In Vitro Methylation of Specific Regions in Recombinant DNA Constructs by Excision and Religation

Abstract

The first imprinted genes were identified in the early 1990s (e.g., refs. 1 1,2) and there are now over 40 mammalian genes known to be regulated by genomic imprinting (for an up-to-date list, see ref. 3). The details of the mechanism that discriminates between the active and silent alleles of these genes, based on their parent of origin, may differ from one imprinted gene to the next, but must include some form of epigenetic mark that distinguishes alleles that have passed through the male or female germline (4-7). The addition of methyl groups to cytosine residues of CpG dinucleotides might provide such a mark, since regions of differential methylation have been identified in the vicinity of many of the known imprinted genes (8,9). Moreover, analysis of imprinted gene expression in a methyltransferase knockout (Dnmt1 ^-/-) mouse has shown that the imprint is lost in a number of cases, resulting in either two silent alleles (Igf2, Igf2r and Kvlqt) or two expressed alleles (H19, p57 ^kip2, Snrpn, and Xist) (4,10–12). Although there may be exceptions (for instance, imprinted expression of Mash2 is maintained in Dmnt1 ^-/- embryos; ref. 13), differential methylation is likely to be an important aspect of the imprinting mechanism that is relevant to most of the imprinted genes in mammals. There is accumulating evidence that methylation is also important for the imprinting of plant genes (reviewed in ref. 14).