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症有关，但环境神经毒性剂仍然是许多此类疾病病因的关键因素。例如，接触诸如Maneb和Paraquat之类的农药增加了患帕金森氏病的风险。不幸的是，没有评估化合物神经毒性潜力的最佳体外模型系统。结果，暴露于特征性较差的化合物是导致环境引起的疾病发展的重要原因。对体外模型和端点测定的需求令人信服，这些需求具有成本效益，准确，预测性和敏感性，也可以适合高通量筛选。特别是，有必要开发同质的体外筛选，这些筛选可用于定量的高通量筛选，以查询大量化合物的库，例如“ TOX21 10K”化学库。我们将利用位点特异性核酸酶基因组工程和无脚印的“基因编辑”中的核心优势，以产生报告基因细胞系，我们将在其中将报道基因基因插入内源性毒物响应基因serpine1 and ddit3和DDIT3通过同源重组（即创建newot sening were segnity wery wery），从而创建成本量，以创建成本量的snefoins wery，并快速地进行了成本，快速地，有效地，有效地有效。有效地有效。 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 DDIT3的ATG靶向矢量将包含一个含有嘌呤霉素耐药性和胸苷激酶基因的PIGGYBAC选择模块，以通过纯摩霉素的耐药性选择候选靶向克隆，并选择通过千斤顶表达coccision-onallylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylylyly compoggybac troxslovir discoss。结果，内源性serpine1和ddit3基因座将包含其各自的记者，而不会产生任何其他异物DNA序列。为了验证报道细胞的功能，我们将分别使用报告基因（荧光素酶或β-内酰胺酶）和QRT-PCR分别比较目标LUX和BLA报告基因的诱导性能与相应的未靶向（野生型）等位基因的感应特性。如果记者的诱导幅度落在每个记者对每个测试化合物的相应内源基因观察到的诱导量的20％之内，则取得成功。细胞系的验证将为筛选神经毒素的高通量筛查提供初始工具，并将代表强大的平台技术，以创建用于神经毒理学筛查的细胞系。从长远来看，第二阶段研究将旨在创建一系列记者细胞系，该细胞系在各种线路中采样多个基因座的响应，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，毒性响应性的记者线条，并在费用的服务毒性测试中使用目录或定制细胞系作为对学术和行业研究人员的服务，并开放了新的机会，以检查各种细胞环境中特定途径对特定途径的影响。