Suppression of keratin 18 gene expression in bovine blastocysts by RNA interference
Karen Goossens A D , Dawit Tesfaye B , Franca Rings B , Karl Schellander B , Michael Hölker B , Mario Van Poucke A , Alex Van Zeveren A , Isabel Lemahieu C , Ann Van Soom C and Luc J. Peelman AA Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium.
B Institute of Animal Science, Animal Breeding and Husbandry Group, University of Bonn, Endenicher Allee 15, 53115 Bonn, Germany.
C Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium.
D Corresponding author. Email: karen.goossens@ugent.be
Reproduction, Fertility and Development 22(2) 395-404 https://doi.org/10.1071/RD09080
Submitted: 31 March 2009 Accepted: 13 July 2009 Published: 4 January 2010
Abstract
The expression of the cytoskeleton protein Keratin 18 (KRT18) starts at the onset of bovine blastocyst formation. KRT18 is solely expressed in the trophectoderm and can therefore be used as a marker for trophectodermal differentiation. In the present study, the expression of KRT18 was suppressed by RNA interference to probe its functional importance in bovine blastocyst formation. Microinjection of KRT18 double-stranded RNA into the cytoplasm of zygotes resulted in reduced KRT18 mRNA (76% reduction) and protein expression at the blastocyst stage and a lower developmental competence (41% reduction in the percentage of blastocyst formation) compared with non-injected and phosphate-buffered saline (PBS)-injected controls. KRT18 downregulation was associated with reduced mRNA expression of KRT8, the binding partner of KRT18, but had no effect on the expression of KRT19, CDH1 and DSP, other genes involved in intermediate filament and cytoskeleton formation. The results of the present study demonstrated that KRT18 knockdown in preimplantation embryos results in reduced blastocyst formation, but no further morphological aberrations were observed with regard to the biological function of KRT18. These observations could be due to the function of KRT18 being replaced by that of another gene, the surviving blastocysts expressing the minimum level of KRT18 required for normal blastocyst development or the possibility that further aberrations may occur later in development.
Acknowledgements
The authors thank Petra Van Damme and Dominique Vander Donckt en Linda Impe for their excellent technical assistance, Ruben Van Gansbeke for his help with the layout of the figures and sample collection. This work was supported by Ghent University, with additional research funds provided by the Department of Nutrition, Genetics and Ethology (Ghent University).
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