Split-GFP tagging and live imaging of hair cell proteins

毛细胞蛋白的 Split-GFP 标记和实时成像

• 批准号: 10623203
• 负责人: Jung-Bum Shin
• 金额: $ 20.19万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10623203
• 关键词: Address; Adopted; Affect; Behavior; Cell physiology; Cells; Cellular biology; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; DNA; Development; Diffusion; Electroporation; Equilibrium; Fluorescence; Fluorescence Microscopy; Gene Expression; Genes; Goals; Hair; Hair Cells; Hearing; Image; Investigation; Kinetics; Knock-in; Knock-in Mouse; Labyrinth; Length; Link; Mediating; Methods; Modernization; Molecular; Morphologic artifacts; Motor; Movement; Mus; Pillar Cell; Property; Proteins; Role; Sensory Receptors; System; Techniques; Testing; Transfection; Transgenic Mice; USH1C gene; Virus; fluorescence imaging; gene gun; genome editing; genomic locus; hearing impairment; insight; interest; live cell imaging; nanoscale; overexpression; reconstitution; tool; transmission process

Over the past few decades, the hair cell field has gained great insight into the inner workings of hair cells, the sensory receptors that mediate our sense of hearing and balance. The molecular identity of many factors important for hair cell development and function, and their significance for hearing loss, have been elucidated. Despite this progress, the study of hair cells presents many challenges. In particular live-cell imaging and tracking of proteins, important pillars of cell biology studies, have been especially difficult, mainly due to limited means to transfect and culture hair cells. Considering that the essence of hair cell function is micro- and nanoscale movement, investigations of the dynamic properties of proteins is crucial for a complete understanding of hair cell function. The goal of this proposal therefore is to develop a tool that makes imaging of protein movements in living hair cells more accessible. The gold standard technique for tracking protein movement is live-cell imaging of tagged proteins. Traditionally, this is achieved by expression of exogenous DNA constructs fused to fluorescent proteins, delivered to cells by gene gun, electroporation or viruses. Transgenic mice also have been employed. These approaches, however, are potentially confounded by overexpression artifacts and low transduction/transfection efficiency. Knock-in (KI) of genetically-encoded fluorescent tags into the endogenous gene loci has become more tractable through modern genome editing methods, but KI of large DNA segments, such as the full-length GFP coding sequence, remains difficult and has the potential to affect gene expression. To address these challenges, we plan to develop a mouse tool kit that streamlines fluorescent tagging of proteins for localization and live cell imaging. To this end, we adopted the Split-GFP strategy. In this two- component system, the endogenous gene of interest is genetically tagged with a small part of GFP (“GFP11”). Co-expressing the remaining (non-fluorescent) portion of GFP (“GFP1-10”) reconstitutes GFP fluorescence, allowing imaging by fluorescence microscopy. Due to the small size of the GFP11 fragment (48 bps), CRISPR- mediated KI into the endogenous loci is highly efficient. In preliminary studies, we confirmed that the Split-GFP approach faithfully recapitulates the endogenous localization of the cuticular plate protein LMO7, by delivering AAV-packaged GFP1-10 into the hair cells of Lmo7-GFP11 KI mice. To circumvent AAV-mediated delivery, we propose to generate a transgenic mouse line that expresses the GFP1-10 fragment. Founders have already been obtained and germline transmission was confirmed (SA1). This system will be tested on live-imaging tasks of increasing difficulty: In SA2, we will study the diffusion kinetics and movement of LMO7 in the hair cell. In SA3, challenging the sensitivity limits of this system, we will investigate the dynamics of the tip link motor complex component USH1C in the hair bundle.

在过去的几十年里，毛细胞领域对毛细胞的内部运作有了深入的了解， 介导我们的听觉和平衡感的感觉受体许多因素的分子特性。 对于毛细胞发育和功能的重要性及其对听力损失的重要性已被阐明。 尽管取得了这些进展，毛细胞的研究仍面临许多挑战，特别是活细胞成像和。 蛋白质是细胞生物学研究的重要支柱，追踪蛋白质尤其困难，主要是由于有限的 考虑到毛细胞功能的本质是微观和培养，即转染和培养毛细胞。 纳米级运动，蛋白质动态特性的研究对于完整的研究至关重要 因此，该提案的目标是开发一种进行成像的工具。 活毛细胞中的蛋白质运动更容易。 追踪蛋白质运动的黄金标准技术是标记蛋白质的活细胞成像。 这是通过与荧光蛋白融合的外源 DNA 构建体的表达来实现的，并通过 然而，也使用了基因枪、电穿孔或转基因小鼠。 可能会因过度表达伪影和低转导/转染效率而混淆。 (KI) 基因编码的荧光标签进入内源基因位点变得更容易处理 现代基因组编辑方法，但是大 DNA 片段的 KI，例如全长 GFP 编码序列， 仍然很困难并且有可能影响基因表达。 为了应对这些挑战，我们计划开发一个鼠标工具包，以简化荧光标记 为此，我们采用了 Split-GFP 策略。 在组件系统中，感兴趣的内源基因用一小部分 GFP（“GFP11”）进行基因标记。 共表达 GFP 的剩余（非荧光）部分（“GFP1-10”）可重建 GFP 荧光， 由于 GFP11 片段较小（48 bps），因此可以通过荧光显微镜进行成像。 在初步研究中，我们证实 Split-GFP 介导的 KI 进入内源基因座的效率很高。 该方法通过提供 AAV 包装的 GFP1-10 进入 Lmo7-GFP11 KI 小鼠的毛细胞中 为了规避 AAV 介导的传递，我们。 创始人已经提议生成表达 GFP1-10 片段的转基因小鼠系。 已获得并确认了种系传播（SA1），该系统将在实时成像上进行测试。 难度越来越大的任务：在SA2中，我们将研究LMO7在毛细胞中的扩散动力学和运动。 在 SA3 中，为了挑战该系统的灵敏度极限，我们将研究尖端连杆电机的动力学 发束中的复杂成分USH1C。