Retrovirus genomes introduced into mouse zygotes by microinjection of cloned DNA, or into morula stage pre-implantation mouse embryos by infection with Moloney murine leukaemia virus (M-MuLV), became de novo methylated and were blocked in expression. No restriction of virus expression and no de novo methylation were observed when post-implantation mouse embryos were infected with virus. Efficient de novo methylation activity may be an important characteristic of gene regulation in early mouse embryos.

We have investigated the block to expression of Moloney murine leukemia virus in murine embryonal carcinoma (EC) cells. Infected EC cells were found to contain up to 100 integrated proviral genomes. However, expression of virus as measured by XC plaque and virus-specific RNA synthesis did not occur at significant levels, in contrast to productively infected differentiated cells. Analysis of the DNA in the infected EC cells revealed that the proviral genomes were highly methylated, as shown by their resistance to cleavage by Sma I. Integrated proviral genomes in infected differentiated cells were readily cut by Sma I and thus were not methylated at these sites. Transfection ofDNA from infected EC cells to cells permissive for virus expression failed to induce virus expression. The proviral genomes, however, were potentially infectious because they induced XC plaques when the recipient cells for transfection were treated with 5-azacytidine. This drug is believed to interfere with DNA methylation. We conclude that expression ofproviral genomes introduced into EC cells is suppressed and that this inactivation can be correlated with the de novo methylation of the viral DNA. De novo methylation activity thus may be a characteristic of early embryonic cells.In eukaryotes, methylation of cytosine residues at the 5' position has been extensively studied as a possible mechanism involved in the control ofgene expression and these studies have shown that an inverse correlation exists between gene expression and the level of methylation (1-3).As a means to study gene expression in mammals, we have introduced the murine retrovirus Moloney murine leukemia virus (M-MuLV) into the germ line of mice. A number of substrains, each carrying one M-MuLV genome at a different chromosomal position, have been derived and the expression of the M-MuLV genome has been shown to differ between some of them (4). A previous report (5) showed that the germ line-transmitted copy of the viral genome was methylated and noninfectious, whereas the somatically acquired copies, derived from the germ-line copy, were hypomethylated and infectious. Furthermore, cloning of the germ-line gene, which removes the methyl group from the cytosine bases, rendered the germ-line copy infectious (6). Because the proviral DNA introduced into the mouse embryos to derive the Mov-substrains was nonmethylated, de novo methylation of the integrated proviral genome must have occurred at some point during derivation ofthe substrains. This may have occurred either during the development of the infected embryo or as a consequence of transmitting the proviral genome through the germ line to the next generation.To (20) were used for virus production. All cells were grown as described (21) in Dulbecco's modified Eagle's medium with 10% fetal calf serum (heat-inactivated at 56WC for 30 min before use).Infection of the Cells with M-MuLV. Two protocols were used to infect cells with M-MuLV. (i) F9 EC cells were infected by cocultivation with mitomycin C-treated (10 ,g/ml ...

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