Kinoshita Riko, Homma Yuta, Fukuda Mitsunori
Lab of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Miyagi, Japan.
Methods Mol Biol. 2021;2293:243-256. doi: 10.1007/978-1-0716-1346-7_17.
The Rab family small GTPases are key regulators of intracellular membrane traffic that are conserved in all eukaryotic cells. Rabs are thought to regulate various steps of membrane traffic, including the budding, transport, tethering, docking, and fusion of vesicles or organelles. Approximately 60 different Rabs have been identified in mammals, and each Rab is thought to localize to a specific membrane compartment and regulate its trafficking in a timely manner. Although a few mammalian Rabs have been thoroughly studied, the precise function of the majority of them remains poorly understood. In a recent study, we established a comprehensive collection of Rab-knockout (KO) renal epithelial cells (i.e., Madin-Darby canine kidney [MDCK] II cells) by using Cas9-mediated genome editing technology to analyze the function of each Rab or closely related Rabs in cell viability (or growth), organelle morphology, and epithelial morphogenesis. In this chapter, we describe the procedures for generating Rab-KO MDCK II cells in detail.
Rab家族小GTP酶是细胞内膜运输的关键调节因子，在所有真核细胞中都保守存在。Rabs被认为可调节膜运输的各个步骤，包括囊泡或细胞器的出芽、运输、拴系、对接和融合。在哺乳动物中已鉴定出约60种不同的Rabs，并且每种Rab被认为定位于特定的膜区室并及时调节其运输。尽管少数哺乳动物Rabs已得到深入研究，但它们中的大多数的精确功能仍知之甚少。在最近的一项研究中，我们通过使用Cas9介导的基因组编辑技术建立了Rab基因敲除（KO）肾上皮细胞（即Madin-Darby犬肾[MDCK] II细胞）的综合文库，以分析每种Rab或密切相关的Rabs在细胞活力（或生长）、细胞器形态和上皮形态发生中的功能。在本章中，我们详细描述了生成Rab-KO MDCK II细胞的程序。
