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

项目总结 可以高效、灵敏、同时测量多个信号的多路细胞分析 同一单元中的路径需要具有大动态范围的正交探头，其测量结果可以是 很快就能拿到。荧光素酶是基因编码的、经济高效的、多功能的候选酶，可以满足这些要求 要求。常用的双荧光素酶报告分析目前检测到一个荧光素酶与 细胞途径，以及第二荧光素酶，为了归一化目的有效地连接到控制途径 从而只对单个路径进行一次活性测量。要增加细胞信令的数量 可以使用荧光素酶记者同时探测的途径，我们计划探索 荧光素酶在本提案中的多路传输。前期工作论证了扩展多路复用的可行性 六个荧光素酶，可以有效地报告五个细胞信号通路和一个对照 将双荧光素酶检测的效价提高了五倍。为了确保较低的实验变异，我们采用了 一条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，导致在每个被转染者中每个转录单位的化学计量比相等 手机。为了证明概念的正确性，我们设计了一种专门用于探测途径通量的荧光素酶分析方法。 通过五条已知的细胞信号通路的转录反应元件来对抗一种成分 标准化促进剂提出并分析了siRNA、配体和化合物的作用 同时对它们的靶通路和其他四个细胞通路进行治疗。在此基础上 在初步了解的基础上，我们建议进一步探索荧光素酶的多重作用。四个互补 将采取以下方法：1.增加已经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.包括第三组底物消耗荧光素酶， 也就是精氨酸荧光素酶。3.确定针对辅酶链氨嘧啶和/或精氨酸荧光素酶组的猝灭剂。 4.开发简化的装配流水线，尽可能少的克隆步骤来合成缝合 在单个多路荧光素酶报告载体中结合多达12个荧光素酶报告单元，并结合 能够容纳包含所有12个大DNA插入货物的专门化的质粒骨架 荧光素酶报道单位。此外，为了演示多重荧光素酶检测的概念验证，使用 最多12个荧光素酶报道单元，我们将产生一组多路荧光素酶记者，可以报道 一些最常见的细胞信号通路的转录读数将是 对上调和下调监管进行了测试。因此，这项工作有望通过增加荧光素酶的 提供底物特定的、可猝灭的和光谱可分离的荧光素酶。这个 这项提案的最终目标是提供一个框架，以探索更多底物消耗荧光素酶 包括荧光素酶基团特定的猝灭剂在内的基团，因此多路传输可以进一步扩展。

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.