FIGURE 4.

DNA methylation in the 5′ region of Olfr17 in GFP positive neurons. (A) Olfactory mucosa cells were dissociated from P2-GFP mice and submitted to fluorescence activated cell sorting (FACS) in order to isolate GFP+ neurons. The flow cytometry dot plot on the right was obtained when olfactory neurons from the P2-GFP mice were sorted, and the green dots within the square represent GFP+ neurons. (B) Schematic representation of the olfactory neurons sorted by FACS, showing that the cells from heterozygous knock-in mice contain one transgenic allele (129 Olfr17 allele) and one B6 Olfr17 allele. The vertical lines indicate the positions of the CpGs (–128, –83, and +33) in the promoter region and exon1. The 129 allele contains the three CpGs and the B6 allele contains only CpG –83. (C) Comparison of the Olfr17 methylation frequencies in pre-sorted and sorted neurons in both alleles. In the pre-sorted cells, CpG-83 in the B6 allele showed 0% methylation frequency. Student’s t-test, n = 3, *P < 0.05. (D) Distribution of the methylated CpGs in each sequenced clone from the 129 allele shown in the graph in (C). Open and closed circles indicate unmethylated and methylated CpGs, respectively.