Abstract
During mouse meiosis, gene expression and homologous synapsis are intimately linked. Chromosomes that fail to synapse at the zygotene–pachytene transition become transcriptionally silenced by a process called Meiotic Silencing of Unsynapsed Chromatin (MSUC), and this silencing (or defects in it) may in turn cause germ cell losses and infertility. Gene transcription at the chromosomal level can be readily observed using RNA fluorescence in-situ hybridisation (FISH), and this approach allows one to directly compare expression at a specific locus with the synaptic status of the chromosome domain on which it resides. Here we describe a protocol for carrying out RNA FISH on male meiotic cells, together with detail on the important controls and common problems associated with this technique.
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References
Turner, J.M. (2007). Meiotic sex chromosome inactivation. Development 134, 1823–1831.
Handel, M.A. (2004). The XY body: a specialized meiotic chromatin domain. Exp. Cell Res. 296, 57–63.
Turner, J.M., Mahadevaiah, S.K., Ellis, P.J., Mitchell, M.J., and Burgoyne P.S. (2006) Pachytene asynapsis drives meiotic sex chromosome inactivation and leads to substantial postmeiotic repression in spermatids. Dev. Cell 10, 521–529.
Turner, J.M., Mahadevaiah, S.K., Fernandez-Capetillo, O., Nussenzweig, A., Xu, X., Deng, C.X., and Burgoyne, P.S. (2005) Silencing of unsynapsed meiotic chromosomes in the mouse. Nat. Genet. 37, 41–47.
Baarends, W.M., Wassenaar, E., van der Laan, R., Hoogerbrugge, J., Sleddens-Linkels, E., Hoeijmakers, J.H., de Boer, P., and Grootegoed, J.A. (2005) Silencing of unpaired chromatin and histone H2A ubiquitination in mammalian meiosis. Mol. Cell Biol. 25, 1041–1053.
Schimenti, J. (2005) Synapsis or silence. Nat. Genet. 37, 11–13.
van Raamsdonk, C.D., and Tilghman, S.M. (2001) Optimizing the detection of nascent transcripts by RNA fluorescence in situ hybridization. Nucleic Acids Res. 29, E42.
Clemson C.M., Hall L.L., Byron M., McNeil J., and Lawrence J.B. (2006) The X chromosome is organized into a gene-rich outer rim and an internal core containing silenced nongenic sequences. Proc. Natl. Acad. Sci.USA 103, 7688–7693.
Chaumeil, J., Okamoto, I., Heard, E. (2004) X-chromosome inactivation in mouse embryonic stem cells: analysis of histone modifications and transcriptional activity using immunofluorescence and FISH. Methods Enzymol. 376, 405–419.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Mahadevaiah, S.K., Costa, Y., Turner, J.M.A. (2009). Using RNA FISH to Study Gene Expression During Mammalian Meiosis. In: Keeney, S. (eds) Meiosis. Methods in Molecular Biology, vol 558. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-103-5_25
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DOI: https://doi.org/10.1007/978-1-60761-103-5_25
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-102-8
Online ISBN: 978-1-60761-103-5
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