Construction of Sphingolipid Remodeled Cells by Genome Editing
Construction of Sphingolipid Remodeled Cells by Genome Editing
复制标题
通过基因组编辑构建鞘脂重塑细胞
DOI：
10.1007/978-1-0716-2910-9_10
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Homma Y
中科院分区：
文献类型：
--
作者：
Yamaji T;Homma Y
鞘脂在真核生物中广泛表达，具有多种功能。鞘脂的关键特征是其分子种类的多样性。鞘磷脂（SM）和鞘糖脂（GSL）是质膜鞘脂的主要成分，由神经酰胺和极性头基组成。SM是哺乳动物细胞中最丰富的鞘脂种类，而GSL具有多种聚糖作为头基。神经酰胺中的各种脂肪酸也有助于鞘脂种类的多样性。为了分析每种鞘脂的细胞功能，精确的基因操作是必不可少的。基因组编辑技术的最新发展促进了培养细胞中基因的完全破坏。本章描述了使用成簇规则间隔短回文重复序列（CRISPR）/Cas9系统构建各种鞘脂相关基因敲除HeLa细胞的方案，以及使用放射性同位素和薄层色谱法确认其脂质组成变化的方案。这种鞘脂重塑细胞组是分析鞘脂种类的细胞功能的有用工具，并作为脂质分析的参考。
Sphingolipids are ubiquitously expressed in eukaryotes and play various functional roles. The key characteristic of sphingolipids is their diversity of molecular species. Sphingomyelin (SM) and glycosphingolipids (GSLs) are the major components of sphingolipids in the plasma membrane, which are composed of ceramide and a polar head-group. SM is the most abundant sphingolipid species in mammalian cells, while GSLs have a wide variety of glycans as head groups. Various fatty acids in ceramide also contribute to the diversity of sphingolipid species. To analyze the cellular function of each sphingolipid species, precise gene manipulation is essential. Recent developments in genome editing technologies have facilitated complete gene disruption in cultured cells. This chapter describes protocols for the construction of various sphingolipid-related gene knockout HeLa cells using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and for confirmation of changes in their lipid composition using radioisotopes and thin layer chromatography. This sphingolipid-remodeled cell panel is a useful tool for analyzing the cellular functions of sphingolipid species and as a reference for lipid analysis.