Abstract
A common problem in research laboratories that study the mammalian embryo after nuclear transfer is the limited supply of material. For this reason, new methods are continually developed, and existing methods for cells in culture are adapted to suit this peculiar experimental model. Among them is the fluorescent immunodetection.
Fluorescent immuno-detection on fixed embryos is an invaluable technique to detect and locate proteins, especially nuclear ones such as modified histones, in single embryos thanks to its specificity and its sensitivity. Moreover, with specific fixation procedures that preserve the 3D shape of the embryos, immunostaining can now be performed on whole-mount embryos. Target proteins are detected by specific binding of first antibody usually nonfluorescent, and revealed with a second antibody conjugated with a fluorochrome directed specifically against the host animal in which the first antibody was produced. The result can then be observed on a microscope equipped with fluorescent detection. Here, we describe the 3D fluorescent immunodetection of epigenetic modifications in mouse embryos. This procedure can be used on nuclear transferred embryos but also on in vivo-collected, in vitro-developed and in vitro-fertilized ones.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Maalouf WE, Liu Z, Brochard V et al (2009) Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term. BMC Dev Biol 9:11
Le Bourhis D, Beaujean N, Ruffini S et al (2010) Nuclear remodeling in bovine somatic cell nuclear transfer embryos using MG132-treated recipient oocytes. Cell Reprogram 12: 729–738
Liu Z, Wan H, Wang E et al (2012) Induced pluripotent stem-induced cells show better constitutive heterochromatin remodeling and developmental potential after nuclear transfer than their parental cells. Stem Cells Dev 21: 3001–3009
Yang CX, Liu Z, Fleurot R et al (2013) Heterochromatin reprogramming in rabbit embryos after fertilization, intra-, and inter-species SCNT correlates with preimplantation development. Reproduction 145:149–159
Schneider R, Grosschedl R (2007) Dynamics and interplay of nuclear architecture, genome organization, and gene expression. Genes Dev 21:3027–3043
Bernstein BE, Meissner A, Lander ES (2007) The mammalian epigenome. Cell 128:669–681
Mason K, Liu Z, Aguirre-Lavin T et al (2012) Chromatin and epigenetic modifications during early mammalian. Anim Reprod Sci 134: 45–55
Aguirre-Lavin T, Adenot P, Bonnet-Garnier A et al (2012) 3D-FISH analysis of embryonic nuclei in mouse highlights several abrupt changes of nuclear organization during preimplantation development. BMC Dev Biol 12:30
Andrey P, Kiêu K, Kress C et al (2010) Statistical analysis of 3D images detects regular spatial distributions of centromeres and chromocenters in animal and plant nuclei. PLoS Comput Biol 6:e1000853
Beaujean N, Taylor J, Gardner J et al (2004) Effect of limited DNA methylation reprogramming in the normal sheep embryo on somatic cell nuclear transfer. Biol Reprod 71:185–193
Yang J, Yang S, Beaujean N et al (2007) Epigenetic marks in cloned rhesus monkey embryos: comparison with counterparts produced in vitro. Biol Reprod 76:36–42
Pichugin A, Le Bourhis D, Adenot P et al (2010) Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos. Reproduction 139:129–137
Liu Z, Hai T, Dai X et al (2012) Early patterning of cloned mouse embryos contributes to post-implantation development. Dev Biol 368: 304–311
Salvaing J, Aguirre-Lavin T, Boulesteix C et al (2012) 5-Methylcytosine and 5-hydroxymethylcytosine spatiotemporal profiles in the mouse zygote. PLoS One 7:e38156
Acknowledgement
All the present and past members from the lab should be acknowledged for their hard work, especially Lydia Ruddick and Bénédicte Sanseau who corrected this book chapter. We are also grateful to Pierre Adenot and Renaud Fleurot for Confocal and Apotome microscopy on the MIMA2 platform (Microscopie et Imagerie des Microorganismes, Animaux et aliments). Work in the lab is supported by the REVIVE Labex.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Boulesteix, C., Beaujean, N. (2015). Fluorescent Immunodetection of Epigenetic Modifications on Preimplantation Mouse Embryos. In: Beaujean, N., Jammes, H., Jouneau, A. (eds) Nuclear Reprogramming. Methods in Molecular Biology, vol 1222. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1594-1_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-1594-1_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-1593-4
Online ISBN: 978-1-4939-1594-1
eBook Packages: Springer Protocols