Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells
Jiyoung Lee1,2,
Kimiko Inoue2,3,
Ryuichi Ono1,2,
Narumi Ogonuki3,
Takashi Kohda1,2,
Tomoko Kaneko-Ishino2,4,
Atsuo Ogura2,3,* and
Fumitoshi Ishino1,2,*
1 Gene Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
2 CREST, Japan Science and Technology Corporation (JST), 4-1-8 Hon-machi, Kawaguchi, Saitama 332-0012, Japan
3 Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo 162-8640, Japan
4 Tokai University, School of Health Sciences, Bohseidai, Isehara, Kanagawa 259-1193, Japan
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Fig. 1. Development of embryos derived from enucleated oocytes injected with primordial germ cell (PGC) nuclei. (A) Culture and development of PGC transferred cells from day 11.5 to day 13.5. *After 72 hours in culture; **some embryos were cultured for 48 hours and transferred into the oviducts of day 1 pseudopregnant females. (B) Photographs of day 12.5 (top) and day 11.5 PGC clone embryos (middle) at dpc 10.5 (left) and 11.5 (right). Embryos produced by in vitro fertilization (IVF, bottom) were used as controls to compare the developmental stages of the PGC clone embryos.
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Fig. 2. PGC clone embryo expression ratios and imprinting status. Relative expression levels in PGC clone embryos at day 9.5 were estimated by quantitative RT-PCR. The expression levels of IVF control embryos are shown as 1. Allelic expression was determined by DNA polymorphism analysis between C57BL6 (Mus musculus musculus) and JF1 (Mus musculus molossinus). Blue and red bars show paternal and maternal allelic expression profiles, respectively, that are similar to normal imprinted states. In the 11.5 PGC clone embryos, allele-specific expression of Pegs (A-D,J,L) and Megs (E-I,K) starts to convert to two of the so-called default states: biallelic expression (white bars) or non-expression (black bars). The timing of this erasing process differs with the individual imprinting genes, but the lack of distinction between male (sample number written in blue) and female (red) germ cells indicates that the erasure process is simultaneous in both germ lines. The ratios of imprinted gene expression in erased PGC clones and Dnmt1 c/c embryos (light-gray bars) were essentially the same. Placental expression was examined in the case of Mash2. (M) The numbers of genomic imprinted genes showing an imprinted monoallelic expression pattern.
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Fig. 3. DMR methylation and expression of H19 and Peg10 in the day 11.5 PGC clone embryos. (A) DNA methylation of H19 DMR. (B) DNA methylation of Peg10 DMR. DNA methylation was analyzed by bisulfite genomic sequencing. Paternal and maternal alleles were distinguished by DNA polymorphism between B6 and JF1 in the DMR sequences. Black ovals indicate methylated CpGs and white ovals indicate unmethylated CpGs. (C) Expression rates from originally repressed alleles. Expression rates were calculated by comparing the intensity of RT-PCR products by RFLP methods in H19 or by comparing the numbers of subclones containing DNA polymorphic sites by DNA sequencing in Peg10.
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Fig. 4. DNA methylation of Nnat (previously known as Peg5; A) H19 (B) and Peg10 (C) in day 10.5 to day 12.5 PGCs. The DNA demethylation status of three imprinted genes that had fast, intermediate and slow erasing of imprinted expression, were analyzed using bisulfite-treated genomic DNA from PGCs isolated from the genital ridges of day 10.5 to day 12.5 embryos. The results were consistent with the DNA methylation status of day 11.5 and day 12.5 PGC clones shown in Fig. 3.
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Fig. 5. Possible scheme for genomic imprinting memory erasure in PGCs. The erasure process is divided into two patterns, which proceed to biallelic expression as one default state (four Pegs, and H19 and Meg3) or non-expression as the other default state (four Megs, and Igf2 and Dlk1). In the former pattern, status conversion timing depends on individual genes; in the latter pattern, however, conversion occurred almost simultaneously in all genes examined.
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© The Company of Biologists Ltd 2002
