Fig 2: Transgenic mice (Tg) phenotypes. (A) Schematic illustration of polymerase chain reaction (PCR) primer annealing sites to confirm Tg cassette insertion sites. “Tg internal” legends indicate the junctions between Miwi2 promoter and asDnmt3L. “Tg head” shows the junction between genomic DNA and the Miwi2 promoter at Ch18qB3 and E1 sites, and “Tg tail” shows the junction between the tail of the Tg cassette and genomic DNA at each insertion site. The primer sequences are shown in Supplemental Table S4. The PCR products were subjected to agarose gel electrophoresis with Tris-acetate EDTA buffer (right panel). (B) Morphology of testes of adult mice with transgenes inserted at the E1 locus (Tg-E), B3 locus (Tg-B), and B3 and E1 loci (Tg-BE). Black bar indicates a 2 mm scale. (C) Weights of the testes of Tg-E, Tg-B, and Tg-BE mice. Each dot represents the weight of each testis. The range of the whisker lines represents the minimum and maximum values; outliers were excluded. The box ranges indicate the interquartile range (IQR), the horizontal bars in the box indicate the median, and the “X” marks indicate the mean weights. (98.4, 90.8, 32.0, and 29.8 mg in non-Tg, Tg-E, Tg-B, and Tg-BE mice, respectively; t-test; [*] P = 0.15; [**] P < 0.01; [***] P = 0.72). (D) DNMT3L expression in mice testes at E16.5 was analyzed using western blotting. Upper panel shows DNMT3L, and lower panel shows ß-actin. Each lane was loaded with protein extracts from one-half of a testis. The band intensity of DNMT3L was normalized to that of ß-actin, as well as to that of DNMT3L in non-Tg mice. Analysis was performed using a GelAnalyzer 2010. (E) Hematoxylin and eosin-stained histological sections showing developmental differences between non-Tg, Tg-E, Tg-B, and Tg-BE mouse testes.

Fig 3: Small RNAs from Miwi2P-asDnmt3L in fetal testes. (A) Numbers of small RNAs annotated as from the Miwi2P-asDnmt3L transgene among total reads in the testes of nontransgenic (Tg) mice and mice with transgene cassettes inserted at the E1 locus (Tg-E), B3 locus (Tg-B), and B3 and E1 loci (Tg-BE) at E16.5. Blue bars show the small RNAs from the sense Miwi2 promoter and antisense Dnmt3L (transgene sense strand), while the orange bars show the small RNAs from the antisense Miwi2 promoter and sense Dnmt3L (transgene antisense strand). (B) Size distribution of transgene-annotated small RNAs. The upper side represents the transgene sense strand where blue and purple indicate small RNAs with and without uracil as their first nucleotide, respectively. The lower side represents the transgene antisense strand where orange and purple indicate small RNAs with and without adenine at the tenth position, respectively. (C) Schematic diagram showing the directions of small RNAs. Red arrow indicates the expected transcript of asDnmt3L driven by the Miwi2 promoter. Light blue, blue, and gray arrows indicate transgene sense strands, while yellow, orange, and brown arrows indicate transgene antisense strands. (D) Ratio of transgene-annotated small RNAs in the fetal testis of Tg mice. The colors of the pie charts correspond to the colors of the arrows in C. The shaded orange color contains piRNAs from the transgene and the endogenous Dnmt3L. (E) Mapping of piRNAs of the Tg-B mice onto the transgene cassette sequence. The upper and lower sides represent the sense strand in blue and the antisense strand in orange, respectively.

Fig 4: Bisulfite sequencing and northern blot of DNMT3L de novo DNA methylation targets. (A) Methylation levels of retrotransposon genes (L1A, LINE1 type A; L1Tf, LINE1 Tf; and IAP, IAP1?1) in germ cells of non-Tg and mice with transgenes inserted at the E1 locus (Tg-E), B3 locus (Tg-B), and B3 and E1 loci (Tg-BE). (B) Methylation levels of paternally imprinted genes (Rasgrf1, H19, and Dlk1). (C) Methylation levels of Snrpn, a maternally imprinted gene, representing sorted cell purity. Black and white circles indicate methylated and unmethylated CpGs, respectively. (D) Northern blotting of LINE1 and IAP in testes of 3 wk in the non-Tg and Tg mice. To compare the amount of RNA loaded, the filters were stained with methylene blue after transfer to show the amount of 28S and 18S.

Fig 5: Bisulfite sequencing of Dnmt3L promoter. The top panel shows a diagram of the Dnmt3L promoter and its methylation positions (white circles). Methylation levels of the Dnmt3L promoter in germ cells of 0-d-old (P0) nontransgenic (Non-Tg) mice and of mice with a transgene cassette inserted at the B3 locus (Tg-B) as well as in those of 10-d-old (P10) non-Tg mice and mice with transgene cassettes inserted at the E1 locus (Tg-E) and at both the E1 and B3 loci (Tg-BE). Black and white circles indicate methylated and unmethylated CpGs, respectively.