Timing of establishment of paternal methylation imprints in the mouse
Section snippets
Methylation of H19 and Snrpn in the male germ line
We isolated germ cell populations of greater than 90% purity from e10.5 [21], e12.5, and e17.5 gonads [20] or from cauda epididymis [22] as previously described. DNA was prepared as described under Materials and methods and samples were treated with bisulfite before PCR was carried out on the target genes. The structure of the DMR and their locations relative to the transcriptional start sites of the genes are depicted in Fig. 1. The CpG’s assayed in each DMR are indicated below the cognate DMR
Discussion
The Rasgrf1 DMR is located 30 kb upstream of the body of the gene, immediately 5′ to a cluster of unique 40-bp repeats [8]. In contrast to H19, methylation of the Rasgrf1 DMR is required for expression of the paternal allele rather than its repression. Deletion of the adjacent repeats leads to a variable loss of DMR methylation and silencing of the gene to different extents [12]. These results suggested that the DMR and associated repeats may form part of the germ-line imprint for this gene [12]
Mice
Hsd:ICR(CD-1), C57BL/6, and CBA mice were purchased from Harlan UK Ltd. (Oxon, England). Natural matings were used to produce embryos for the isolation of PGCs and prospermatogonia. The day we observed a plug was taken as e0.5.
Isolation of germ cell DNA
For germ cells from embryonic stages, embryos were harvested at e10.5, e12.5, and e17.5. Germ cells from e10.5 embryos were harvested using the MiniMacs system and TG-1 antibody as described previously [21]. From e12.5 onward, the male gonads can be distinguished by the
Acknowledgments
We thank J. Trasler for advice on harvesting oocyte DNA and R. Black and F. Liu for technical support. This work was supported by grants from NSF China and the Max-Planck Society to G.L.X. and grants from the BBSRC (G12997), the Northern Ireland HPSS R&D Office (RRG 6.7), and the Royal Society (RSRG 20735) to C.P.W.
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