How to Study Hox Gene Expression and Function in Mammalian Oocytes and Early Embryos
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 wordsMammalian oocyte Early embryo RT-qPCR RNA silencing Whole-mount immuno-fluorescence
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.
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