Collaborative Research: Multimodal Mapping of Gene Isoforms by Electromicrofluidic Manipulation

细胞从相同的遗传物质产生不同版本的基因。这种能力对于生物体在各种情况下的功能至关重要。然而，目前研究不同基因版本的方法有限，无法精确分析它们在生物体内的表达。此外，研究不同的蛋白质版本具有挑战性，因为现有工具可能无法区分它们。虽然有一些技术可以测量基因版本的丰度，但它们需要隔离和分离细胞，这会导致有关细胞相互作用和环境的重要信息丢失。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，该技术将电力、工程凝胶和微芯片上的细胞培养结合起来，自动研究单细胞水平的基因版本。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，例如发育、生长和修复。 除了创建一种新颖的生物传感方法外，该研究还将整合多个教育前沿，包括研究生课程开发、通过学生设计的项目进行主动学习、社交媒体传播、本科生和少数族裔研究机会以及外展计划。该研究旨在建立一个基于 EMF 的生物传感平台，用于全面的基因表达谱分析。该平台结合了电对准增强的各向异性凝胶聚合以及原位免疫标记和扩增，能够同时分析多种基因表达模式。研究人员将探索适合研究蛋白质亚型和（核糖核酸）RNA 剪接变体的不同凝胶层配置。这种方法可以进行准确的分析，同时最大限度地减少转移过程中的样品损失和污染。通过实施电排列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。这种增强有助于原位单细胞分析，从而能够研究空间协调和细胞异质性。此外，EMF 技术的使用可实现检测程序的自动化，提高重现性并促进该平台的更广泛采用。最初，将开发 3D 凝胶测定来分析蛋白质亚型和 RNA 剪接变体。随后，这些检测将被整合到一个多功能平台中，能够同时检测多种蛋白质亚型和 RNA 剪接变体。该项目将重点绘制迁移细胞单层中的神经源性基因座缺口同源蛋白 (NOTCH) 家族成员，以评估它们在集体细胞迁移中的调节功能。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力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.