The mammalian genome undergoes significant changes in its cytosine methylation content during development. The majority of genomic CpG dinucleotides are methylated in differentiated cells of the germ and somatic lineages. In contrast, genomes of primordial germ cells (PGCs) and preimplantation embryos are relatively poorly methylated. The purpose of these rearrangements in genomic methylation are not known, although they are probably part of a more general process of epigenetic reprogramming that is associated with the generation of germline and embryo stem cells. The increases in methylation during gametogenesis and post-implantation embryogenesis are mediated by the combined actions of de novo methyltransferases and the DNMT1 maintenance methyltransferase. It is less clear how the reductions in methylation in PGCs and preimplantation embryos are regulated, although it is likely that DNMT1 plays a major role. To study the roles of DNMT1 in the developmental changes in genomic methylation, we generated the homozygous mutant Dnmt1tet/tet ES cell line using standard techniques in targeted mutagenesis. In the absence of doxycycline (DOX), Dnmt1tet/tet cells express a higher level of DNMT1 due to the strong effect of the tTA transactivator protein. In the presence of 2μg/ml DOX, DNMT1 levels fall to undetectable levels in 72 hours. Upon the removal of DOX, DNMT1 levels rise to pretreatment levels. Genomic methylation falls and rises in response to the addition and removal of DOX, respectively. Methylation on differentially methylated domains (DMDs) of imprinted genes is permanently lost with the DOX-induced fall in global (DMD and non-DMD) methylation, whereas non-DMD methylation rapidly returns to pre-treatment levels upon removal of DOX and re-expression of DNMT1. These observations are consistent with a specific germline requirement of de novo methyltransferase activity to establish DMD methylation. The DOX-induced loss of global methylation is prevented by expression of wild-type DNMT1 protein from an exogenous expression plasmid. In contrast, the expression of certain mutant DNMT1 proteins in Dnmt1tet/tet ES cells failed to prevent the DOX-induced loss of DMD and/or non-DMD methylation. Based on these observations, we conclude that the Dnmt1tet/tet ES cells can be used as a tool to study the specific roles of DNMT1 in the maintenance of methylation on different categories of DMD sequences. Research supported by NIH HD044133 to JRC. (poster) Copyright 2009 by The Society for the Study of Reproduction. Copyright 2009 by The Society for the Study of Reproduction.