Innovative approach for high-volume production of endogenous reporter cells

描述（由申请人提供）：基因表达报告分析将生物信息学数据与基因特异性生物分析连接起来，以监测配体、化合物、抗体、sirna、miRNAs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控、途径发现、靶点验证、药物发现和细胞靶点功效的台式或高通量筛选。在体内使用全动物成像，它们提供了药物传递和体内靶点疗效的时间和定量分析。最常用的报告基因检测将易于监测的报告基因（如荧光蛋白或荧光素酶）置于克隆启动子和转录调控元件的下游。在外源性报告细胞中，载体被引入受体细胞，在那里表达要么短暂地进行，要么“随机”并稳定地整合到基因组中。外源性报告基因测定是高度不可预测的，往往不能索引基因表达到已知的调节因子。它们可能会提供错误的数据，误导研究调查，从而浪费时间、费用和未实现的发现和开发机会。相比之下，在内源性报告基因中，报告基因在其正常染色体位点引入，利用天然启动子、调控元件、局部染色体修饰和微rna调控序列可靠、准确地指示基因调控。然而，很少有内源性人类基因表达报告细胞检测方法可用，灵敏度限制了对许多基因的准确监测。更重要的是，目前用于生产检测的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，这使得大多数实验室无法实现。在I期研究中，我们建议将Xactagen先前在报告细胞敏感性方面的研究进展与大规模生产、储存和检索内源性人类基因表达报告细胞的创新方法结合起来。我们的假设是，我们将具有广泛的基因覆盖范围，对监测表达不良基因的敏感性，对已知基因调节因子的可靠反应，以及从我们的文库中检索特定报告细胞的能力，以便在订购后4-6周内交付给研究人员。我们的具体目标是：

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.