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定的生物测定联系起来，以监测配体、化合物、抗体、siRNAs、miRNAs和多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、通路发现、靶标验证、药物发现和细胞靶标功效的台式或高通量筛选。在活体内与整个动物成像一起使用，它们提供了药物输送和体内靶向效果的时间和定量分析。最常用的报告基因分析方法是在克隆的启动子和转录调控元件下游放置一个容易监测的报告基因，如荧光蛋白或荧光素酶。在外源报告中，载体被引入受体细胞，在那里表达要么瞬时进行，要么在“随机”和稳定地整合到基因组之后进行。外源报告基因分析是高度不可预测的，而且通常不能将基因表达与已知的调节因子联系起来。它们可能提供错误的数据并误导研究调查，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，报告基因被引入到其正常的染色体位置，利用天然启动子、调控元件、局部染色体修饰和微RNA调控序列来可靠和准确地索引基因调控。然而，可用的内源性人类基因表达报告细胞分析很少，而且敏感性限制了对许多基因的准确监测。更糟糕的是，目前用于生产化验的基因捕获和基因靶向方法需要在资源、时间和成本上进行大量投资，使得大多数实验室无法实现这些方法。在第一阶段的研究中，我们建议将Xactagen在报告细胞敏感性方面的先前研究进展与大规模生产、存储和提取内源性人类基因表达报告细胞的创新方法相结合。我们的假设是，我们将拥有广泛的基因覆盖范围，对即使表达较低的基因的表达也有监测的敏感性，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： (1)建立19,200个排列的基因捕获报告细胞(NIH-H292人粘液表皮样癌细胞系)的中试文库；(2)鉴定载体插入位点，并表征基因/转录单位的表达；(3)检索10个内源性报告细胞并验证其功能。这项创新的研究使人内源性基因表达报告细胞库的生产在几周内而不是几年内具有成本效益和极快的速度。因此，更多的研究人员将能够接触到内源性人类报告细胞，并能够专注于细胞培养和体内应用，而不是生产。此外，研究人员可以通过其不可预测的基因表达指数来避免外源报告细胞。最终，科学家将以更少的时间和更低的成本做出更多可行的研究和医学发现。 与公共卫生相关：目前生产内源性基因表达报告细胞的方法费力且成本高昂。这项研究的最终目标是在几周内而不是几年内提供具有成本效益的、高度加速的人类内源性报告细胞库的生产。在不同的人类细胞系和干细胞中生产的数十万容易获得、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开发的生产率。

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.