Abstract
Transposable elements (TEs) constitute a large proportion of the genome in multiple organisms. Therefore, anti-transposable element machineries are essential to maintain genomic integrity. PIWI-interacting RNAs (piRNAs) are a major force to repress TEs in Drosophila ovaries. Ovarian somatic cells (OSC), in which nuclear piRNA regulation is functional, have been used for research on piRNA pathway as a cell culture system to elucidate the molecular mechanisms underlying the piRNA pathway. Analysis of piRNA pathway using a reporter system to monitor the gene regulation or overexpression of specific genes would be a powerful approach. Here, we present the technical protocol to establish stable cell lines using the piggyBac system, adopted for OSCs. This easy, consistent, and timesaving protocol may accelerate research on the piRNA pathway.
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References
Ozata DM, Gainetdinov I, Zoch A, O'Carroll D, Zamore PD (2019) PIWI-interacting RNAs: small RNAs with big functions. Nat Rev Genet 20(2):89–108. https://doi.org/10.1038/s41576-018-0073-3
Iwasaki YW, Siomi MC, Siomi H (2015) PIWI-interacting RNA: its biogenesis and functions. Annu Rev Biochem 84:405–433. https://doi.org/10.1146/annurev-biochem-060614-034258
Senti KA, Brennecke J (2010) The piRNA pathway: a fly's perspective on the guardian of the genome. Trends Genet 26(12):499–509. https://doi.org/10.1016/j.tig.2010.08.007
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(44):16325–16330. https://doi.org/10.1073/pnas.0607435103
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(7268):1296–1299. https://doi.org/10.1038/nature08501
Saito K (2014) RNAi and overexpression of genes in ovarian somatic cells. Methods Mol Biol 1093:25–33. https://doi.org/10.1007/978-1-62703-694-8_3
Murano K, Iwasaki YW, Ishizu H, Mashiko A, Shibuya A, Kondo S, Adachi S, Suzuki S, Saito K, Natsume T, Siomi MC, Siomi H (2019) Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing. EMBO J 38(17):e102870. https://doi.org/10.15252/embj.2019102870
Cary LC, Goebel M, Corsaro BG, Wang HG, Rosen E, Fraser MJ (1989) Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses. Virology 172(1):156–169. https://doi.org/10.1016/0042-6822(89)90117-7
Fraser MJ, Cary L, Boonvisudhi K, Wang HG (1995) Assay for movement of lepidopteran transposon IFP2 in insect cells using a baculovirus genome as a target DNA. Virology 211(2):397–407. https://doi.org/10.1006/viro.1995.1422
Chen K, Birkinshaw RW, Gurzau AD, Wanigasuriya I, Wang R, Iminitoff M, Sandow JJ, Young SN, Hennessy PJ, Willson TA, Heckmann DA, Webb AI, Blewitt ME, Czabotar PE, Murphy JM (2020) Crystal structure of the hinge domain of Smchd1 reveals its dimerization mode and nucleic acid-binding residues. Sci Signal 13(636):eaaz5599. https://doi.org/10.1126/scisignal.aaz5599
Ding S, Wu X, Li G, Han M, Zhuang Y, Xu T (2005) Efficient transposition of the piggyBac (PB) transposon in mammalian cells and mice. Cell 122(3):473–483. https://doi.org/10.1016/j.cell.2005.07.013
Yusa K, Zhou L, Li MA, Bradley A, Craig NL (2011) A hyperactive piggyBac transposase for mammalian applications. Proc Natl Acad Sci U S A 108(4):1531–1536. https://doi.org/10.1073/pnas.1008322108
Ishizu H, Sumiyoshi T, Siomi MC (2017) Use of the CRISPR-Cas9 system for genome editing in cultured drosophila ovarian somatic cells. Methods 126:186–192. https://doi.org/10.1016/j.ymeth.2017.05.021
Batki J, Schnabl J, Wang J, Handler D, Andreev VI, Stieger CE, Novatchkova M, Lampersberger L, Kauneckaite K, Xie W, Mechtler K, Patel DJ, Brennecke J (2019) The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation. Nat Struct Mol Biol 26(8):720–731. https://doi.org/10.1038/s41594-019-0270-6
Ishii T, Ishikawa M, Fujimori K, Maeda T, Kushima I, Arioka Y, Mori D, Nakatake Y, Yamagata B, Nio S, Kato TA, Yang N, Wernig M, Kanba S, Mimura M, Ozaki N, Okano H (2019) In vitro modeling of the bipolar disorder and schizophrenia using patient-derived induced pluripotent stem cells with copy number variations of PCDH15 and RELN. eNeuro 6(5):ENEURO.0403-0418. https://doi.org/10.1523/eneuro.0403-18.2019
Acknowledgments
We thank Dr. Haruhiko Siomi for the critical reading of the manuscript. OSC_Reporter_UAS_traffic jam_BoxB_d2eGFP_t2a_Blast, used for construction of donor plasmid, was a kind gift from Dr. Julius Brennecke (Addgene plasmid # 128010; http://n2t.net/addgene:128010; RRID:Addgene_128010). YWI is supported by funding from JSPS KAKENHI Grant Numbers 22H02547, 21H00259 and 18H02421, JST PRESTO Grant Number JPMJPR20E2. KM is supported by funding from JSPS KAKENHI Grant Number 20H03439.
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Takeuchi, C., Murano, K., Ishikawa, M., Okano, H., Iwasaki, Y.W. (2022). Generation of Stable Drosophila Ovarian Somatic Cell Lines Using the piggyBac System. In: Parrish, N.F., Iwasaki, Y.W. (eds) piRNA. Methods in Molecular Biology, vol 2509. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2380-0_9
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DOI: https://doi.org/10.1007/978-1-0716-2380-0_9
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