Published online 5 March 2004
Nucleic Acids Research, 2004, Vol. 32, No. 4 1566-1576
© 2004 Oxford University Press
Influence of in vitro manipulation on the stability of methylation patterns in the Snurf/Snrpn-imprinting region in mouse embryonic stem cells
Institute of Genetics, University of Cologne, Weyertal 121, D-50931 Cologne, Germany
*To whom correspondence should be addressed at present address: Centre for Addiction and Mental Health, The Krembil Epigenetics Laboratory, 250 College Street, Toronto, Ontario, M5T 1R8, Canada. Tel: +1 416 5358501 4809; Fax: +1 416 979 4666; Email: axel_schumacher{at}camh.net
Recent work on embryonic stem (ES) cells showed that stem cell-derived tissues and embryos, cloned from ES cell nuclei, often fail to maintain epigenetic states of imprinted genes. This deregulation is frequently associated with in vitro manipulations and culture conditions which might affect the cells potential to develop into normal fetuses. Usually, epigenetic instability is reported in differentially methylated regions of mostly growth-related imprinted genes. However, little is known about the epigenetic stability of genes that function late in organogenesis. Hence, we set out to investigate the epigenetic stability of neuronal genes and analyzed DNA methylation patterns in the Snurf/Snrpn imprinted cluster in several cultured mouse ES cell lines. We also determined the effects of in vitro stress factors such as consecutive passaging, trypsination, mechanical handling, single cell cloning, centrifugation, staurosporine-induced neurogenesis and the insertion of viral (foreign) DNA into the host genome. Intriguingly, none of these in vitro manipulations interfered with the stability of the methylation patterns in the analyzed neuronal genes. These data imply that, in contrast to growth-related genes like Igf2, H19, Igf2r or Grb10, the methylation imprints of the analyzed neuronal genes in the Snurf/Snrpn cluster may be particularly stable in manipulated ES cells.
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