- Email: [email protected]
Allele-specific expression of imprinted genes in mouse migratory primordial germ cells
Mechanisms of Development 115 (2002) 157–160 www.elsevier.com/locate/modo
Gene expression pattern
Allele-specific expression of imprinted genes in mouse migratory primordial germ cells Piroska E. Szabo´ a, Karin Hu¨bner b, Hans Scho¨ler b, Jeffrey R. Mann a,* a
b
Division of Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010-3011, USA Center for Animal Transgenesis and Germ Cell Research, University of Pennsylvania, New Bolton Center, 382 West Street Road, Kennett Square, PA 19348-1692, USA Received 12 February 2002; received in revised form 27 March 2002; accepted 27 March 2002
Abstract In somatic cells, imprinted genes are expressed monoallelically according to parent-of-origin. In contrast, in 11.5 days post-coitum primordial germ cells (PGCs), and later stage germ cells, these same genes are expressed biallelically, suggesting that imprints inherited from the gametes are largely erased by this stage. To determine when in germ cell development this biallelic expression phenomenon commences, we isolated migrating PGCs by flow cytometry and determined the allele-specific expression of four imprinted genes – Snrpn, Igf2, H19 and Igf2r. The first three genes were expressed monoallelically, while the latter gene was expressed biallelically. These results show that inherited imprints regulating monoallelic expression are largely intact in migrating PGCs. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Imprinting; Epigenetics; Gene expression; Primordial germ cell; Transgene; Green fluorescent protein; Germ line; Germ cell; Reverse transcriptionpolymerase chain reaction; Flow cytometry
1. Results and discussion A transgenic mouse line was made in which the transgene consisted of the Pou domain, class 5, transcription factor 1 (Pou5f1, or Oct4) promoter and enhancers driving the egfp reporter gene. Pou5f1 expression marks the totipotent cell lineage (Pesce et al., 1998). The degree of primordial germ cell (PGC) purity in EGFP 1 cells sorted by flow cytometry was assessed using PGC-specific markers. At 9.5 days postcoitum (dpc), a discrete population of EGFP 1 cells was obtained, and nearly all (96%) were PGCs (Fig. 1C,D). At 10.5 dpc, EGFP 1 cells were even more discrete (Fig. 2A). At 11.5 dpc, EGFP 1 cells were positive for the PGC marker, stage-specific embryonic antigen (SSEA1) (Fig. 2B). Fluorescence was restricted to the gonad at subsequent stages (Fig. 2C–E). These results show that 9.5 and 10.5 dpc migratory PGCs can be enriched to near-homogeneity using the method described. Reverse transcription-polymerase chain reaction (RTPCR) single nucleotide primer extension assays for quantifying allele-specific expression were performed on sorted EGFP 1 and EGFP 2 cells. Results are shown in Fig. 3. At * Corresponding author. Tel.: 11-626-301-8813. E-mail address: [email protected] (J.R. Mann).
11.5 dpc, or at the early post-migratory stage, the expression of all four imprinted genes was biallelic in EGFP 1 PGCs, and monoallelic in EGFP 2 somatic cells, confirming the results of a previous study on manually purified PGCs (Szabo´ and Mann, 1995b). However, 1 day earlier, in 10.5 dpc migratory PGCs, the expression of the small nuclear ribonucleoprotein N (Snrpn) gene, H19, and the insulinlike growth factor 2 (Igf2) gene, was markedly skewed, with the expression of the latter two genes being very close to monoallelic. At 9.5 dpc, Snrpn was expressed monoallelically. We did not assay Igf2 expression at 9.5 dpc, assuming that this would be essentially monoallelic, as was H19 expression. These results demonstrate that the inherited epigenetic mechanisms regulating the monoallelic expression of these three, and probably many other imprinted genes, are retained during PGC specification, and for sometime thereafter. The insulin-like growth factor 2 receptor (Igf2r) gene provided the exceptional result. It was monoallelically and biallelically expressed in somatic cells and PGCs, respectively, at all stages examined. In intersubspecific hybrid embryos as studied here, Igf2r is also biallelically expressed at earlier stages – during cleavage, in the blastocyst inner cell mass, and in the primitive ectoderm of the 6.5 dpc egg cylinder (Szabo´ and Mann, 1995a). Therefore, Igf2r appears always to be expressed
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00087-4
158
P.E. Szabo´ et al. / Mechanisms of Development 115 (2002) 157–160
biallelically in the totipotent lineage. However, we note that a transgenically modified Igf2r allele is paternally downregulated in the 6.5 dpc primitive ectoderm, as assessed by LacZ activity (Lerchner and Barlow, 1997).
