Exploring the limits of luciferase multiplexing to assay multiple cellular signaling pathways at once

项目摘要 可高效、灵敏、同时测量多种信号传导的多重细胞分析 相同细胞中的通路需要具有大动态范围的正交探针， 迅速获得。荧光素酶是基因编码的、具有成本效益的、多用途的候选物，其实现这些功能。 要求.常用的双荧光素酶报告基因测定目前检测一个荧光素酶偶联到一个荧光素酶。 细胞途径，以及为了标准化目的与对照途径偶联的第二荧光素酶，有效地 从而导致对单个通路仅进行一次活性测量。为了增加细胞信号的数量 途径，可以同时探测使用荧光素酶报告，我们计划探索的限制， 荧光素酶多重化。初步工作证明了通过扩展多路复用的可行性 针对六种能够有效报告五种细胞信号通路和一种控制的酶， 使双荧光素酶测定的效力增加五倍。为了确保低实验变异，我们采用 一个灵活的合成组装克隆管道，将所有六个荧光素酶报告单元缝合在一起， 载体，导致在每个转染的载体中每个转录单位的相等化学计量比的转染。 cell.为了证明概念，我们设计了一种针对探针途径通量定制的荧光素酶测定法 通过五种已知细胞信号传导途径的转录反应元件， 标准化启动子提出并分析了siRNA、配体和化合物的作用 治疗的目标途径和其他四个细胞途径在同一时间。基于此 初步的知识，我们建议进一步探索荧光素酶多路复用。四个相辅相成 将采取以下办法：1.增加已经探索的两种底物的消耗量 我们可以在一个实验中检测到的酶。2.包括第三组底物消耗型内切酶， 即vargulin酶。3.鉴定针对腔肠素和/或vargulin荧光素酶基团的猝灭剂。 4.开发一个精简的组装流水线，尽可能少的克隆步骤来合成缝合 在单个多重荧光素酶报告子载体中一起包含多达12个荧光素酶报告子单元，并掺入 能够容纳大DNA插入物货物的特化质粒骨架， 荧光素酶报告单位。此外，为了证明使用荧光素酶的多重荧光素酶测定的概念验证， 多达十二个荧光素酶报告单元，我们将产生一组多重荧光素酶报告子， 转录读数的一些最常见的已知细胞信号传导途径，将是 进行了上下调节测试因此，这项工作有望通过增加荧光素酶的表达来扩大荧光素酶的多路复用。 可用的底物特异性的、可淬灭的和光谱可分离的荧光素酶库。的 该建议的最终目标是提供一个框架，以探索额外的底物消耗荧光素酶 包括荧光素酶基团特异性猝灭剂的基团，使得多重化可以进一步扩展。

Project summary Multiplexed cellular assays that can efficiently, sensitively, and simultaneously measure multiple signaling pathways in the same cells require orthogonal probes with large dynamic ranges whose measurements can be obtained quickly. Luciferases are genetically encoded, cost-effective, versatile candidates that fulfill these requirements. Commonly used dual luciferase reporter assays currently detect one luciferase coupled to a cellular pathway, and a second luciferase coupled to a control pathway for normalization purposes, effectively resulting in just one activity measurement for a single pathway. To increase the number of cellular signaling pathways that can be simultaneously probed using luciferase reporters, we plan to explore the limits of luciferase multiplexing in this proposal. Preliminary work demonstrates feasibility by expanding multiplexing towards six luciferases that can report on five cellular signaling pathways and one control, effectively increasing the potency of the dual luciferase assay five-fold. To ensure low experimental variation, we adopted a flexible synthetic assembly cloning pipeline that stitches together all six luciferase reporter units into a single vector, resulting in the transfection of equal stoichiometric ratios of each transcriptional unit in each transfected cell. To demonstrate proof of concept, we engineered a luciferase assay tailored to probe pathway fluxes through transcriptional response elements of five known cellular signaling pathways against a constitutive promoter for normalization proposes and assayed the effects of siRNA, ligand, and chemical compound treatments on their target pathways and the four other cellular pathways at the same time. Based on this preliminary knowledge, we propose to explore luciferase multiplexing even further. Four complementary approaches will be pursued: 1. Increase the number of the two already explored substrate-consuming luciferases we can detect in a single experiment. 2. Include a third group of substrate-consuming luciferases, namely vargulin luciferases. 3. Identify quenchers against the coelenterazine and/or vargulin luciferase groups. 4. Develop a streamlined assembly pipeline with as few cloning steps as possible to synthetically stitch together up to twelve luciferase reporter units in a single multiplex luciferase reporter vector, and incorporate a specialized plasmid backbone capable of accommodating large DNA insert cargo encompassing all twelve luciferase reporter units. Furthermore, to demonstrate proof-of-concept for multiplex luciferase assaying using up to twelve luciferase reporter units, we will generate a set of multiplex luciferase reporters that can report on transcriptional readouts for a number of the most commonly known cellular signaling pathways that will be tested for up- and downregulation. Hence, this work promises to expand luciferase multiplexing by growing the available repertoire of substrate-specific, quenchable and spectrally separable luciferase enzymes. The ultimate goal of this proposal is to provide a framework to explore additional substrate-consuming luciferase groups including luciferase group-specific quenchers, so that multiplexing can be expanded even further.