Figure 5
The forced expression of maternal Dnmt1s protein prevents loss of imprinting due to the absence of maternal Dnmt1o protein. A. Schematic of construct used to force expression of an epitope-tagged version of Dnmt1s in fully grown mouse oocytes of the TgEL transgenic mouse line. ZP3 = 6.6-kb mouse zona pellucida 3 gene promoter [23]. Dnmt1s = full-length Dnmt1s cDNA containing coding sequence for N-terminal Anti-Xpress epitope tag. BGH = bovine growth hormone polyadenylation and transcription termination sequence. B. Summary of bisulfite genomic sequencing of two normally imprinted genes in mice of various genotypes. Dnmt1Δ1o/+ alleles were analyzed in a DNA sample isolated from an E13.5 embryo derived from a cross between a homozygous Dnmt1Δ1o/Δ1ofemale and a Cast-7 male. Dnmt1Δ1o/+, TgEL alleles were analyzed in DNA samples isolated either from an E13.5 embryo (Embryo #4) or from an adult (Adult #1) derived from crosses between a Dnmt1Δ1o/Δ1o, TgEL female and a Cast-7 male. C. Analysis of H19 and Snurf/Snrpn gene expression in mice of various genotypes. Allele-specific expression of H19 was determined by restriction endonuclease digestion of RT-PCR products. Allele-specific expression of Snurf/Snrpn was determined by SnuPE assay. Lanes 1–4: Dnmt1Δ1o/+, TgEL E13.5 embryos, Embryo #1 through Embryo #4. Lane 5–6: Dnmt1Δ1o/+ embryos. Lanes 7–10: Dnmt1Δ1o/+, TgEL adults, Adult #1 through Adult #4. Lane 11: inbred Cast-7. Lane 12: inbred FVB/N. Lane 13: F1 hybrid from cross between an FVB/N female mouse and a Cast-7 male mouse.