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

项目概要 多重细胞检测可以高效、灵敏、同时测量多种信号传导 相同细胞中的通路需要具有大动态范围的正交探针，其测量可以是 很快就获得了。荧光素酶是基因编码的、具有成本效益的、多功能的候选物，可以满足这些要求 要求。目前常用的双荧光素酶报告基因检测检测一种与一种荧光素酶偶联的荧光素酶。 细胞途径，以及与控制途径偶联的第二荧光素酶以用于标准化目的，有效地 导致对单一途径仅进行一次活性测量。增加细胞信号传导的数量 可以使用荧光素酶报告基因同时探测的途径，我们计划探索其局限性 该提案中的荧光素酶多重分析。初步工作证明了通过扩展复用的可行性 六种荧光素酶可以有效地报告五种细胞信号传导途径和一种对照 将双荧光素酶测定的效力提高五倍。为了确保较低的实验变异，我们采用 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成一个单一的 载体，导致每个转染中每个转录单位的化学计量比相等的转染 细胞。为了证明概念，我们设计了一种专门用于探测通路通量的荧光素酶测定 通过五种已知细胞信号传导途径的转录反应元件对抗组成型 标准化启动子提出并分析了 siRNA、配体和化合物的效果 同时对其目标途径和其他四种细胞途径进行治疗。基于此 初步了解后，我们建议进一步探索荧光素酶复用。四者互补 将采取的方法： 1. 增加已探索的两种底物消耗的数量 我们可以在一次实验中检测到荧光素酶。 2. 包括第三组消耗底物的荧光素酶， 即vargulin荧光素酶。 3. 鉴定针对腔肠素和/或 vargulin 荧光素酶组的猝灭剂。 4. 开发简化的组装流程，以尽可能少的克隆步骤进行综合缝合 在单个多重荧光素酶报告载体中组合多达十二个荧光素酶报告单位，并合并 专门的质粒主链能够容纳包含所有十二个的大DNA插入货物 荧光素酶报告单位。此外，为了证明使用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，我们将生成一组多重荧光素酶报告单元，可以报告 一些最常见的细胞信号传导途径的转录读数 进行上调和下调测试。因此，这项工作有望通过生长荧光素酶来扩大荧光素酶复用 底物特异性、可淬灭和光谱可分离的荧光素酶的可用库。的 该提案的最终目标是提供一个框架来探索额外的底物消耗荧光素酶 基团包括荧光素酶基团特异性猝灭剂，因此可以进一步扩展多重检测。

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.