DNA methylation analysis of transgenes driven by C. intestinalis gene promoters. (A) Bisulfite sequencing of transgenic green fluorescent protein (GFP) gene bodies driven by Ciona promoters. Black bar represents the position of the analyzed area. Methylated CpG sites (solid), unmethylated CpG sites (open) and CpG sites that were mutated or failed to be sequenced (dashed line) are shown. Genomic DNA from larvae and juveniles was examined. (B) Same as (A), but line E sperm DNA was examined by bisulfite sequencing. A region-spanning promoter, GFP and a 3′ untranslated region (UTR) in the transgene were analyzed by five primer pairs, displayed as 1, 2, 3, 4 and 5. A gene body methylation pattern with a boundary of unmethylated and methylated regions is established.