Abstract
Fluorescence in situ hybridization (FISH) is a molecular cytogenetic technique. It identifies the location of DNA loci and RNAs, including nascent RNAs in the process of being transcribed, within individual cells. Great advances in fluorescent dye technology and technique sensitivity, combined with developments in light microscopy and imaging software have made it widely accessible and have expanded the range of applications in basic research as well as in diagnostics. Being able to perform RNA hybridization, DNA hybridization, and protein immunofluorescence consecutively on the same sample is an invaluable tool to study RNA expression in relation to their gene loci and to map RNA and DNA in relation to nuclear or cellular structures. This has contributed to enormous progress in understanding basal mechanisms of male and female meiosis in different animal model systems. In this chapter we describe in detail the protocols for FISH based techniques applied to study gene expression dynamics and nuclear architecture of chicken oocytes during meiotic prophase I. These techniques can be easily performed in any molecular and cell biology laboratory and be adapted to different systems and to different phases of gametogenesis.
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References
Gall JG, Pardue ML (1969) Formation and detection of RNA-DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci U S A 63(2):378–383
Rudkin GT, Stollar BD (1977) High resolution detection of DNA-RNA hybrids in situ by indirect immunofluorescence. Nature 265(5593):472–473
Baker M (2012) RNA imaging in situ. Nat Methods 9:787–790
Hu L, Ru K, Zhang L, Huang Y, Zhu X, Liu H, Zetterberg A, Cheng T, Miao W (2014) Fluorescence in situ hybridization (FISH): an increasingly demanded tool for biomarker research and personalized medicine. Biomark Res 2(1):3. doi:10.1186/2050-7771-2-3
Lichter P, Cremer T, Borden J, Manuelidis L, Ward DC (1988) Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum Genet 80(3):224–234
Speicher MR, Gwyn Ballard S, Ward DC (1996) Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 12(4):368–375. doi:10.1038/ng0496-368
Baarends WM, Wassenaar E, van der Laan R, Hoogerbrugge J, Sleddens-Linkels E, Hoeijmakers JH, de Boer P, Grootegoed JA (2005) Silencing of unpaired chromatin and histone H2A ubiquitination in mammalian meiosis. Mol Cell Biol 25(3):1041–1053. doi:10.1128/MCB.25.3.1041-1053.2005
Guioli S, Lovell-Badge R, Turner JM (2012) Error-prone ZW pairing and no evidence for meiotic sex chromosome inactivation in the chicken germ line. PLoS Genet 8(3):e1002560. doi:10.1371/journal.pgen.1002560
Turner JM, Mahadevaiah SK, Ellis PJ, Mitchell MJ, Burgoyne PS (2006) Pachytene asynapsis drives meiotic sex chromosome inactivation and leads to substantial postmeiotic repression in spermatids. Dev Cell 10(4):521–529. doi:10.1016/j.devcel.2006.02.009
Turner JM, Mahadevaiah SK, Fernandez-Capetillo O, Nussenzweig A, Xu X, Deng CX, Burgoyne PS (2005) Silencing of unsynapsed meiotic chromosomes in the mouse. Nat Genet 37(1):41–47. doi:10.1038/ng1484
Gaginskaya E, Kulikova T, Krasikova A (2009) Avian lampbrush chromosomes: a powerful tool for exploration of genome expression. Cytogenet Genome Res 124(3–4):251–267. doi:10.1159/000218130
Handyside AH (2012) Molecular origin of female meiotic aneuploidies. Biochim Biophys Acta 1822(12):1913–1920. doi:10.1016/j.bbadis.2012.07.007
Mahadevaiah SK, Costa Y, Turner JM (2009) Using RNA FISH to study gene expression during mammalian meiosis. Methods Mol Biol 558:433–444. doi:10.1007/978-1-60761-103-5_25
Griffin DK, Haberman F, Masabanda J, O’Brien P, Bagga M, Sazanov A, Smith J, Burt DW, Ferguson-Smith M, Wienberg J (1999) Micro- and macrochromosome paints generated by flow cytometry and microdissection: tools for mapping the chicken genome. Cytogenet Cell Genet 87(3–4):278–281
Acknowledgments
We wish to thank Dr. J. Turner and Dr. S. Mahadevaiah for their contribution to developing the methods to study chicken oogenesis, by sharing their expertise in mouse meiosis, their protocols and reagents; Dr. S. Samson for critical reading of the manuscript.
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Guioli, S., Lovell-Badge, R. (2016). RNA FISH, DNA FISH and Chromosome Painting of Chicken Oocytes. In: Nezis, I. (eds) Oogenesis. Methods in Molecular Biology, vol 1457. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3795-0_14
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DOI: https://doi.org/10.1007/978-1-4939-3795-0_14
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