Fig. 1. Characterization of EGFP 1 cells in 8.5 and 9.5 dpc embryos. All embryos were obtained from CD1 Swiss C £ TgOG2 F matings and hence were hemizygous for the transgene. (A) Flow cytometry of dispersed 8.5 dpc embryo sections. A total of 990 EGFP 1 cells were sorted as indicated – events above line – from 28 embryo sections and were 0.16% of the total number of cells. (B) Staining for the PGC marker, alkaline phosphatase 2 (AKPII), of cells sorted in (A). AKPII 1 cells are stained red, and unstained cells are indicated by arrowheads – 68 of 108 (62%) counted were positive. (C) Flow cytometry of 9.5 dpc embryo sections. A total of 5073 EGFP 1 cells were sorted as indicated – events above line – from 42 embryo sections and were 0.07% of the total number of cells. (D) AKPII staining of EGFP 1 cells sorted in (C) – 370 of 387 (96%) counted were positive. This is the same level of purity achieved for post-migratory germ cells using flow cytometry and an independently derived Pou5f1-egfp transgenic line (Ueda et al., 2000; Yoshimizu et al., 1999). All EGFP 2 cells were AKPII 2 (data not shown). Axes of graphs are marked at 0, 10 1, 10 2, 10 3 and 10 4 intervals. SSC, side scatter.
Fig. 2. Characterization of EGFP 1 cells of 10.5 dpc and later stage embryos. All embryos were obtained from CD1 Swiss C £ TgOG2 F matings. (A) 10.5 dpc. Flow cytometry. EGFP 1 cells above the line are sorted. (B) 11.5 dpc. Flow cytometry. EGFP 1 cells are also positive for the germ cell marker SSEA1. (C) 13.5 dpc. Fluorescence micrograph. EGFP is restricted to the female and male gonads, with fluorescent cords visible in the male. Low-level incident visible light was used to visualize the mesonephros – the larger structure to which each gonad is attached. (D) 15.5 dpc. Flow cytometry, isolated female gonads. (E) 15.5 dpc. Flow cytometry, isolated male gonads. Axes of graphs are marked at 0, 10 1, 10 2, 10 3 and 10 4 intervals.
P.E. Szabo´ et al. / Mechanisms of Development 115 (2002) 157–160
159
bred to homozygosity for the transgene and used for the studies described here. 2.2. PGC-enriched embryo fragments used in flow cytometry 8.5 dpc or ,five somites – the posterior portion as delineated by the node; 9.5 and 10.5 dpc – as described (Cooke et al., 1993); 11.5 dpc – for SSEA1 labeling, all of the embryo between the forelimb and hindlimb buds; for gene expression studies, isolated gonads; 15.5 dpc – isolated gonads. Isolates were placed into 0.15 ml of trypsinEDTA, incubated for 20 min at 37 8C, then dissociated into a single cell suspension by trituration. A total of 0.3 ml of 25% (v/v) fetal bovine serum in medium M2 (Wood et al., 1987) was added before flow cytometry. 2.3. Cell labeling and flow cytometry
Fig. 3. Allele-specific expression of imprinted genes in migrating PGCs. Sorted cells were analyzed using the RT-PCR single nucleotide primer extension (SNuPE) assay. For each gene, the top and bottom rows of bands represent the presumptive inactive and active allele, respectively, with the parental derivation given at the right. The value above each pair of bands is the percent amount of RNA contributed by the presumptive inactive allele to the total RNA present for that gene. For example, for 10.5 dpc EGFP 1 cells, 15 and 85% of the total amount of Snrpn RNA was derived from the maternal and paternal alleles, or the presumptive inactive and active alleles, respectively. For the 11.5 dpc sample, when PGCs are at the early post-migratory stage, only cells of the isolated genital ridge were subjected to flow cytometry. Thus, EGFP 2 cells were also derived from the genital ridge.
The present results, and previous observations of hypomethylation of imprinted genes in 13.5 dpc female and male germ cells (Brandeis et al., 1993; Ueda et al., 2000), are consistent with a large scale loss of methylation imprints in germ cells at the end of the migratory period. This would be indicative of a shift in the epigenetic program of the germ line at this stage. It might be regulated by a developmental clock, or be induced by somatic cells within the genital ridge. An investigation of the methylation profiles of imprinted genes in migratory PGCs is underway.
Labeling of dissociated live cells with SSEA1 was carried out as described (Harlow and Lane, 1988). First antibody— monoclonal antibody MC-480 (SSEA1) (Developmental Studies Hybridoma Bank, University of Iowa, IA); second antibody—goat anti-mouse IgM-APC conjugate (Caltag). AKPII staining was carried out using ADCELLe slides (Erie Scientific) and reagents as described (Cooke et al., 1993). 2.4. Allele-specific expression Embryos for PGC isolation were obtained from matings between TgOG2 prepubertal females and CAST/Ei males (The Jackson Laboratory). Cells were sorted directly into 0.25 ml of RNAzol B (Tel-Test), then RNA isolated as described (Szabo´ and Mann, 1995b). Allele-specific expression was quantified using RT-PCR single nucleotide primer extension assays as described (Szabo´ and Mann, 1995b). Acknowledgements We thank Lucy Brown, Claudio Spalla, and Jim Bolen for flow cytometry. This work was supported by NIH grant 2RO1GM48103-04 and NSF grant BIR-9220534. References
2. Methods 2.1. Production of a transgenic line expressing EGFP in the germ line The construct used was identical to that described— GOF18DPE (Yeom et al., 1996)—except that the egfp reporter (Clontech) was substituted for the lacZ reporter. A number of transgenic lines were made by injecting (CBA/CaJ £ C57BL/6J)F2 zygotes. One line, TgOG2, was
Brandeis, M., Kafri, T., Ariel, M., Chaillet, J.R., McCarrey, J., Razin, A., Cedar, H., 1993. The ontogeny of allele-specific methylation associated with imprinted genes in the mouse. EMBO J. 12, 3669–3677. Cooke, J.E., Godin, I., Ffrench-Constant, C., Heasman, J., Wylie, C.C., 1993. Culture and manipulation of primordial germ cells. Methods Enzymol. 225, 37–58. Harlow, E., Lane, D., 1988. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Lerchner, W., Barlow, D.P., 1997. Paternal repression of the imprinted mouse Igf2r locus occurs during implantation and is stable in all tissues of the post-implantation mouse embryo. Mech. Dev. 61, 141–149.
160
P.E. Szabo´ et al. / Mechanisms of Development 115 (2002) 157–160
Pesce, M., Gross, M.K., Scho¨ ler, H.R., 1998. In line with our ancestors: Oct-4 and the mammalian germ. Bioessays 20, 722–732. Szabo´ , P.E., Mann, J.R., 1995a. Allele-specific expression and total expression levels of imprinted genes during early mouse development: implications for imprinting mechanisms. Genes Dev. 9, 3097–3108. Szabo´ , P.E., Mann, J.R., 1995b. Biallelic expression of imprinted genes in the mouse germ line: implications for erasure, establishment, and mechanisms of genomic imprinting. Genes Dev. 9, 1857–1868. Ueda, T., Abe, K., Miura, A., Yuzuriha, M., Zubair, M., Noguchi, M., Niwa, K., Kawase, Y., Kono, T., Matsuda, Y., Fujimoto, H., Shibata, H., Hayashizaki, Y., Sasaki, H., 2000. The paternal methylation imprint of the mouse H19 locus is acquired in the gonocyte stage during foetal testis development. Genes Cells 5, 649–659.
Wood, M.J., Whittingham, D.G., Rall, W.F., 1987. The low temperature preservation of mouse oocytes and embryos. In: Monk, M. (Ed.). Mammalian Development: A Practical Approach, IRL Press, Oxford, pp. 255–280. Yeom, Y.I., Fuhrmann, G., Ovitt, C.E., Brehm, A., Ohbo, K., Gross, M., Hu¨ bner, K., Scho¨ ler, H.R., 1996. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894. Yoshimizu, T., Sugiyama, N., De Felice, M., Yeom, Y.I., Ohbo, K., Masuko, K., Obinata, M., Abe, K., Scholer, H.R., Matsui, Y., 1999. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev. Growth Differ. 41, 675–684.
Gene expression pattern
Allele-specific expression of imprinted genes in mouse migratory primordial germ cells Piroska E. Szabo´ a, Karin Hu¨bner b, Hans Scho¨ler b, Jeffrey R. Mann a,* a
b
Division of Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, CA 91010-3011, USA Center for Animal Transgenesis and Germ Cell Research, University of Pennsylvania, New Bolton Center, 382 West Street Road, Kennett Square, PA 19348-1692, USA Received 12 February 2002; received in revised form 27 March 2002; accepted 27 March 2002
Abstract In somatic cells, imprinted genes are expressed monoallelically according to parent-of-origin. In contrast, in 11.5 days post-coitum primordial germ cells (PGCs), and later stage germ cells, these same genes are expressed biallelically, suggesting that imprints inherited from the gametes are largely erased by this stage. To determine when in germ cell development this biallelic expression phenomenon commences, we isolated migrating PGCs by flow cytometry and determined the allele-specific expression of four imprinted genes – Snrpn, Igf2, H19 and Igf2r. The first three genes were expressed monoallelically, while the latter gene was expressed biallelically. These results show that inherited imprints regulating monoallelic expression are largely intact in migrating PGCs. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Imprinting; Epigenetics; Gene expression; Primordial germ cell; Transgene; Green fluorescent protein; Germ line; Germ cell; Reverse transcriptionpolymerase chain reaction; Flow cytometry
1. Results and discussion A transgenic mouse line was made in which the transgene consisted of the Pou domain, class 5, transcription factor 1 (Pou5f1, or Oct4) promoter and enhancers driving the egfp reporter gene. Pou5f1 expression marks the totipotent cell lineage (Pesce et al., 1998). The degree of primordial germ cell (PGC) purity in EGFP 1 cells sorted by flow cytometry was assessed using PGC-specific markers. At 9.5 days postcoitum (dpc), a discrete population of EGFP 1 cells was obtained, and nearly all (96%) were PGCs (Fig. 1C,D). At 10.5 dpc, EGFP 1 cells were even more discrete (Fig. 2A). At 11.5 dpc, EGFP 1 cells were positive for the PGC marker, stage-specific embryonic antigen (SSEA1) (Fig. 2B). Fluorescence was restricted to the gonad at subsequent stages (Fig. 2C–E). These results show that 9.5 and 10.5 dpc migratory PGCs can be enriched to near-homogeneity using the method described. Reverse transcription-polymerase chain reaction (RTPCR) single nucleotide primer extension assays for quantifying allele-specific expression were performed on sorted EGFP 1 and EGFP 2 cells. Results are shown in Fig. 3. At * Corresponding author. Tel.: 11-626-301-8813. E-mail address: [email protected] (J.R. Mann).
11.5 dpc, or at the early post-migratory stage, the expression of all four imprinted genes was biallelic in EGFP 1 PGCs, and monoallelic in EGFP 2 somatic cells, confirming the results of a previous study on manually purified PGCs (Szabo´ and Mann, 1995b). However, 1 day earlier, in 10.5 dpc migratory PGCs, the expression of the small nuclear ribonucleoprotein N (Snrpn) gene, H19, and the insulinlike growth factor 2 (Igf2) gene, was markedly skewed, with the expression of the latter two genes being very close to monoallelic. At 9.5 dpc, Snrpn was expressed monoallelically. We did not assay Igf2 expression at 9.5 dpc, assuming that this would be essentially monoallelic, as was H19 expression. These results demonstrate that the inherited epigenetic mechanisms regulating the monoallelic expression of these three, and probably many other imprinted genes, are retained during PGC specification, and for sometime thereafter. The insulin-like growth factor 2 receptor (Igf2r) gene provided the exceptional result. It was monoallelically and biallelically expressed in somatic cells and PGCs, respectively, at all stages examined. In intersubspecific hybrid embryos as studied here, Igf2r is also biallelically expressed at earlier stages – during cleavage, in the blastocyst inner cell mass, and in the primitive ectoderm of the 6.5 dpc egg cylinder (Szabo´ and Mann, 1995a). Therefore, Igf2r appears always to be expressed
0925-4773/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(02)00087-4
158
P.E. Szabo´ et al. / Mechanisms of Development 115 (2002) 157–160
biallelically in the totipotent lineage. However, we note that a transgenically modified Igf2r allele is paternally downregulated in the 6.5 dpc primitive ectoderm, as assessed by LacZ activity (Lerchner and Barlow, 1997).
Fig. 1. Characterization of EGFP 1 cells in 8.5 and 9.5 dpc embryos. All embryos were obtained from CD1 Swiss C £ TgOG2 F matings and hence were hemizygous for the transgene. (A) Flow cytometry of dispersed 8.5 dpc embryo sections. A total of 990 EGFP 1 cells were sorted as indicated – events above line – from 28 embryo sections and were 0.16% of the total number of cells. (B) Staining for the PGC marker, alkaline phosphatase 2 (AKPII), of cells sorted in (A). AKPII 1 cells are stained red, and unstained cells are indicated by arrowheads – 68 of 108 (62%) counted were positive. (C) Flow cytometry of 9.5 dpc embryo sections. A total of 5073 EGFP 1 cells were sorted as indicated – events above line – from 42 embryo sections and were 0.07% of the total number of cells. (D) AKPII staining of EGFP 1 cells sorted in (C) – 370 of 387 (96%) counted were positive. This is the same level of purity achieved for post-migratory germ cells using flow cytometry and an independently derived Pou5f1-egfp transgenic line (Ueda et al., 2000; Yoshimizu et al., 1999). All EGFP 2 cells were AKPII 2 (data not shown). Axes of graphs are marked at 0, 10 1, 10 2, 10 3 and 10 4 intervals. SSC, side scatter.
Fig. 2. Characterization of EGFP 1 cells of 10.5 dpc and later stage embryos. All embryos were obtained from CD1 Swiss C £ TgOG2 F matings. (A) 10.5 dpc. Flow cytometry. EGFP 1 cells above the line are sorted. (B) 11.5 dpc. Flow cytometry. EGFP 1 cells are also positive for the germ cell marker SSEA1. (C) 13.5 dpc. Fluorescence micrograph. EGFP is restricted to the female and male gonads, with fluorescent cords visible in the male. Low-level incident visible light was used to visualize the mesonephros – the larger structure to which each gonad is attached. (D) 15.5 dpc. Flow cytometry, isolated female gonads. (E) 15.5 dpc. Flow cytometry, isolated male gonads. Axes of graphs are marked at 0, 10 1, 10 2, 10 3 and 10 4 intervals.
P.E. Szabo´ et al. / Mechanisms of Development 115 (2002) 157–160
159
bred to homozygosity for the transgene and used for the studies described here. 2.2. PGC-enriched embryo fragments used in flow cytometry 8.5 dpc or ,five somites – the posterior portion as delineated by the node; 9.5 and 10.5 dpc – as described (Cooke et al., 1993); 11.5 dpc – for SSEA1 labeling, all of the embryo between the forelimb and hindlimb buds; for gene expression studies, isolated gonads; 15.5 dpc – isolated gonads. Isolates were placed into 0.15 ml of trypsinEDTA, incubated for 20 min at 37 8C, then dissociated into a single cell suspension by trituration. A total of 0.3 ml of 25% (v/v) fetal bovine serum in medium M2 (Wood et al., 1987) was added before flow cytometry. 2.3. Cell labeling and flow cytometry
Fig. 3. Allele-specific expression of imprinted genes in migrating PGCs. Sorted cells were analyzed using the RT-PCR single nucleotide primer extension (SNuPE) assay. For each gene, the top and bottom rows of bands represent the presumptive inactive and active allele, respectively, with the parental derivation given at the right. The value above each pair of bands is the percent amount of RNA contributed by the presumptive inactive allele to the total RNA present for that gene. For example, for 10.5 dpc EGFP 1 cells, 15 and 85% of the total amount of Snrpn RNA was derived from the maternal and paternal alleles, or the presumptive inactive and active alleles, respectively. For the 11.5 dpc sample, when PGCs are at the early post-migratory stage, only cells of the isolated genital ridge were subjected to flow cytometry. Thus, EGFP 2 cells were also derived from the genital ridge.
The present results, and previous observations of hypomethylation of imprinted genes in 13.5 dpc female and male germ cells (Brandeis et al., 1993; Ueda et al., 2000), are consistent with a large scale loss of methylation imprints in germ cells at the end of the migratory period. This would be indicative of a shift in the epigenetic program of the germ line at this stage. It might be regulated by a developmental clock, or be induced by somatic cells within the genital ridge. An investigation of the methylation profiles of imprinted genes in migratory PGCs is underway.
Labeling of dissociated live cells with SSEA1 was carried out as described (Harlow and Lane, 1988). First antibody— monoclonal antibody MC-480 (SSEA1) (Developmental Studies Hybridoma Bank, University of Iowa, IA); second antibody—goat anti-mouse IgM-APC conjugate (Caltag). AKPII staining was carried out using ADCELLe slides (Erie Scientific) and reagents as described (Cooke et al., 1993). 2.4. Allele-specific expression Embryos for PGC isolation were obtained from matings between TgOG2 prepubertal females and CAST/Ei males (The Jackson Laboratory). Cells were sorted directly into 0.25 ml of RNAzol B (Tel-Test), then RNA isolated as described (Szabo´ and Mann, 1995b). Allele-specific expression was quantified using RT-PCR single nucleotide primer extension assays as described (Szabo´ and Mann, 1995b). Acknowledgements We thank Lucy Brown, Claudio Spalla, and Jim Bolen for flow cytometry. This work was supported by NIH grant 2RO1GM48103-04 and NSF grant BIR-9220534. References
2. Methods 2.1. Production of a transgenic line expressing EGFP in the germ line The construct used was identical to that described— GOF18DPE (Yeom et al., 1996)—except that the egfp reporter (Clontech) was substituted for the lacZ reporter. A number of transgenic lines were made by injecting (CBA/CaJ £ C57BL/6J)F2 zygotes. One line, TgOG2, was
Brandeis, M., Kafri, T., Ariel, M., Chaillet, J.R., McCarrey, J., Razin, A., Cedar, H., 1993. The ontogeny of allele-specific methylation associated with imprinted genes in the mouse. EMBO J. 12, 3669–3677. Cooke, J.E., Godin, I., Ffrench-Constant, C., Heasman, J., Wylie, C.C., 1993. Culture and manipulation of primordial germ cells. Methods Enzymol. 225, 37–58. Harlow, E., Lane, D., 1988. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Lerchner, W., Barlow, D.P., 1997. Paternal repression of the imprinted mouse Igf2r locus occurs during implantation and is stable in all tissues of the post-implantation mouse embryo. Mech. Dev. 61, 141–149.
160
P.E. Szabo´ et al. / Mechanisms of Development 115 (2002) 157–160
Pesce, M., Gross, M.K., Scho¨ ler, H.R., 1998. In line with our ancestors: Oct-4 and the mammalian germ. Bioessays 20, 722–732. Szabo´ , P.E., Mann, J.R., 1995a. Allele-specific expression and total expression levels of imprinted genes during early mouse development: implications for imprinting mechanisms. Genes Dev. 9, 3097–3108. Szabo´ , P.E., Mann, J.R., 1995b. Biallelic expression of imprinted genes in the mouse germ line: implications for erasure, establishment, and mechanisms of genomic imprinting. Genes Dev. 9, 1857–1868. Ueda, T., Abe, K., Miura, A., Yuzuriha, M., Zubair, M., Noguchi, M., Niwa, K., Kawase, Y., Kono, T., Matsuda, Y., Fujimoto, H., Shibata, H., Hayashizaki, Y., Sasaki, H., 2000. The paternal methylation imprint of the mouse H19 locus is acquired in the gonocyte stage during foetal testis development. Genes Cells 5, 649–659.
Wood, M.J., Whittingham, D.G., Rall, W.F., 1987. The low temperature preservation of mouse oocytes and embryos. In: Monk, M. (Ed.). Mammalian Development: A Practical Approach, IRL Press, Oxford, pp. 255–280. Yeom, Y.I., Fuhrmann, G., Ovitt, C.E., Brehm, A., Ohbo, K., Gross, M., Hu¨ bner, K., Scho¨ ler, H.R., 1996. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894. Yoshimizu, T., Sugiyama, N., De Felice, M., Yeom, Y.I., Ohbo, K., Masuko, K., Obinata, M., Abe, K., Scholer, H.R., Matsui, Y., 1999. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev. Growth Differ. 41, 675–684.