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Affinity Purification of Intraflagellar Transport (IFT) Proteins in Mice Using Endogenous Streptavidin/FLAG Tags

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Cilia

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2725))

Abstract

Biological complexity is achieved through elaborate interactions between relatively few individual components. Affinity purification (AP) has allowed these networks of protein-protein interactions that regulate key biological processes to be interrogated systematically. In order to perform these studies at the required scale, easily transfectable immortalized cell lines have typically been used. Gene-editing now affords the systematic creation of isogenic mouse models carrying endogenous tags for affinity proteomics. This may allow protein-protein interactions to be characterized in the appropriate tissue for a particular biological process or disease phenotype under physiological conditions, and for interaction landscapes to be compared across tissues. Here we demonstrate application to intraflagellar transport (IFT) proteins, which are WD40-domain-containing proteins that are essential for the formation and function of all types of cilia. We describe a method to generate mice with an endogenous C-terminal streptavidin/FLAG tag, using Ift80 as an example, and demonstrate the successful implementation of AP in this model. This method can easily be adapted for N- and C-terminal tagging of many other proteins in vivo.

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Acknowledgments

This work was funded by a Wellcome Trust Collaborative Award in Science (210585/Z/18/Z) to DJ, PLB, MU, and KB. This research was also supported by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London.

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Author notes

  1. Authors Tina Beyer, Tiago Martins, Jeshmi Jeyabalan Srikaran, Marian Seda, Emma Peskett, Franziska Klose, Katrin Junger have equally contributed to this chapter.

Authors and Affiliations

  1. Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany

    Tina Beyer, Franziska Klose, Katrin Junger, Marius Ueffing & Karsten Boldt

  2. UCL Great Ormond Street Institute of Child Health, University College London, London, UK

    Tiago Martins, Jeshmi Jeyabalan Srikaran, Marian Seda, Emma Peskett, Philip L. Beales & Dagan Jenkins

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  1. Tina Beyer
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  2. Tiago Martins
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  3. Jeshmi Jeyabalan Srikaran
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  4. Marian Seda
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  5. Emma Peskett
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  6. Franziska Klose
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  7. Katrin Junger
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  8. Philip L. Beales
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  9. Marius Ueffing
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  10. Karsten Boldt
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Corresponding author

Correspondence to Dagan Jenkins .

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Editors and Affiliations

  1. Medical Research Council Toxicology Unit, School of Biological Sciences, University of Cambridge, Cambridge, UK

    Vito Mennella

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Beyer, T. et al. (2024). Affinity Purification of Intraflagellar Transport (IFT) Proteins in Mice Using Endogenous Streptavidin/FLAG Tags. In: Mennella, V. (eds) Cilia. Methods in Molecular Biology, vol 2725. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3507-0_12

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  • DOI: https://doi.org/10.1007/978-1-0716-3507-0_12

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3506-3

  • Online ISBN: 978-1-0716-3507-0

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