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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有关，但环境神经毒物仍然是许多此类疾病的病因学的关键因素。例如，接触甘露醇和百草枯等杀虫剂会增加患帕金森氏症的风险。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，接触特性不佳的化合物是导致环境诱发疾病的一个重要因素。对体外模型和终点分析的迫切需求尚未得到满足，这些模型和终点分析具有成本效益、准确性、预测性和敏感度，也适用于高通量筛查。特别是，需要开发可用于定量高通量筛选的同质体外筛选，以查询大的化合物文库，例如‘Tox21 10K’化学文库。我们将利用我们在定点核酸酶基因组工程和无足迹基因编辑方面的核心优势，生产报告细胞系，通过同源重组(即创建敲门突变)将报告基因插入内源性毒物反应基因SERPINE1和DDIT3中，以创建具有成本效益的快速神经毒性检测系统。简而言之，我们在内部设计了一种SERPINE1外显子2特异的位点特异性核酸酶，以驱动萤火虫荧光素酶T2A-肾型荧光素酶(Lux)报告直接插入SERPINE1启动子ATG下游，从而使Lux基因在天然基因组环境中置于SERPINE1启动子的控制下；类似地，我们将在DDIT3的ATG后面敲入TEM1-β内酰胺酶报告。靶向载体将包含一个含有嘌呤霉素抗性和胸苷激酶基因的iggyBac选择模块，以允许通过对嘌呤霉素的抗性来选择候选靶向克隆，并选择在通过更昔洛韦抗性瞬时表达仅可切除的iggyBac转座酶时完全去除表达模块。因此，内源SERPINE1和DDIT3基因座将包含它们各自的报告基因，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(荧光素酶或β-内酰胺酶)和qRT-PCR方法比较靶向Lux和BLA报告基因的诱导特性与相应的非靶向(野生型)等位基因的诱导特性。如果对记者的诱导幅度落在每个被检测化合物的每个记者对应的内源基因观察到的诱导的20%以内，就会成功。细胞系的验证将为神经毒物的高通量筛选提供初步工具，并将代表着为创建神经毒物筛选细胞系而开发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数据库，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；我们还将能够销售定制的敲入、毒物反应报告细胞系，以及在收费毒性测试中使用目录或定制细胞系，作为对学术和行业调查人员的服务，并为在各种细胞环境中检查制剂对特定途径的影响打开新的机会。