHM可以上调和下调，因为SHM速率与基因表达水平成比例。在未来的研究中，我们计划使用该系统来筛选对成瘾药物如可卡因、苯丙胺、甲基苯丙胺、MDMA（摇头丸）或治疗药物如利他林和百忧解不敏感的单胺转运蛋白突变体。该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、调控等。

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.