Collaborative Research: Multimodal Mapping of Gene Isoforms by Electromicrofluidic Manipulation

合作研究：通过电微流体操作进行基因异构体的多模式图谱

• 批准号: 2303927
• 负责人: Shih-Kang Fan
• 金额: $ 30万
• 依托单位: Kansas State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2303927&HistoricalAwards=false
• 关键词: Collaborative; Research; Multimodal; Mapping; Gene

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.

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，无法准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。虽然有一些技术可以用来测量基因版本的丰度，但它们需要分离和分离细胞，这导致丢失了关于细胞相互作用和环境的重要信息。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，它将电力、工程凝胶和细胞培养结合在一个微芯片上，在单细胞水平上自动研究基因版本。这项技术将为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提供洞察力，如发育、生长和修复。除了创造一种新的生物传感方法，这项研究还将整合到多个教育领域，包括研究生项目开发、通过学生设计的项目进行主动学习、社交媒体传播、本科生和少数族裔的研究机会以及推广计划。该研究旨在建立一个基于电动势的生物传感平台，用于全面的基因表达谱分析。该平台结合了电排列增强的各向异性凝胶聚合和原位免疫标记和扩增，从而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异构体和(核糖核酸)RNA剪接变体的不同凝胶层配置。这种方法可实现准确的分析，同时最大限度地减少转移过程中的样品损失和污染。通过实施电取向增强的各向异性凝胶聚合，限制了分子的横向扩散，从而提高了空间分辨率和灵敏度。这种增强促进了原位单细胞分析，使空间协调和细胞异质性的研究成为可能。此外，电动势技术的使用使分析过程自动化，提高了重复性，促进了该平台的更广泛采用。首先，将开发3D凝胶分析来分析蛋白质异构体和RNA剪接变体。随后，这些分析将被集成到一个多功能平台中，能够同时检测多种蛋白质异构体和RNA剪接变体。该项目将专注于绘制迁移细胞单层中的神经起源基因座缺口同源蛋白(Noch)家族成员图，以评估它们在集体细胞迁移中的调节功能。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。