A construct consisting of the E2A late promoter of Ad2 DNA and the CAT indicator gene was integrated in the non-methylated or in the 5´-CCGG-3´ premethylated form into the genome of mice, and the state of methylation was analyzed by HpaII cleavage of DNA from various organs of the transgenic animals [125]. In general, the transgenic construct remained stably integrated. In the founder animal, the non-methylated construct became de novo methylated at all or at most of the 5´-CCGG-3´ sites. Pre-imposed methylation patterns were stable for up to four generations beyond the founder animal. However, in the DNA from testes of two founder animals and two F1-males, the premethylated, transgenic DNA was demethylated by an unknown mechanism. In all other organs, the transgenic DNA preserved the pre-imposed 5´-CCGG-3´ methylation patterns. Differences in these transmission modes were not seen depending on whether the transgene was inherited maternally or paternally [125]. There are studies to support the notion that genetic background in mice can have a decisive influence on the type of de novo methylation patterns imposed on a foreign DNA transgene and on their stability [126-128]. The molecular mechanisms involved in the “modifier gene” effects are not understood. We addressed this problem by introducing into the genomes of different mouse strains (DBA/2, 129/sv FVB/N or C57BL/6, CB20 or Balb/c) a construct, which consisted of the E2A late promoter of Ad2 DNA and the chloramphenicol acetyltransferase (CAT) gene as reporter. The patterns of de novo transgene methylation were transmitted to the offspring and remained stable for 11 backcross generations, regardless of the heterozygosity in the recipient mouse strain and the presence of presumptive modifier genes. In seven additional mouse strains carrying the same transgene in different chromosomal locations, strain-specific alterations of methylation patterns were not observed [129].
We also investigated the stability of DNA methylation patterns in the Snurf/Snrpn imprinted gene cluster in mouse embryonal stem cell lines cultured under different experimental conditions, like prolonged passaging, trypsinization, mechanical handling, single cell cloning, staurosporin-induced neurogenesis [130] or the insertion of foreign (viral) DNA into the ES cell genome. None of these in vitro manipulations affected the stability of the methylation patterns in the analyzed gene cluster [131]. Growth-related genes, IGF2, H19, IGF2R, or GRB10, are known to respond by altered imprint patterns. The analyzed neuronal gene cluster, however, exhibited stable patterns of DNA methylation under the experimental conditions chosen.
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