Abstract
Transcriptional and epigenetic dynamics of the genome occur during early development in mammals. It has been difficult to study these dynamics due to the limitation of materials and the difficulty of handling. In this chapter, we describe our attempt to apply a combination of immunofluorescence (IF), and RNA and DNA fluorescent in situ hybridization (FISH) in preimplantation mouse embryos. We have concentrated on refining these techniques to study the dynamics of X chromosome inactivation in mouse embryos. The techniques and general underlying principles described here should be applicable to other mammals of interest.
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References
Heard E, Disteche CM (2006) Dosage compensation in mammals: fine-tuning the expression of the X chromosome. Genes Dev 206:1848–1867. https://doi.org/10.1101/gad.1422906.1848
Chaumeil J, Le Baccon P, Wutz A, Heard E (2006) A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced. Genes Dev 20(16):2223–2237. https://doi.org/10.1101/gad.380906
Chaumeil J, Okamoto I, Guggiari M, Heard E (2002) Integrated kinetics of X chromosome inactivation in differentiating embryonic stem cells. Cytogenet Genome Res 99(1-4):75–84. https://doi.org/10.1159/000071577
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. https://doi.org/10.1016/S0076-6879(03)76027-3
Chaumeil J, Augui S, Chow JC, Heard E (2008) Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation. Methods Mol Biol 463:297–308
Okamoto I, Otte AP, Allis CD, Reinberg D, Heard E (2004) Epigenetic dynamics of imprinted X inactivation during early mouse development. Science 303(5658):644–649. https://doi.org/10.1126/science.1092727
Okamoto I, Arnaud D, Le Baccon P et al (2005) Evidence for de novo imprinted X-chromosome inactivation independent of meiotic inactivation in mice. Nature 438(7066):369–373. https://doi.org/10.1038/nature04155
Patrat C, Okamoto I, Diabangouaya P et al (2009) Dynamic changes in paternal X-chromosome activity during imprinted X-chromosome inactivation in mice. Proc Natl Acad Sci U S A 106(13):5198–5203
Nora EP, Lajoie BR, Schulz EG et al (2013) Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485(7398):381–385. https://doi.org/10.1038/nature11049
Okamoto I, Patrat C, Thépot D et al (2011) Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development. Nature 472(7343):370–374. https://doi.org/10.1038/nature10184
Hogan BL, Beddington R, Costantini F, Facy E (1994) Manipulating the mouse embryo. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Lawrence JB, Singer RH (1985) Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucleic Acids Res 13(5):1777–1799
Ranisavljevic N, Okamoto I, Heard E, Ancelin K (2017) RNA FISH to study zygotic genome activation in early mouse embryos. Methods Mol Biol 1605:103–124
Acknowledgments
We thank E. Heard for critical reading of the manuscript.
This work was supported by funds from MEXT and JST-ERATO and by JSPS KAKENHI Grant Number JP25291076 to I.O.
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Okamoto, I. (2018). Combined Immunofluorescence, RNA FISH, and DNA FISH in Preimplantation Mouse Embryos. In: Sado, T. (eds) X-Chromosome Inactivation. Methods in Molecular Biology, vol 1861. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8766-5_12
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DOI: https://doi.org/10.1007/978-1-4939-8766-5_12
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