Novel reporter cell lines for neurotoxicant assays

用于神经毒物测定的新型报告细胞系

• 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
• 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

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，接触代森锰和百草枯等农药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，接触特征较差的化合物是环境诱发疾病发生的一个重要因素。对于体外模型和终点检测的迫切需求尚未得到满足，这些模型和终点检测必须具有成本效益、准确、预测性和敏感性，并且也适合高通量筛选。特别是，需要开发可用于定量高通量筛选的同质体外筛选，以查询大型化合物库，例如“Tox21 10K”化学库。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毒物反应基因SERPINE1和DDIT3（即，创建敲入突变），以创建一个经济高效的快速神经毒性测试系统。 简而言之，我们设计了 SERPINE1 外显子 2 特异性位点特异性。内部核酸酶驱动萤火虫荧光素酶 T2A-海肾荧光素酶 (lux) 报告基因直接插入 SERPINE1 启动子 ATG 下游，以便 lux 基因将置于天然基因组环境中 SERPINE1 启动子的控制之下；在 DDIT3 的 ATG 后面敲入 TEM1-β 内酰胺酶报告基因，靶向载体将包含一个包含嘌呤霉素抗性和的 PiggyBac 选择模块。胸苷激酶基因，以允许通过嘌呤霉素抗性选择候选目标克隆，并通过更昔洛韦抗性选择在仅切除的piggyBac转座酶瞬时表达后完全去除表达模块。因此，内源 SERPINE1 和 DDIT3 基因座将包含其各自的报告基因，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 检测，将靶向 lux 和 BLA 报告基因的诱导特性与其相应的非靶向（野生型）等位基因的诱导特性进行比较。如果报告基因的诱导幅度落在针对所测定的每种测试化合物的每个报告基因的相应内源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，并将代表用于创建用于神经毒理学筛选的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创建一个报告细胞系库，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；我们还能够向学术和行业研究人员提供敲入、毒物反应报告系的定制创建以及在按服务付费毒性测试中使用目录或定制细胞系的服务，并开辟新的机会用于检查药物对各种细胞环境中特定途径的影响。