Harnessing Somatic Hypermutation for Drug Addiction Research

描述(由申请人提供)：定点和随机突变是研究蛋白质结构和功能的不可或缺的工具，包括那些在药物成瘾中起关键作用的蛋白质。然而，体外诱变过程劳动强度大，产量低。体细胞超突变(SHM)是B淋巴细胞产生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基因。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，当建立适当的选择方法时，可以快速筛选出这些突变。在某些基因座(例如免疫球蛋白V区的基因座)，SHM的比率比细胞基因组的其余部分高得多(高达106倍)。然而，在高SHM的基因座上插入GOI没有简便的方法。不能进行靶向同源重组，因为这些基因座上的序列是不同的，并且不同细胞之间的序列不同。因此，我们建议建立新的B淋巴瘤细胞系，使GOI容易插入到高SHM的基因座上。数以百万计的突变体可以在细胞系中产生和表达。可以对GOI突变体的某些特性进行筛选和选择，以供进一步研究。诱变过程可以重复多次，以使GOI进化到所需的特性。我们将通过将DNA构建随机整合到细胞基因组中来生成细胞系，该DNA构建包含一个插入引导标记和一个突变报告基因。我们将进行一次预筛选和两轮筛选，以分离出报告基因在单细胞分裂中突变的细胞。这样的细胞应该在SHM率很高的基因座上整合报告基因。插入引导标记将允许GOI在高SHM基因座容易和特定地插入。此外，GOI将由可诱导的启动子控制，因此SHM可以被上调和下调，因为SHM率与基因表达水平成正比。在未来的研究中，我们计划使用这个系统来筛选对可卡因、苯丙胺、甲基苯丙胺、MDMA(摇头丸)或治疗性药物如利他林和百忧解不敏感的单胺转运体突变。该细胞系可用于研究许多其他蛋白质的结构、功能、药物结合部位、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.