Global Chromatin Reprogramming in Fetal Germ Cells Figure 8. Global changes of H3K79me3 in fetal germ cells. Gonad preparations were stained with anti-H3K79me3 antibody. DAPI was used to visualize nuclei (blue). Other details are as in legend to Figure 1. doi:10.1371/journal.pone.0023848.g008 cells at specific loci and at a genome-wide scale to precisely reveal the patterning effect of chromatin on de novo DNA methylation. Similarly to fetal male germ cells, dynamic chromatin changes may also occur in the growing oocytes of the adult mouse before and during the establishment of de novo methylation in the female germ line. Materials and Methods Ethics statement The experiments involving mice had been approved by the IACUC of the City of Hope under protocol ID 91023. Housing and care of the animals has been consistent with the Public Health Service Policy, the NIH ‘‘Guide for the Care and Use of Laboratory Animals’’ and the Animal Welfare Act. Embryo isolation and staging Female CF1 (Charles River Laboratories) mice were mated with male OG2 [13] mice. Genital ridges were dissected at 11.5 dpc. Gonads were dissected from male and female fetuses at 13.5, 15.5 and 17.5 dpc. Male and female gonads were distinguished by their distinct morphology at 13.5, 15.5 and 17.5 dpc. At 11.5 dpc the sex of the embryo was detemined by PCR. Gonads were incubated at 37 C for 15 m in Trypsin-EDTA and triturated to achieve a single cell suspension containing germ cells and somatic cells. Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen, Carlsbad, CA) supplemented with 20% FBS was added to inactivate trypsin. Sex-specific PCR At 11.5 dpc sex of the embryo was determined by PCR amplification of two genes in a single reaction. The Sry amplicon PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23848