Collaborative Research: Multimodal Mapping of Gene Isoforms by Electromicrofluidic Manipulation

细胞从相同的遗传物质中产生不同版本的基因。这种能力对生物体在各种情况下的运作至关重要。然而，目前研究不同基因版本的方法是有限的，不能精确地分析它们在生物体中的表达。此外，研究不同的蛋白质版本是具有挑战性的，因为现有的工具可能无法区分它们。虽然有技术可以测量基因版本的丰度，但它们需要分离和分离细胞，这导致了关于细胞相互作用和环境的重要信息的丢失。在这个项目中，研究人员的目标是开发一种名为电微流体（EMF）的新技术，该技术将电、工程凝胶和微芯片上的细胞培养结合起来，在单细胞水平上自动研究基因版本。这项技术将深入了解这些不同的基因版本如何在涉及多个细胞的复杂生物过程中发挥作用，如发育、生长和修复。除了创造一种新的生物传感方法外，该研究还将整合几个教育前沿，包括研究生课程开发，通过学生设计的项目主动学习，社交媒体传播，本科生和少数民族研究机会以及外展计划。本研究旨在建立一个基于电磁场的生物传感平台，用于全面的基因表达谱分析。该平台结合了电校准增强的各向异性凝胶聚合和原位免疫标记和扩增，能够同时分析多种基因表达模式。研究人员将探索不同的凝胶层结构，适合研究蛋白质异构体和（核糖核酸）RNA剪接变异体。这种方法允许准确的分析，同时最大限度地减少转移过程中的样品损失和污染。通过实现电对准增强的各向异性凝胶聚合，分子的横向扩散受到限制，从而提高了空间分辨率和灵敏度。这种增强有利于原位单细胞分析，使空间协调和细胞异质性的研究成为可能。此外，EMF技术的使用使分析程序自动化，提高了可重复性并促进了平台的更广泛采用。最初，3D凝胶分析将用于分析蛋白质异构体和RNA剪接变异体。随后，这些检测将集成到一个多功能平台，能够同时检测多种蛋白质异构体和RNA剪接变体。该项目将重点定位迁移细胞单层中的神经源性位点notch同源蛋白（notch）家族成员，以评估其在细胞集体迁移中的调节功能。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Cells produce different versions of genes from the same genetic material. This ability is crucial for the functioning of organisms in various situations. However, current methods for studying different gene versions are limited and cannot precisely analyze their expression within the organism. Additionally, studying different protein versions is challenging because existing tools may not distinguish between them. While there are techniques available to measure the abundance of gene versions, they require isolating and separating cells, which results in the loss of important information about cell interactions and the environment. In this project, the investigators aim to develop a new technology called electromicrofluidics (EMF), which combines electricity, engineered gels, and cell culture on a microchip to automatically study gene versions at the single-cell level. This technology will provide insights into how these different gene versions function in complex biological processes involving multiple cells, such as development, growth, and repair. In addition to creating a novel biosensing approach, the research will be integrated across several educational fronts, including graduate program development, active learning through student-designed projects, social media dissemination, undergraduate and minority research opportunities,and outreach programs.The research aims to establish an EMF based biosensing platform for comprehensive gene expression profiling. This platform incorporates electroalignment-enhanced anisotropic gel polymerization and in situ immunolabeling and amplification to enable simultaneous analysis of multiple gene expression modes. The investigators will explore different gel layer configurations suitable for studying protein isoforms and (ribonucleic acid) RNA splicing variants. This approach allows for accurate profiling while minimizing sample loss and contamination during transfer. By implementing electroalignment-enhanced anisotropic gel polymerization, the lateral diffusion of molecules is constrained, resulting in improved spatial resolution and sensitivity. This enhancement facilitates in situ single-cell analysis, enabling the study of spatial coordination and cell heterogeneity. Furthermore, the use of EMF technology automates the assay procedures, enhancing reproducibility and facilitating wider adoption of the platform. Initially, 3D gel assays will be developed to analyze protein isoforms and RNA splice variants. Subsequently, these assays will be integrated into a versatile platform capable of simultaneously detecting multiple protein isoforms and RNA splicing variants. The project will focus on mapping the Neurogenic locus notch homolog protein (NOTCH) family members in migrating cell monolayers to evaluate their regulatory functions in collective cell migration.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.