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Novel reporter cell lines for neurotoxicant assays

用于神经毒物测定的新型报告细胞系

基本信息

批准号：
9034411
负责人：
ERIC M OSTERTAG
金额：
$ 22.5万
依托单位：
HERA TESTING LABORATORIES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-18 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9034411
关键词：
Novel reporter cell lines neurotoxicant

项目摘要

DESCRIPTION (provided by applicant): While it has been demonstrated that gene mutations are associated with an increasing number of neurodegenerative syndromes, environmental neurotoxicants remain a key factor in the etiology of many such diseases. For example, exposure to pesticides such as maneb and paraquat increase the risk of developing Parkinson's disease. Unfortunately, there is no optimal in vitro model system to assess the neurotoxic potential of compounds. As result, exposure to poorly characterized compounds represents a significant contributor to the development of environmentally-induced diseases. There is a compelling unmet need for in vitro models and endpoint assays that are cost-effective, accurate, predictive, and sensitive that would also be amenable to high throughput screening. In particular, there is a need to develop homogeneous in vitro screens that can be used in quantitative high-throughput screening to query large libraries of chemical compounds such as the 'Tox21 10K' chemical library. We will take advantage of our core strengths in site-specific nuclease genome engineering and Footprint-Free" Gene Editing to produce reporter cell lines in which we will insert reporter genes into the endogenous toxicant-responsive genes SERPINE1 and DDIT3 by homologous recombination (i.e., create knockin mutations) in order to create a cost- effective, rapid system for neurotoxicity testing. Briefly, we engineer a SERPINE1 exon 2-specific site-specific nuclease in-house to drive the targeted insertion of firefly luciferase T2A-renilla luciferase (lux) reporter immediately downstream of the SERPINE1 initiator ATG so that the lux gene will be placed under the control of SERPINE1 promoter within the native genomic context; similarly we will knock in a TEM1-beta lactamase reporter behind the ATG of DDIT3. The targeting vectors will contain a piggyBac selection module containing the puromycin resistance and thymidine kinase genes, to allow selection for candidate targeted clones via puromycin resistance and to select for complete removal of the expression module upon transient expression of excision-only piggyBac transposase via ganciclovir resistance. As a result, the endogenous SERPINE1 and DDIT3 loci will contain their respective reporters without any other foreign DNA sequences will be created. To validate the function of the reporter cells, we will compare the induction properties of the targeted lux and BLA reporters with that of their corresponding untargeted (wild type) alleles using reporter (luciferase or beta-lactamase) and qRT-PCR assays, respectively. Success will be achieved if the magnitude of induction for the reporters falls within 20% of the induction observed for each reporter's corresponding endogenous gene for each test compound assayed. Validation of the cell lines will provide an initial tool for screening high-throughput screening for neurotoxicants and will represent the development of a powerful platform technology for the creation of cell lines for neurotoxicology screening. In the long term, Phase II studies would be aimed at creating a bank of reporter cell lines that sample the responses of multiple loci in a variety of lines that we will be able to markt as catalog items; we would also be able to market custom creation of knock-in, toxicant-responsive reporter lines and the use of either catalog or custom cell lines in fee-for-service toxicity testing as a service to academic and industry investigators, and open new opportunities for examining the impact of agents on specific pathways in a wide variety of cellular contexts.

描述（由申请人提供）：虽然已经证明基因突变与越来越多的神经退行性综合征相关，但环境神经毒物仍然是许多此类疾病病因学的关键因素。例如，接触农药如代森锰和百草枯会增加患帕金森病的风险。不幸的是，没有最佳的体外模型系统来评估化合物的神经毒性潜力。因此，暴露于表征不佳的化合物是环境诱发疾病发展的重要因素。对于成本有效、准确、预测性和灵敏的体外模型和终点测定存在迫切的未满足的需求，其也将适合于高通量筛选。特别地，需要开发可用于定量高通量筛选的均质体外筛选，以查询化合物的大型文库，例如“Tox 21 10 K”化学文库。我们将利用我们在位点特异性核酸酶基因组工程和“无足迹”基因编辑方面的核心优势来生产报告细胞系，其中我们将通过同源重组将报告基因插入内源性毒物应答基因SERPINE 1和DDIT 3中（即，创建敲入突变），以便创建用于神经毒性测试的具有成本效益的快速系统。简而言之，我们在内部工程化SERPINE 1外显子2特异性位点特异性核酸酶，以驱动萤火虫荧光素酶T2 A-海肾荧光素酶（lux）报告基因的靶向插入紧接SERPINE 1起始子ATG的下游，使得lux基因将在天然基因组背景内置于SERPINE 1启动子的控制下;类似地，我们将在DDIT 3的ATG后面敲入TEM 1-β内酰胺酶报告基因。靶向载体将包含含有嘌呤霉素抗性和胸苷激酶基因的piggyBac选择模块，以允许通过嘌呤霉素抗性选择候选靶向克隆，并在通过更昔洛韦抗性瞬时表达仅切除的piggyBac转座酶时选择完全去除表达模块。因此，内源性SERPINE 1和DDIT 3基因座将含有其各自的报告基因，而不会产生任何其他外源DNA序列。为了验证报告细胞的功能，我们将分别使用报告基因（荧光素酶或β-内酰胺酶）和qRT-PCR试验比较靶向lux和BLA报告基因与其相应非靶向（野生型）等位基因的诱导特性。如果报告基因的诱导幅度福尔斯在测定的每种供试化合物的每种报告基因相应内源性基因观察到的诱导的20%以内，则将获得成功。细胞系的验证将为筛选神经毒物的高通量筛选提供初始工具，并将代表用于创建神经毒理学筛选细胞系的强大平台技术的发展。从长远来看，第二阶段研究的目标是建立一个报告细胞系库，这些细胞系可以对各种细胞系中多个基因座的反应进行采样，我们将能够将其标记为目录项目;我们还可以销售定制的敲门砖，毒性反应性报告细胞系以及目录或定制细胞系在费用-服务毒性测试作为一项服务，学术和行业的调查，并打开新的机会，检查代理商的影响，在特定的途径，在各种各样的细胞环境。