Generation of Reporter Cell Lines for Endogenous Expression Analysis of Peroxisomal Proteins.
Generation of Reporter Cell Lines for Endogenous Expression Analysis of Peroxisomal Proteins.
复制标题
用于过氧化物酶体蛋白内源表达分析的报告细胞系的生成。
DOI：
10.1007/978-1-0716-3048-8_18
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Silva BSC
中科院分区：
文献类型：
--
作者：
Silva BSC
过氧化物酶体是一种多功能、普遍存在的动态细胞器。它们负责多种代谢和生理功能，并通过膜接触位点与其他细胞器（包括ER、线粒体、脂滴和溶酶体）通信。然而，尽管它们对健康细胞功能很重要，但值得注意的是，人们对过氧化物酶体和过氧化物酶体蛋白在人体细胞中的生理条件下如何调节知之甚少。在这里，我们提出了一种方法来产生报告细胞系，以测量内源性表达的过氧化物酶体蛋白的兴趣。通过CRISPR介导的将容易检测的蛋白质编码标签框内敲入相关基因组基因座中，可以在不同条件下以高通量方式定量细胞群体中目标蛋白质的内源水平。这对于从根本上理解过氧化物酶体蛋白是如何调节的具有重要意义，并可能揭示调节过氧化物酶体蛋白表达以改善细胞性能的治疗潜力。
Peroxisomes are multifunctional, ubiquitous, and dynamic organelles. They are responsible for diverse metabolic and physiological functions and communicate with other organelles, including the ER, mitochondria, lipid droplets, and lysosomes, through membrane contact sites. However, despite their importance for healthy cell function, remarkably, little is known about how peroxisomes and peroxisomal proteins are regulated under physiological conditions in human cells. Here, we present a method to generate reporter cell lines to measure endogenous expression of peroxisomal proteins of interest. By CRISPR-mediated knock-in of an easily detectable protein-coding tag in-frame into the relevant genomic loci, endogenous levels of the protein of interest in a cell population can be quantified in a high-throughput manner under different conditions. This has important implications for the fundamental understanding of how peroxisomal proteins are regulated and may reveal the therapeutic potential of modulating peroxisomal protein expression to improve cell performance.