Methods for Establishing Rab Knockout MDCK Cells

Riko Kinoshita, Yuta Homma, Mitsunori Fukuda

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages243-256
Number of pages14
DOIs
Publication statusPublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2293
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • CRISPR/Cas9
  • Epithelial cells
  • Gene knockout
  • MDCK II cells
  • Membrane traffic
  • Polarized trafficking
  • Rab GTPase

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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