Germline Stem Cells pp 205-216

Part of the Methods in Molecular Biology book series (MIMB, volume 1463) | Cite as

Identification of Mouse piRNA Pathway Components Using Anti-MIWI2 Antibodies

  • Takamasa Hirano
  • Hidetoshi Hasuwa
  • Haruhiko Siomi
Protocol

Abstract

The mouse testis has served as a popular model system to study a wide range of biological processes, including germ cell development, meiosis, epigenetic changes of chromatin, transposon silencing, and small RNA-mediated epigenetic modifications. PIWI-interacting RNAs (piRNAs) are a class of small RNAs that are almost exclusively expressed in animal gonads. They repress transposons by forming effector complexes with PIWI proteins to maintain genome integrity of the germline. Here we describe detailed procedures of how to produce monoclonal antibodies against a mouse nuclear PIWI protein, MIWI2, which functions in de novo DNA methylation of target transposon loci. We then describe how to use the antibodies to isolate associated complexes and to detect MIWI2 immunohistochemically.

Key words

piRNA MIWI2 Monoclonal antibody Inclusion body Mouse embryonic testes 

References

  1. 1.
    Biémont C, Vieira C (2006) Genetics: junk DNA as an evolutionary force. Nature 443:521–524CrossRefPubMedGoogle Scholar
  2. 2.
    Lynch M (2007) The origins of genome architecture. Sinauer Associates, Sunderland, MAGoogle Scholar
  3. 3.
    Slotkin RK, Martienssen R (2007) Transposable elements and the epigenetic regulation of the genome. Nat Rev Genet 8:272–285CrossRefPubMedGoogle Scholar
  4. 4.
    Siomi MC, Sato K, Pezic D, Aravin AA (2011) PIWI-interacting small RNAs: the vanguard of genome defence. Nat Rev Mol Cell Biol 12:246–258CrossRefPubMedGoogle Scholar
  5. 5.
    Iwasaki YW, Siomi MC, Siomi H (2015) PIWI-interacting RNA: its biogenesis and functions. Annu Rev Biochem 84:405–433CrossRefPubMedGoogle Scholar
  6. 6.
    Yamanaka S, Siomi MC, Siomi H (2014) piRNA clusters and open chromatin structure. Mobile DNA 5:22CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Pillai RS, Chuma S (2012) piRNAs and their involvement in male germline development in mice. Dev Growth Differ 54:78–92CrossRefPubMedGoogle Scholar
  8. 8.
    Deng W, Lin H (2002) miwi, a murine homolog of piwi, encodes a cytoplasmic protein essential for spermatogenesis. Dev Cell 2:819–830CrossRefPubMedGoogle Scholar
  9. 9.
    Kuramochi-Miyagawa S, Kimura T, Ijiri TW et al (2004) Mili, a mammalian member of piwi family gene, is essential for spermatogenesis. Development 131:839–849CrossRefPubMedGoogle Scholar
  10. 10.
    Carmell MA, Girard A, van de Kant HJG et al (2007) MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline. Dev Cell 12:503–514CrossRefPubMedGoogle Scholar
  11. 11.
    Aravin AA, Sachidanandam R, Bourc’his D et al (2008) A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. Mol Cell 31:785–799CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    De Fazio S, Bartonicek N, Di Giacomo M et al (2011) The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements. Nature 480:259–263CrossRefPubMedGoogle Scholar
  13. 13.
    Reuter M, Berninger P, Chuma S et al (2011) Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing. Nature 480:264–267CrossRefPubMedGoogle Scholar
  14. 14.
    Kuramochi-Miyagawa S, Watanabe T, Gotoh K et al (2008) DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes. Genes Dev 22:908–917CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Ishizuka A, Siomi MC, Siomi H (2002) A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes Dev 16:2497–2508CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Hirano T, Iwasaki YW, Lin ZY-C et al (2014) Small RNA profiling and characterization of piRNA clusters in the adult testes of the common marmoset, a model primate. RNA 20:1223–1237CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Carmell MA, Xuan Z, Zhang MQ, Hannon GJ (2002) The Argonaute family: tentacles that reach into RNAi, developmental control, stem cell maintenance, and tumorigenesis. Genes Dev 16:2733–2742CrossRefPubMedGoogle Scholar
  18. 18.
    Swarts DC, Makarova K, Wang Y et al (2014) The evolutionary journey of Argonaute proteins. Nat Struct Mol Biol 21:743–753CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Aravin AA, van der Heijden GW, Castañeda J et al (2009) Cytoplasmic compartmentalization of the fetal piRNA pathway in mice. PLoS Genet 5:e1000764CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Takamasa Hirano
    • 1
    • 2
  • Hidetoshi Hasuwa
    • 1
  • Haruhiko Siomi
    • 1
  1. 1.Department of Molecular BiologyKeio University School of MedicineTokyoJapan
  2. 2.Division for Mammalian DevelopmentNational Institute of GeneticsMishimaJapan

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