Innovative approach for high-volume production of endogenous reporter cells

大批量生产内源报告细胞的创新方法

基本信息

批准号：
8315758
负责人：
Robert E Finney
金额：
$ 22.34万
依托单位：
XACTAGEN, LLC.
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-06-05 至 2014-06-04
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8315758
关键词：
Animals Antibodies Bar Codes Bioinformatics Biological Assay Biological Products Cell Culture Techniques Cell Line Cells Chemical Agents Clone Cells DNA biosynthesis Data Databases Development Drug Delivery Systems Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Recombination Genetic Transcription Genome Goals Human Human Cell Line Human Genome Image Investigation Investments Laboratories Libraries Ligands Luciferases Malignant Epithelial Cell Medical Research Methods MicroRNAs Modification Molecular Biology Molecular and Cellular Biology Monitor Mucoepidermoid Carcinoma Pathway interactions Peptides Pharmacologic Substance Phase Production Productivity Proteins Qualifying Regulator Genes Regulatory Element Reporter Reporter Genes Research Research Personnel Resistance Resources Ribonucleic Acid Regulatory Sequences Scientist Screening procedure Services Site Small Interfering RNA Stem cells Technology Testing Time Transcript United States National Institutes of Health Validation antibiotic G 418 base cellular targeting commercialization cost cost effective drug discovery experience genome database high throughput screening in vivo indexing innovation phase 1 study phase 2 study programs promoter response vector

项目摘要

DESCRIPTION (provided by applicant): Gene-expression reporter assays bridge bioinformatics data with gene-specific bioassays to monitor effects of ligands, compounds, antibodies, siRNAs, miRNAs, and peptides on gene expression. In cell culture, they provide for bench top or high throughput screening applicable to gene regulation, pathway discovery, target validation, drug discovery, and cellular target efficacy. Used in vivo with whole animal imaging, they provide for temporal and quantitative analysis of drug delivery and in vivo target efficacy. The most commonly used reporter-gene assays place an easily monitored reporter gene such as a fluorescent protein or luciferase downstream to cloned promoter and transcriptional regulatory elements. In an exogenous reporter, the vector is introduced into recipient cells where expression proceeds either transiently, or after "random" and stable integration into the genome. Exogenous reporter-gene assays are highly unpredictable, and often incapable of indexing gene expression to known regulatory agents. They can provide erroneous data and mislead research investigations, thereby costing time, expense and unrealized discovery and development opportunities. In contrast, in an endogenous reporter, reporter genes are introduced at their normal chromosomal locus where native promoters, regulatory elements, local chromosomal modifications and micro-RNA regulatory sequences are utilized to reliably and accurately index gene regulation. However, very few endogenous human gene- expression reporter cell assays are available and sensitivity limits accurate monitoring of many genes. Compounding this, current gene trapping and gene targeting methods for producing assays require large investments in resources, time, and cost, making them unattainable for most laboratories. In phase I studies, we propose to combine Xactagen's prior research advances in reporter cell sensitivity with innovative methods to mass produce, store and retrieve endogenous, human gene expression reporter cells. Our hypothesis is that we will have extensive gene coverage, sensitivity to monitor expression of even poorly expressed genes, reliable responses to known gene regulators, and the ability to retrieve specific reporter cells from our libraries for delivery to researchers within 4-6 weeks of ordering. Our specific aims are: (1) Generate a pilot library of 19,200 arrayed gene-trapped reporter cells (NIH-H292 human mucoepidermoid carcinoma cell line), (2) Identify vector insertion sites, and characterize gene/transcription unit representation, and (3) Retrieve 10 endogenous reporter cells and verify functionality. This innovative research provides for cost-effective and highly expedited production of human endogenous gene expression reporter cell libraries in weeks as opposed to years. As a result, more researchers will have access to endogenous human reporter cells and will be able to focus on cell culture and in vivo applications, rather than on production. Additionally, researchers can avoid exogenous reporter cells with their unpredictable indexing of gene expression. Ultimately, scientists will make more viable research and medical discoveries in less time and at far less cost. PUBLIC HEALTH RELEVANCE: Current methods for production of endogenous gene expression reporter cells are laborious and cost- prohibitive. The ultimate goal of this research i to provide cost-effective and highly expedited production of human endogenous reporter cell libraries - in weeks as opposed to years. Hundreds of thousands of readily available, highly sensitive, easy to use, and affordable gene expression reporter cells produced in diverse human cell lines and stem cells are expected to increase productivity of scientific research and drug discovery.

描述（由申请人提供）：基因表达报告基因测定法与基因特异性生物测定桥桥接生物信息学数据，以监测配体，化合物，抗体，siRNA，miRNA和肽对基因表达的影响。在细胞培养中，它们提供了适用于基因调节，途径发现，靶标验证，药物发现和细胞靶疗效的高吞吐量筛选。它们与整个动物成像一起在体内使用，提供了对药物递送和体内靶疗效的时间和定量分析。最常用的记者基因测定法放置了一个易于监测的记者基因，例如下游的荧光蛋白或荧光素酶，对克隆的启动子和转录调节元件。在外源报告中，将载体引入到受体细胞中，其中表达瞬时或“随机”和稳定的整合到基因组之后。外源报告基因测定法是高度不可预测的，通常无法将基因表达索引到已知的调节剂。他们可以提供错误的数据和误导研究调查，从而耗费时间，费用以及未实现的发现和开发机会。相反，在内源性报告基因中，在其正常染色体基因座上引入了报告基因，其中天然启动子，调节元件，局部染色体修饰和微-RNA调节序列可用于可靠，准确地索引基因调节。但是，很少有内源性人基因表达报告基因细胞分析可用，灵敏度限制了许多基因的准确监测。复杂化这种测定的当前基因捕获和基因靶向方法需要大量的资源，时间和成本投资，这对于大多数实验室而言无法实现。在第一阶段的研究中，我们建议将Xactagen的先前研究进展与报告细胞敏感性的进展与创新方法，以大规模生产，存储和检索内源性的人类基因表达报告基细胞。我们的假设是，我们将具有广泛的基因覆盖范围，敏感性，以监测甚至表达较差的基因的表达，对已知基因调节剂的可靠反应以及从我们的库中检索特定的报告基因在订购4-6周内向研究人员输送到研究人员的能力。。我们的具体目的是：（1）生成一个由19,200个被捕获的基因捕获的报道细胞（NIH-H292人粘膜表皮样细胞）的试验库，（2）识别矢量插入位点，并表征基因/转录单位表示，并且（3）检索10个内源性报告基因细胞并验证功能。这项创新的研究为数周而不是几年而言，为人类内源性基因表达者的细胞库提供了成本效益且高度加快的人类内源性基因表达细胞库。结果，越来越多的研究人员将可以使用内源性人类记者细胞，并能够专注于细胞培养和体内应用，而不是生产。此外，研究人员可以避免使用其基因表达的不可预测的索引。最终，科学家将在更少的时间和成本更少的时间内进行更可行的研究和医疗发现。公共卫生相关性：当前生产内源基因表达细胞细胞的方法是费力且成本效益的。这项研究的最终目标是提供具有成本效益且高度加快人类内生细胞库的高度生产 - 几周而不是几年。预计在多种人类细胞系和干细胞中产生的数十万可用，高度敏感，易于使用和负担得起的基因表达报告细胞有望提高科学研究和药物发现的生产力。