利用基因组编辑的人类干细胞融合快速筛选体内新的蛋白质功能。

IF 2.7 3区 生物学 Q3 CELL BIOLOGY
Samantha L Smith, Yuichiro Iwamoto, Aadhithya Manimaran, David G Drubin
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引用次数: 0

摘要

基因组编辑使荧光蛋白编码序列能够整合到基因组中,从而使框架内融合蛋白在其天然基因调控序列的控制下表达。虽然这项技术克服了与基因过表达相关的对蛋白质化学计量改变敏感的生物过程(如网格蛋白介导的内吞作用(CME))有充分记录的人工产物,但与酵母菌等更简单的生物相比,编辑后生动物细胞的基因组需要大量的时间成本。编辑两个或多个基因以在单个细胞系中表达多个荧光融合蛋白已被证明是揭示不同蛋白质之间空间动态关系(因此是功能关系)的有力策略,但在同一细胞系中编辑每个基因可能需要数月时间。在这里,通过利用细胞融合,我们在基因组编辑的人类细胞中快速生成表达荧光融合蛋白成对排列的细胞,以揭示以前未检测到的蛋白质-细胞器相互作用。我们将表达CME和肌动蛋白骨架蛋白框架内融合的人诱导多能干细胞(hiPSCs)与表达荧光标记细胞器标记的hiPSCs融合在一起,发现了CME蛋白、分支肌动蛋白丝网络和溶酶体之间新的相互作用。[媒体:见文][媒体:见文][媒体:见文][媒体:见文][媒体:见文][媒体:见文][媒体:见文][媒体:见文][媒体:见文][媒体:见文][媒体:见文]。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Harnessing fusion of genome-edited human stem cells to rapidly screen for novel protein functions in vivo.

Genome editing has enabled the integration of fluorescent protein coding sequences into genomes, resulting in expression of in-frame fusion proteins under the control of their natural gene regulatory sequences. While this technique overcomes the well-documented artifacts associated with gene overexpression for biological processes sensitive to altered protein stoichiometry, such as clathrin-mediated endocytosis (CME), editing genomes of metazoan cells incurs a significant time cost compared to simpler organisms, such as yeast. Editing two or more genes to express multiple fluorescent fusion proteins in a single cell line has proven to be a powerful strategy for uncovering spatio-dynamic, and therefore functional, relationships among different proteins, but it can take many months to edit each gene within the same cell line. Here, by utilizing cell fusions, we quickly generated cells expressing pairwise permutations of fluorescent fusion proteins in genome-edited human cells to reveal previously undetected protein-organelle interactions. We fused human induced pluripotent stem cells (hiPSCs) that express in-frame fusions of CME and actin cytoskeleton proteins with hiPSCs that express fluorescently tagged organelle markers, uncovering novel interactions between CME proteins, branched actin filament networks, and lysosomes. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

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来源期刊
Molecular Biology of the Cell
Molecular Biology of the Cell 生物-细胞生物学
CiteScore
6.00
自引率
6.10%
发文量
402
审稿时长
2 months
期刊介绍: MBoC publishes research articles that present conceptual advances of broad interest and significance within all areas of cell, molecular, and developmental biology. We welcome manuscripts that describe advances with applications across topics including but not limited to: cell growth and division; nuclear and cytoskeletal processes; membrane trafficking and autophagy; organelle biology; quantitative cell biology; physical cell biology and mechanobiology; cell signaling; stem cell biology and development; cancer biology; cellular immunology and microbial pathogenesis; cellular neurobiology; prokaryotic cell biology; and cell biology of disease.
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