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结构和功能不可或缺的工具，包括那些在药物成瘾中至关重要的蛋白质。然而，体外诱变过程是非常劳动密集型和低通量的。体细胞超突变（SHM）是B淋巴细胞产生多种抗体的主要机制之一，可用于随机突变编码非抗体蛋白的外源感兴趣基因（GOI）。由于突变在每个细胞中都是独立发生的，因此很容易产生数百万个随机突变，并且在建立适当的选择方法时可以快速筛选。在某些位点（如免疫球蛋白V区位点）的SHM率比细胞基因组的其余部分高得多（高达106倍）。然而，在高SHM位点没有方便的方法插入GOI。由于这些位点上的序列是异质的，并且在细胞之间是不同的，因此不能进行靶向同源重组。因此，我们建议产生新的b淋巴瘤细胞系，使GOI易于插入高SHM位点。数以百万计的突变体可以在细胞系中产生和表达。GOI突变体的某些特性可以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。我们将通过将包含插入引导标记和突变报告基因的DNA结构随机整合到细胞基因组中来生成细胞系。我们将进行一次预筛选和两轮筛选，以分离单细胞分裂中报告基因突变的细胞。这样的细胞应该在非常高的SHM率位点上整合了报告基因。插入引导标记将允许在高SHM位点上容易和特异性地插入GOI。此外，GOI将由诱导启动子控制，因此SHM可以调高或调低，因为SHM率与基因表达水平成正比。在未来的研究中，我们计划使用该系统筛选对上瘾药物如可卡因、安非他明、甲基苯丙胺、摇头丸（摇头丸）或治疗药物如利他林和百忧解不敏感的单胺转运体突变体。这些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.