Skip to main content
SpringerLink
Log in

Methods for Establishing Rab Knockout MDCK Cells

  • Protocol
  • First Online:
Rab GTPases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2293))

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 149.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Pereira-Leal JB, Seabra MC (2001) Evolution of the Rab family of small GTP-binding proteins. J Mol Biol 313:889–901

    Article  CAS  Google Scholar 

  2. Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107–117

    Article  CAS  Google Scholar 

  3. Fukuda M (2008) Regulation of secretory vesicle traffic by Rab small GTPases. Cell Mol Life Sci 65:2801–2813

    Article  CAS  Google Scholar 

  4. Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Nat Rev Mol Cell Biol 10:513–525

    Article  CAS  Google Scholar 

  5. Hutagalung AH, Novick PJ (2011) Role of Rab GTPases in membrane traffic and cell physiology. Physiol Rev 91:119–149

    Article  CAS  Google Scholar 

  6. Nottingham RM, Pfeffer SR (2009) Defining the boundaries: Rab GEFs and GAPs. Proc Natl Acad Sci 106:14185–14186

    Article  Google Scholar 

  7. Barr F, Lambright DG (2010) Rab GEFs and GAPs. Curr Opin Cell Biol 22:461–470

    Article  CAS  Google Scholar 

  8. Fukuda M (2011) TBC proteins: GAPs for mammalian small GTPase Rab? Biosci Rep 31:159–168

    Article  CAS  Google Scholar 

  9. Ishida M, Oguchi ME, Fukuda M (2016) Multiple types of guanine nucleotide exchange factors (GEFs) for Rab small GTPases. Cell Struct Funct 41:61–79

    Article  CAS  Google Scholar 

  10. Lamber EP, Siedenburg AC, Barr FA (2019) Rab regulation by GEFs and GAPs during membrane traffic. Curr Opin Cell Biol 59:34–39

    Article  CAS  Google Scholar 

  11. Fukuda M, Kanno E, Ishibashi K, Itoh T (2008) Large scale screening for novel Rab effectors reveals unexpected broad Rab binding specificity. Mol Cell Proteomics 7:1031–1042

    Article  CAS  Google Scholar 

  12. Kanno E, Ishibashi K, Kobayashi H, Matsui T, Ohbayashi N, Fukuda M (2010) Comprehensive screening for novel Rab-binding proteins by GST pull-down assay using 60 different mammalian Rabs. Traffic 11:491–507

    Article  CAS  Google Scholar 

  13. Feig LA (1999) Tools of the trade: use of dominant-inhibitory mutants of Ras-family GTPases. Nat Cell Biol 1:E25–E27

    Article  CAS  Google Scholar 

  14. Oguchi ME, Etoh K, Fukuda M (2018) Rab20, a novel Rab small GTPase that negatively regulates neurite outgrowth of PC12 cells. Neurosci Lett 662:324–330

    Article  CAS  Google Scholar 

  15. Ramalho JS, Anders R, Jaissle GB, Seeliger MW, Huxley C, Seabra MC (2002) Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models. BMC Cell Biol 3:26

    Article  Google Scholar 

  16. Homma Y, Fukuda M (2016) Rabin8 regulates neurite outgrowth in both GEF-activity-dependent and -independent manners. Mol Biol Cell 27:2107–2118

    Article  CAS  Google Scholar 

  17. Homma Y, Kinoshita R, Kuchitsu Y, Wawro PS, Marubashi S, Oguchi ME, Ishida M, Fujita N, Fukuda M (2019) Comprehensive knockout analysis of the Rab family GTPases in epithelial cells. J Cell Biol 218:2035–2050

    Article  CAS  Google Scholar 

  18. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308

    Article  CAS  Google Scholar 

  19. Naito Y, Hino K, Bono H, Ui-Tei K (2015) CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics 31:1120–1123

    Article  CAS  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. Lab of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Miyagi, Japan

    Riko Kinoshita, Yuta Homma & Mitsunori Fukuda

Authors
  1. Riko Kinoshita
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuta Homma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mitsunori Fukuda
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mitsunori Fukuda .

Editor information

Editors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Peggy and Charles Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA

    Guangpu Li

  2. Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, IL, USA

    Nava Segev

Rights and permissions

Reprints and permissions

Copyright information

© 2021 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Kinoshita, R., Homma, Y., Fukuda, M. (2021). Methods for Establishing Rab Knockout MDCK Cells. In: Li, G., Segev, N. (eds) Rab GTPases. Methods in Molecular Biology, vol 2293. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1346-7_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-1346-7_17

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1345-0

  • Online ISBN: 978-1-0716-1346-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation