mono allelic expression at the mouse igf2 h19 locus is controlled by differential allelic dna methylation of the imprinting control region icr because a randomly integrated h19 icr fragment when incorporated into the genome of transgenic mice tgm was allele specifically methylated in somatic but not in germ cells it was suggested that allele discriminating epigenetic signature set within or somewhere outside of the tg h19 icr fragment in germ cells was later translated into a differential dna methylation pattern to test if the chicken β globin hs4 chs4 chromatin insulator might interfere with methylation imprinting establishment at the h19 icr we inserted the h19 icr fragment flanked by a set of floxed chs4 core sequences into a human β globin locus yac and generated tgm insulated icr tgm as controls the chs4 sequences were removed from one side 5 hs4 deleted icr or both sides pseudo wt icr of the insulated icr by in vivo cre loxp recombination the data show that while maternally inherited transgenic h19 icr was not methylated in insulated icr tgm it was significantly methylated upon paternal transmission though the level was lower than in the pseudo wt icr control because this reduced level of methylation was also observed in the 5 hs4 deleted icr tgm we speculate that the phenotype is due to vezf1 dependent demethylation activity rather than the insulator function borne in chs4 collectively although we cannot rule out the possibility that chs4 is incapable of blocking an allele discriminating signal from outside of the transgene the epigenetic signature appears to be marked intrinsically within the h19 icr.