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Hox Genes pp 19-36 | Cite as

How to Study Hox Gene Expression and Function in Mammalian Oocytes and Early Embryos

  • Delphine Paul
  • Caroline Sauvegarde
  • René Rezsohazy
  • Isabelle DonnayEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1196)

Abstract

Mammalian oocytes and early embryos have unique characteristics and can only be obtained in small amounts. As a consequence, the techniques to be used to characterize gene expression and function have to be adapted. It is also important to keep in mind that differences exist between mammalian species. Here we describe a set of techniques useful to analyze gene expression in oocytes and early bovine embryos, including techniques to quantify maternal and embryonic transcripts by RT-qPCR, to evaluate the translation potential of maternal transcripts, to knock down HOX transcripts by injection of siRNA, and to localize HOX proteins by whole-mount immunofluorescence.

Key words

Mammalian oocyte Early embryo RT-qPCR RNA silencing Whole-mount immuno-fluorescence 

Notes

Acknowledgements

We gratefully acknowledge the team of the professor Schellander from the University of Bonn and in particular Franca Rings and David Tesfaye for the training in microinjection and the fruitful discussions about RNAi. We gratefully acknowledge Karen Goossens from the Ghent University, Belgium and Rozenn Dalbiès-Tran from the INRA, Nouzilly, France for the fruitful discussions about qPCR normalization. The authors also deeply thank Nathalie Beaujean from INRA, France, Françoise Gofflot from UCL, Belgium, and Bernard Knoops from UCL, Belgium for their precious advices about the immunofluorescence. We also acknowledge Philippe Bombaerts, Raphael Chiarelli, Nathan Nguyen, Wendy Sonnet, Laure Bridoux, and Emmanuelle Ghys for their assistance in embryo production and Marie-Anne Mauclet for her help with administrative procedures.

References

  1. 1.
    Li L, Zheng P, Dean J (2010) Maternal control of early mouse development. Development 137:859–870PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Evsikov AV, Graber JH, Brockman JM et al (2006) Cracking the egg: molecular dynamics and evolutionary aspects of the transition from the fully grown oocyte to embryo. Genes Dev 20:2713–2727PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Paul D, Bridoux L, Rezsöhazy R et al (2011) HOX genes are expressed in bovine and mouse oocytes and early embryos. Mol Reprod Dev 78:436–449PubMedCrossRefGoogle Scholar
  4. 4.
    Kuijk EW, van Tol LT, Oei CH et al (2008) Differences in early lineage segregation between mammals. Dev Dyn 237:918–927PubMedCrossRefGoogle Scholar
  5. 5.
    Bilodeau-Goeseels S (2011) Cows are not mice: the role of cyclic AMP, phosphodiesterases, and adenosine monophosphate-activated protein kinase in the maintenance of meiotic arrest in bovines oocytes. Mol Reprod Dev 78:734–743PubMedCrossRefGoogle Scholar
  6. 6.
    Rossant J (2011) Developmental biology: a mouse is not a cow. Nature 471:457–458PubMedCrossRefGoogle Scholar
  7. 7.
    Favetta LA, Madan P, Mastromonaco GF et al (2007) The oxidative stress adaptor p66Shc is required for permanent embryo arrest in vitro. BMC Dev Biol 7:132PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Vandesompele J, De Preter K, Pattyn F et al (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3:7Google Scholar
  9. 9.
    Goossens K, Van Poucke M, Van Soom A et al (2005) Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos. BMC Dev Biol 5:7CrossRefGoogle Scholar
  10. 10.
    Ohnemus S, Bobola N, Kanzler B et al (2001) Different levels of Hoxa2 are required for particular developmental processes. Mech Dev 108:135–147PubMedCrossRefGoogle Scholar
  11. 11.
    Goossens K, Vandaele L, Wydooghe E et al (2011) The importance of adequate fixation for immunofluorescent staining of bovine embryos. Reprod Domest Anim 46:1098–1103PubMedCrossRefGoogle Scholar
  12. 12.
    Khan DR, Dube D, Gall L et al (2012) Expression of pluripotency master regulators during two key developmental transitions: EGA and early lineage specification in the bovine embryo. PloS One 7:e34110Google Scholar
  13. 13.
    Shi SR, Cote R, Taylor C (2001) Antigen retrieval techniques: current perspectives. J Mol Histol 49:931–937Google Scholar
  14. 14.
    Yamashita S (2007) Heat-induced antigen retrieval: mechanisms and application to histochemistry. J Histochem Cytochem 41:141–200CrossRefGoogle Scholar
  15. 15.
    Montero C (2003) The antigen–antibody reaction in immunohistochemistry. J Histochem Cytochem 51:1–4PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Delphine Paul
    • 1
  • Caroline Sauvegarde
    • 1
  • René Rezsohazy
    • 1
  • Isabelle Donnay
    • 1
    Email author
  1. 1.Institut des Sciences de la VieUniversité catholique de LouvainLouvain-la-NeuveBelgium

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