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/Δ1o female 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.