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Generation and Analysis of CCM Phenotypes in C. elegans

  • Evelyn Popiel
  • William Brent DerryEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2152)

Abstract

This chapter presents methods for exploiting the powerful tools available in the nematode worm Caenorhabditis elegans to understand the in vivo functions of cerebral cavernous malformation (CCM) genes and the organization of their associated signaling pathways. Included are methods for assessing phenotypes caused by loss-of-function mutations in the worm CCM genes kri-1 and ccm-3, CRISPR-based gene editing techniques, and protocols for conducting high-throughput forward genetic and small molecule screens.

Key words

kri-1 ccm-ccm-3 Cerebral cavernous malformations C. elegans CRISPR High-throughput screen Drug screen Forward genetic screen Suppressor screen 

References

  1. 1.
    Berman JR, Kenyon C (2006) Germ-cell loss extends C. elegans life span through regulation of DAF-16 by kri-1 and lipophilic-hormone signaling. Cell 124:1055–1068CrossRefGoogle Scholar
  2. 2.
    Lant B, Yu B, Goudreault M et al (2015) CCM-3/STRIPAK promotes seamless tube extension through endocytic recycling. Nat Commun 6:6449.  https://doi.org/10.1038/ncomms7449
  3. 3.
    Chapman EM, Lant B, Ohashi Y et al (2019) A conserved CCM complex promotes apoptosis non-autonomously by regulating zinc homeostasis. Nat Commun 10:1791.  https://doi.org/10.1038/s41467-091-09829-zCrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Lant B, Pal S, Chapman EM et al (2018) Interrogating the ccm-3 gene network. Cell Rep 24:2857–2868CrossRefGoogle Scholar
  5. 5.
    Ito S, Greiss S, Gartner A et al (2010) Cell-nonautonomous regulation of C. elegans germ cell death by kri-1. Curr Biol 20:333–338Google Scholar
  6. 6.
    Lant B, Derry WB (2013) Methods for detection and analysis of apoptosis signaling in the C. elegans germline. Methods 61:174–182CrossRefGoogle Scholar
  7. 7.
    Pal S, Lant B, Yu B et al (2017) CCM-3 promotes C. elegans germline development by regulating vesicle trafficking, cytokinesis, and polarity. Curr Biol 27:868–876Google Scholar
  8. 8.
    Chu VT, Weber T, Wefers B (2015) Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol 33:543–548CrossRefGoogle Scholar
  9. 9.
    Paix A, Folkmann A, Rasoloson D (2015) High efficiency, homology-directed genome editing in Caenorhabditis elegans using CRISPR-Cas9 ribonucleoprotein complexes. Genetics 201:47–54CrossRefGoogle Scholar
  10. 10.
    Goudreault M, D’Ambrosio LM, Kean MJ (2009) A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein. Mol Cell Proteomics 8:157–171CrossRefGoogle Scholar
  11. 11.
    Ran FA, Hsu PD, Wright J et al (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8:2281–2308CrossRefGoogle Scholar
  12. 12.
    Kim S, Kim D, Cho SW et al (2014) Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res 24:1012–1019CrossRefGoogle Scholar
  13. 13.
    Lant B, Derry WB (2014) High-throughput RNAi screening for germline apoptosis genes in Caenorhabditis elegans. Cold Spring Harb Protoc 2014:428–434.  https://doi.org/10.1101/pdb.prot080234
  14. 14.
    Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77:71–94PubMedPubMedCentralGoogle Scholar
  15. 15.
    Otten C, Knox J, Boulday G et al (2018) Systematic pharmacological screens uncover novel pathways involved in cerebral cavernous malformations. EMBO Mol Med.  https://doi.org/10.15252/emmm.201809155
  16. 16.
    Evans TC (ed) (2006) Transformation and microinjection. WormBook, ed. The C. elegans Research Community, WormBook.  https://doi.org/10.1895/wormbook.1.108.1
  17. 17.
    Chapman EM (2018) Elucidating the mechanism by which KRI-1/CCM1 regulates apoptosis cell non-autonomously in Caenorhabditis elegans. Dissertation, University of TorontoGoogle Scholar
  18. 18.
    Nilsen TW (2015) Poisoned primer extension. Cold Spring Harb Protoc.  https://doi.org/10.1101/pdb.prot080986

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Developmental and Stem Cell Biology ProgramThe Hospital for Sick Children, Peter Gilgan Centre for Research and LearningTorontoCanada
  2. 2.Department of Molecular GeneticsUniversity of TorontoTorontoCanada

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