Human Molecular Genetics Advance Access published online on September 27, 2007
Human Molecular Genetics, doi:10.1093/hmg/ddm280
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Manipulations of mouse embryos prior to implantation result in aberrant expression of imprinted genes on day 9.5 of development
1 Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA 3 Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
* To whom correspondence should be addressed: Marisa Bartolomei Tel: +1 2158989063; Fax: +1 2155735434; Email: bartolom{at}mail.med.upenn.edu and Richard Schultz Tel: +1 2158987869; Fax: +1 2158988780; Email: rschultz{at}sas.upenn.edu
Received July 31, 2007; Revised September 20, 2007; Accepted September 20, 2007
In vitro culture of mouse embryos results in loss-of-imprinting. The aim of the present study was to examine how two of the techniques commonly used during assisted reproduction, namely embryo culture and embryo transfer, affect genomic imprinting after implantation in the mouse. F1 hybrid mouse embryos were subjected to three experimental conditions: control (unmanipulated), embryo transfer, and in vitro-culture followed by embryo transfer. Concepti were collected on d9.5 of development and allelic expression determination of ten imprinted genes (H19, Snrpn, Igf2, Kcnq1ot1, Cdkn1c, Kcnq1, Mknr3, Ascl2, Zim1, Peg3) was performed. Although control concepti had monoallelic imprinted gene expression in all tissues, both manipulated groups had aberrant expression of one or more imprinted genes in the yolk sac and placenta. Culture further exacerbated the effects of transfer by increasing the number of genes with aberrant allelic expression in extraembryonic, as well as embryonic tissues. Additionally, placentae of both groups of manipulated concepti exhibited reduced levels of Igf2 mRNA and increased levels of Ascl2 mRNA when compared to their unmanipulated counterparts. Furthermore, we show that biallelic expression of Kcnq1ot1 coincided with loss of methylation on the maternal allele of the KvDMR1 locus, a phenotype often associated with the human syndrome Beckwith-Wiedemann. In conclusion, our results show that even the most basic manipulation used during human assisted reproduction, namely, embryo transfer, can lead to misexpression of several imprinted genes during post-implantation development. Additionally, our results serve as a cautionary tale for gene expression studies in which embryo transfer is used.
2 Current address: Animal Science Research Center, Division of Animal Sciences, University of Missouri-Columbia, Columbia, MO
4 Current address: Department of Veterinary and Animal Sciences, University of Massachusetts-Amherst, Amherst, MA
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