Skip to main content

Functional Genomics Assays to Study CFTR Traffic and ENaC Function

  • Protocol
  • First Online:
Cystic Fibrosis

Abstract

As several genomes have been sequenced, post-genomic approaches like transcriptomics and proteomics, identifying gene products differentially expressed in association with a given pathology, have held promise both of understanding the pathways associated with the respective disease and as a fast track to therapy. Notwithstanding, these approaches cannot distinguish genes and proteins with mere secondary pathological association from those primarily involved in the basic defect(s). New global strategies and tools identifying gene products responsible for the basic cellular defect(s) in CF pathophysiology currently being performed are presented here. These include high-content screens to determine proteins affecting function and trafficking of CFTR and ENaC.

J. Almaça and S. Dahimène contributed equally to this work

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Erfle, H., Neumann, B., Liebel, U., Rogers, P., Held, M., Walter, T., et al. (2007) Reverse transfection on cell arrays for high content screening microscopy. Nat Protoc 2, 392–399.

    Article  PubMed  CAS  Google Scholar 

  2. Starkuviene, V., Liebel, U., Simpson, J. C., Erfle, H., Poustka, A., Wiemann, S., et al. (2004) High-content screening microscopy identifies novel proteins with a putative role in secretory membrane traffic. Genome Res 14, 1948–1956.

    Article  PubMed  CAS  Google Scholar 

  3. Starkuviene, V., and Pepperkok, R. (2007) Differential requirements for ts-O45-G and procollagen biosynthetic transport. Traffic 8, 1035–1051.

    Article  PubMed  CAS  Google Scholar 

  4. Pepperkok, R., Simpson, J. C., Rietdorf, J., Cetin, C., Liebel, U., Terjung, S., et al. (2005) Imaging platforms for measurement of membrane trafficking. Methods Enzymol 404, 8–18.

    Article  PubMed  CAS  Google Scholar 

  5. Simpson, J. C., Cetin, C., Erfle, H., Joggerst, B., Liebel, U., Ellenberg, J., et al. (2007) An RNAi screening platform to identify secretion machinery in mammalian cells. J Biotechnol 129, 352–365.

    Article  PubMed  CAS  Google Scholar 

  6. Simpson, J. C., Mateos, A., and Pepperkok, R. (2007) Maturation of the mammalian secretome. Genome Biol 8, 211.

    Article  PubMed  Google Scholar 

  7. Denning, G. M., Ostedgaard, L. S., and Welsh, M. J. (1992) Abnormal localization of cystic fibrosis transmembrane conductance regulator in primary cultures of cystic fibrosis airway epithelia. J Cell Biol 118, 551–559.

    Article  PubMed  CAS  Google Scholar 

  8. Schultz, B. D., Takahashi, A., Liu, C., Frizzell, R. A., and Howard, M. (1997) FLAG epitope positioned in an external loop preserves normal biophysical properties of CFTR. Am J Physiol 273, C2080–C2089.

    PubMed  CAS  Google Scholar 

  9. Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E., et al. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22, 1567–1572.

    Article  PubMed  CAS  Google Scholar 

  10. Erfle, H., and Pepperkok, R. (2007) Production of siRNA- and cDNA-transfected cell arrays on noncoated chambered coverglass for high-content screening microscopy in living cells. Methods Mol Biol 360, 155–161.

    PubMed  CAS  Google Scholar 

  11. Erfle, H., Neumann, B., Rogers, P., Bulkescher, J., Ellenberg, J., and Pepperkok, R. (2008) Work flow for multiplexing siRNA assays by solid-phase reverse transfection in multiwell plates. J Biomol Screen 13, 575–580.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work is supported by TargetScreen2 (EU-FP6-LSH-2005-037365) grant. J.A. is recipient of Ph.D. fellowship SFRH/BD/29134/2006 (FCT, Portugal). The authors wish to thank Beate Neumann and Jutta Bulkescher (Advanced Light Microscopy Core Facility, EMBL) for their expert technical advice.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Margarida D. Amaral .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Almaça, J. et al. (2011). Functional Genomics Assays to Study CFTR Traffic and ENaC Function. In: Amaral, M., Kunzelmann, K. (eds) Cystic Fibrosis. Methods in Molecular Biology, vol 742. Humana Press. https://doi.org/10.1007/978-1-61779-120-8_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-120-8_15

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-119-2

  • Online ISBN: 978-1-61779-120-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics