Model for the establishment and maintenance of transgene DMD methylation during development. The DME1/Igmyc transgene (single arrow) is compared to the DME2/Igmyc transgene (double arrows). The methylation state of the maternal (M) allele is on the top and the methylation state of the paternal allele (P) is on the bottom. The DME2/Igmyc transgene represents the “wild-type” methylation pattern on each allele throughout development, while the “mutant” DME1/Igmyc transgene pattern depicts when and to what extent methylation levels change during development.

Transgene methylation in mutant mouse backgrounds and during preimplantation development. For each panel bisulfite genomic sequencing was performed as described in the legend of Fig. 1D. (A) The DME2/Igmyc transgene was placed into the Dnmt1^Δ1o mutant mouse background, and methylation of the DME2/Igmyc DMD was analyzed in E9.5 transgenic embryos derived from heterozygous Dnmt1^Δ1o/+ (top) or homozygous Dnmt1^Δ1o/Δ1o (bottom) transgenic females. (B) The DME2/Igmyc transgene was placed into the Dnmt3l mutant background, and methylation of the DME2/Igmyc DMD was analyzed in E9.5 transgenic embryos derived from heterozygous Dnmt3l^−/+ (top) or homozygous Dnmt3l^−/− (bottom) transgenic females. (C and D) Bisulfite genomic sequencing of transgene DMD methylation for DME2/Igmyc (C) and DME1/Igmyc (D). Methylation of maternal transgene alleles was compared to paternal transgene alleles. Not all stages were analyzed for the paternal allele.

Igf2r DMD sequences smaller than two unit copies of the TR2+3 repeat are not capable of creating an imprinted transgene locus. (A) Arrows depict the TR2+3 repeats of the Igf2r DMD2. Lines below indicate the DNA fragments used to create several hybrid transgenes. Filled circles represent the number and approximate location of CpGs (not to scale). All transgenes were constructed as described in the legend of Fig. 1. (B to F) Bisulfite genomic sequencing analysis of the DMD region of each transgene. For each panel bisulfite genomic sequencing was performed as described in the legend of Fig. 1D.

The TR2+3 repeat from the Igf2r DMD, but not the Air promoter, is able to create an imprinted transgene locus. (A) Southern blot analysis of DNAs obtained from maternal and paternal carriers of each transgene construct. DNAs were digested with HpaII and hybridized with the probe depicted in Fig. 1B. Band sizes are shown in kilobases (kb). Airprom, Airprom/Igmyc; TR2+3, TR2+3/Igmyc; DME2, DME2/Igmyc. Positions within the DMD are shown in Fig. 1A. (B and C) Summary of bisulfite genomic sequencing analyses performed on DNAs obtained from maternal and paternal carriers of the TR2+3/Igmyc (B) and DME2/Igmyc (C) transgenes (as described in the legend of Fig. 1). M, maternal; P, paternal.

A transgene containing the Snrpn promoter and tandem repeats shows maternal-specific methylation and paternal-specific expression of an eGFP/myc fusion transcript. (A) The transgene is a derivative of the transgene shown in Fig. 1B. The black box indicates Snrpn sequence (exon 1 of Snrpn is shown). Exon 2 of c-myc contains an in-frame fusion of the entire eGFP protein-coding region (GFP). Arrowheads represent primers used for RT-PCR. (B) RT-PCR of cDNAs obtained from adult brain of transgenic mice. M, maternal inheritance; P, paternal inheritance; −, negative control. Paternal-specific eGFP/myc expression (top) and control amplification of the Hprt gene (bottom) are shown. The transgene-specific spliced transcript is shown below. (C) Bisulfite genomic sequencing of maternal (M) and paternal (P) alleles of the transgene (as described in the legend of Fig. 1). Position of sequence amplified by PCR with modified, semi-nested primers is shown in panel A (*).

DMD sequences containing tandem repeats restore maternal-specific methylation to the Ig/myc transgene. (A) Schematics of the mouse Snurf/Snrpn, Kcnq1, and Igf2r DMDs (white boxes). The Snrpn DMD contains the promoter and exon 1 of the Snurf/Snrpn locus, and the Kcnq1 and Igf2r DMDs contain promoters for untranslated antisense transcripts (bent arrows). Arrowheads or arrows within each DMD indicate tandem repeats (different for each DMD). Gray bars represent the regions analyzed in Ig/myc transgenes in this study and previously (21). The Kcnq1 region analyzed in this study included the conserved repeats examined by Mancini-Dinardo et al. plus additional DMD repeats (15). (B) Design of transgenes analyzed in this study. The Cα and Sα regions of the mouse IgA locus (Ig) and the 3′ untranslated region and c-myc exons (1-3) are shown. A black bar depicts the probe for the Southern blots. The DMD sequences depicted by gray bars in panel A were subcloned into an EcoRI site (flanking the black box). (C) Southern blot analysis of DNAs obtained from maternal and paternal carriers of each transgene. DNAs were digested with HpaII and hybridized with the probe shown in panel B. Band sizes are shown in kilobases (kb). SnrpnR, SnrpnR/Igmyc; Kcnq1, Kcnq1/Igmyc. (D) Summary of bisulfite genomic sequencing analyses performed on DNAs obtained from maternal and paternal carriers of the SnrpnR/Igmyc transgene. The top line shows the location of tandem repeats (arrows) and the CpGs analyzed (open circles). Each line below represents one sequenced allele. Filled circles indicate the positions of methylated CpGs. (E) Summary of bisulfite genomic sequencing data obtained from preimplantation stage embryos following maternal or paternal inheritance of the SnrpnR/Igmyc transgene. Mor, morulae; Blast, blastocysts. For the paternal allele only the eight-cell and blastocyst stages were examined. Each bar represents the percentage of CpGs found to be methylated in all of the subclones analyzed. M, maternal; P, paternal.