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PIWI-Interacting RNAs pp 13–23Cite as

Drosophila Germline Stem Cells for In Vitro Analyses of PIWI-Mediated RNAi

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1093))

Abstract

The Drosophila piwi gene has multiple functions in soma and germ cells. An in vitro system provides a powerful tool for elucidating PIWI function in each cell type using stable cell lines originating from germline stem cells (GSCs) and ovarian soma of adult ovaries. We have described methods for the maintenance and expansion of GSCs in an established cell line (fGS/OSS) and an in situ hybridization method for analyzing piwi.

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References

  1. Lin H, Spradling AC (1997) A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. Development 124:2463–2476

    CAS  PubMed  Google Scholar 

  2. Cox DN, Chao A, Baker J, Chang L, Qiao D, Lin H (1998) A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes Dev 12:3715–3727

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Cox DN, Chao A, Lin H (2000) piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells. Development 127:503–514

    CAS  PubMed  Google Scholar 

  4. Thomson T, Lin H (2009) The biogenesis and function of PIWI proteins and piRNAs: progress and prospect. Annu Rev Cell Dev Biol 25:355–376

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Saito K, Inagaki S, Mituyama T, Kawamura Y, Ono Y, Sakota E, Kotani H, Asai K, Siomi H, Siomi MC (2009) A regulatory circuit for piwi by the large Maf gene traffic jam in Drosophila. Nature 461:1296–1299

    Article  CAS  PubMed  Google Scholar 

  6. Megosh HB, Cox DN, Campbell C, Lin H (2006) The role of PIWI and the miRNA machinery in Drosophila germline determination. Curr Biol 16:1884–1894

    Article  CAS  PubMed  Google Scholar 

  7. Sarot E, Payen-Groschêne G, Bucheton A, Pélisson A (2004) Evidence for a piwi dependent RNA silencing of the gypsy endogenous retrovirus by the Drosophila melanogaster flamenco gene. Genetics 166:1313–1321

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Kalmykova AI, Klenov MS, Gvozdev VA (2005) Argonaute protein PIWI controls mobilization of retrotransposons in the Drosophila male germline. Nucleic Acids Res 33:2052–2059

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Vagin VV, Sigova A, Li C, Seitz H, Gvozdev V, Zamore PD (2006) A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313:320–324

    Article  CAS  PubMed  Google Scholar 

  10. Saito K, Nishida KM, Mori T, Kawamura Y, Miyoshi K, Nagami T, Siomi H, Siomi MC (2006) Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev 20:2214–2222

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Brennecke J, Aravin AA, Stark A, Dus M, Kellis M, Sachidanandam R, Hannon GJ (2007) Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128:1089–1103

    Article  CAS  PubMed  Google Scholar 

  12. Forbes AJ, Lin H, Ingham PW, Spradling AC (1996) hedgehog is required for the proliferation and specification of ovarian somatic cells prior to egg chamber formation in Drosophila. Development 122:1125–1135

    CAS  PubMed  Google Scholar 

  13. Decotto E, Spradling AC (2005) The Drosophila ovarian and testis stem cell niches: similar somatic stem cells and signals. Dev Cell 9:501–510

    Article  CAS  PubMed  Google Scholar 

  14. Morris LX, Spradling AC (2011) Long-term live imaging provides new insight into stem cell regulation and germline-soma coordination in the Drosophila ovary. Development 138:2207–2215

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Margolis J, Spradling A (1995) Identification and behavior of epithelial stem cells in the Drosophila ovary. Development 121:3797–3807

    CAS  PubMed  Google Scholar 

  16. Calvi BR, Lilly MA, Spradling AC (1998) Cell cycle control of chorion gene amplification. Gene Dev 12:734–744

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  17. Niki Y, Yamaguchi T, Mahowald AP (2006) Establishment of stable cell lines of Drosophila germ-line stem cells. Proc Natl Acad Sci U S A 103:16325–16330

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Niki Y (2009) Culturing ovarian somatic and germline stem cells of Drosophila. Curr Protoc Stem Cell Biol 10:2E.1.1–2E.1.9

    Google Scholar 

  19. Lau NC, Robine N, Martin R, Chung W-J, Niki Y, Berezikov E, Lai EC (2009) Abundant primary piRNAs, endo-siRNAs and microRNAs in a Drosophila ovary cell line. Genome Res 19:1776–1785

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Currie DA, Milner MJ, Evans CW (1988) The growth and differentiation in vitro leg and wing imaginal disc cells from Drosophila melanogaster. Development 102:805–814

    Google Scholar 

  21. Raap AK, Van de Rijke FM, Dirks RW, Sol CJ, Boom R, Van der Ploeg M (1991) Bicolor fluorescence in situ hybridization to intron- and exon mRNA sequences. Exp Cell Res 197:319–322

    Article  CAS  PubMed  Google Scholar 

  22. O’Neill JW, Bier E (1994) Double-label in situ hybridization using biotin and digoxigenin tagged RNA probes. Biotechniques 17:870–875

    PubMed  Google Scholar 

  23. Tautz D, Pfeiffle C (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98:81–85

    Article  CAS  PubMed  Google Scholar 

  24. Spletter ML, Liu J, Liu J, Su H, Giniger E, Komiyama T, Quake S, Luo L (2007) Lola regulates Drosophila olfactory projection neuron identity and targeting specificity. Neural Dev 2:14

    Article  PubMed Central  PubMed  Google Scholar 

  25. Groves A (1995) III. In situ hybridization on cultured cells. Anderson Lab in situ hybridization protocols. 7 Aug 2012. http://wmc.rodentia.com/docs/Big_In_Situ.html

    Google Scholar 

  26. Roche Applied Science (2008) V. RNA labeling by in vitro transcription of DNA with DIG, biotin, or fluorescein RNA labeling mix. DIG application manual for nonradioactive in situ hybridization, 4th edn. Roche Diagnostics GmbH, Germany, pp 49–52

    Google Scholar 

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Acknowledgment

This work was supported by Grant-in-Aid for Scientific Research (KAKENHI) on Innovative Areas, “Regulatory Mechanism of Gamete Stem Cells” (#24560219) to Y.N.

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Authors and Affiliations

  1. Department of Biology, Faculty of Science, Ibaraki University, Mito, Ibaraki, Japan

    Yuzo Niki, Takuya Sato, Takafumi Yamaguchi, Ayaka Saisho, Hiroshi Uetake & Hidenori Watanabe

Authors
  1. Yuzo Niki
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  2. Takuya Sato
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  3. Takafumi Yamaguchi
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  4. Ayaka Saisho
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  5. Hiroshi Uetake
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  6. Hidenori Watanabe
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Editor information

Editors and Affiliations

  1. The University of Tokyo Dept of Biophysics & Biochemistry, Tokyo, Japan

    Mikiko C. Siomi

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Niki, Y., Sato, T., Yamaguchi, T., Saisho, A., Uetake, H., Watanabe, H. (2014). Drosophila Germline Stem Cells for In Vitro Analyses of PIWI-Mediated RNAi. In: Siomi, M. (eds) PIWI-Interacting RNAs. Methods in Molecular Biology, vol 1093. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-694-8_2

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  • DOI: https://doi.org/10.1007/978-1-62703-694-8_2

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-693-1

  • Online ISBN: 978-1-62703-694-8

  • eBook Packages: Springer Protocols

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