TY - CHAP
T1 - Methods for Establishing Rab Knockout MDCK Cells
AU - Kinoshita, Riko
AU - Homma, Yuta
AU - Fukuda, Mitsunori
N1 - Funding Information:
This work was supported in part by Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (grant number 18K14692 to Y.H.), Grant-in-Aid for Scientific Research(B) from the MEXT (grant number 19H03220 to M.F.), and by Japan Science and Technology Agency (JST) CREST (grant Number JPMJCR17H4 to M.F.). The authors declare no competing financial interests.
Publisher Copyright:
© 2021, The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
PY - 2021
Y1 - 2021
N2 - 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.
AB - 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.
KW - CRISPR/Cas9
KW - Epithelial cells
KW - Gene knockout
KW - MDCK II cells
KW - Membrane traffic
KW - Polarized trafficking
KW - Rab GTPase
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U2 - 10.1007/978-1-0716-1346-7_17
DO - 10.1007/978-1-0716-1346-7_17
M3 - Chapter
C2 - 34453722
AN - SCOPUS:85081971043
T3 - Methods in Molecular Biology
SP - 243
EP - 256
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -