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H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs
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Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs Kyung-Jun Uh a,b , Chi-Hun Park b , Kwang-Hwan Choi a , Jin-Kyu Park a , Yeon-Woo Jeong b , Sangho Roh c , Sang-Hwan Hyun d , Taeyoung Shin b , Chang-Kyu Lee a,∗∗ , Woo Suk Hwang b,∗ a Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science, Seoul National University, Seoul, Republic of Korea b Sooam Biotech Research Foundation, Seoul, Republic of Korea c Dental Research Institute and CLS21, Seoul National University School of Dentistry, Seoul, Republic of Korea d College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea
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Article history: Received 11 June 2013 Received in revised form 25 March 2014 Accepted 28 March 2014 Available online xxx
Keywords: Imprinted gene Embryonic stem cell Parthenogenesis DNA methylation
a b s t r a c t To determine whether the genomic imprinting can be maintained during the process of embryonic stem (ES) cell derivation from pig blastocysts, mRNA and DNA methylation at the IGF2/H19 imprinting control region in putative ES cells derived from in vitro fertilized (IVF) and parthenogenetic (PG) embryos were investigated. In the present study, one IVFand three PG ES-like cell lines were established and analyzed for cellular characteristics such as pluripotent marker expression and differentiation capacity. The results showed that these putative ES cells derived from pig blastocysts fulfilled the general “stemness” criteria. The expression of the H19 gene was significantly greater in PG blastocysts than IVF blastocysts, but there were greater amounts of IGF2 in IVF than PG blastocysts. Of these putative ES cell lines, one PG line had less H19 gene expression than a IVF ES cell line while the other two PG lines had much greater expression of the H19 gene than the IVF line. In contrast, the IGF2 gene was upregulated in the same PG cell line relative to the other two PG cell lines and transcript abundance was similar to IVF ES-like cells. Despite the variable amounts of mRNA among the PG cell lines, the IGF2/H19 gene had a differentially methylated region (DMR) 3 was typically un-methylated in all PG cells, and hemi-methylated in the IVF cells. These findings indicated that the mRNA of H19 and IGF2 genes is susceptible to in vitro environments during the process of ES cell derivation from blastocysts but DNA methylation status at this region was well maintained. These altered gene expressions may not be associated with the methylation of the imprinting control region at this locus. Therefore, with their uni-parental genotype, the pluripotent differentiation potentials of PG ES cells could be a valuable tool for understanding genomic imprinting in embryonic development. © 2014 Elsevier B.V. All rights reserved.
∗ Corresponding authors at: Sooam Biotech Research Foundation, 64, Gyeongin-ro, Guro-gu, Seoul 108-105, Republic of Korea. Tel.: +82 2 2616 5658; fax: +82 2 2616 5672. ∗∗ Corresponding authors at: Department of Agricultural Biotechnology, Seoul National University, 1, Gwanak-ro, Gwanak-gu, Seoul 151-015, Republic of Korea. Tel.: +82 2 880 4805; fax: +82 2 873 4805. E-mail addresses: [email protected] (C.-K. Lee), [email protected] (W.S. Hwang). http://dx.doi.org/10.1016/j.anireprosci.2014.03.020 0378-4320/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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1. Introduction Genomic imprinting is an epigenetic mechanism that results in the expression of certain genes from only one of the two parental chromosomes. The exclusive expression of imprinted genes is dependent on whether these genes originate from either the maternal or paternal allele. To date, more than 80 imprinted genes have been identified in mice and humans and many of these genes are highly conserved across mammalian species, including pigs and cows (Dindot et al., 2004; Bischoff et al., 2009). Parental genomic imprints are deleted and re-established during germ cell development and, once established, the epigenetic imprints are maintained throughout development. Imprinted genes have important roles in early development and growth and influence postnatal behavior. DNA methylation is an important epigenetic marker that regulates parental imprinting for allele-specific gene expression (Reik and Walter, 2001). In mammals, PG embryos with two sets of maternal genomes fail early in development due to a lack of paternal-specific signals for the development of extraembryonic tissues (Barton et al., 1984; Surani et al., 1984). Although these developmental consequences occur within uniparental embryos, embryonic stem (ES) cells derived from these embryos in mice fulfill the criteria of biparental ES cells (Allen et al., 1994). PG ES cells have been successfully achieved in numerous mammalian species including rabbits (Fang et al., 2006), buffalo (Sritanaudomchai et al., 2007), non-human primates (Cibelli et al., 2002; Vrana et al., 2003), and humans (Mai et al., 2007). The PG ES cells provide a valuable source of cells or tissues for cell therapy as a substitute for fertilized ES cells due to their histocompatibility with the oocyte donor (Cibelli et al., 2006). The application of PG ES cells in regenerative medicine would circumvent the immune rejection problem as well as ethical concerns related to destroying fertilized embryos. Moreover, uniparental ES cell lines established from PG or androgenetic (AG) embryos have been a useful tool for understanding genomic imprinting due to their genotype. The comparative study of imprinting among PG, AG, and normal ES cells offers a great opportunity to understand the properties of parental imprinting. Genomic imprinting of uniparental ES cells has been vigorously investigated in mice and humans, however, little is known concerning other species. In the case of pigs, the establishment of putative PG ES cell lines has been reported (Brevini et al., 2010) but an epigenetic analysis has yet to be conducted. Numerous studies have demonstrated that the expression and methylation pattern of imprinted genes in ES cells are susceptible to alterations in in vitro culture (Dean et al., 1998; Humpherys et al., 2001). In vitro culture changes the epigenetic status of imprinted genes during the isolation of PG ES cells from their progenitor embryos and the changes in the expression of several genes, including U2af1rs1, Snrpn, and Igf2r, are correlated with the pluripotency of PG ES cells (Li et al., 2009). Several paternally expressed imprinted genes are activated in PG ES cells, and this phenomenon is associated with a change in the methylation pattern of related differentially methylated regions (DMRs)
(Jiang et al., 2007). It is, however, unclear whether such uniparental pluripotent cells can be used as a model system for studying the mechanisms of epigenetic imprinting events that occur during embryonic development. Therefore, in the present study an allele-specific methylation pattern is maintained in ES cells of pigs and there were investigations as to whether the ES derivation process influences the stability of an epigenetic imprint. For this experiment, one IVF and three porcine PG ES cell lines were established and evaluated for pluripotency following passage number 10. In previous studies, the amount of mRNA and DNA methylation patterns of DMRs at the H19/IGF2 locus have been determined in fertilized and uniparental pre-implantation pig embryos (Park et al., 2009, 2011). Based on these findings, amounts of mRNA and the DNA methylation of DMR3 at the H19/IGF2 locus between PG and IVF ES cell lines were compared. 2. Materials and methods 2.1. Ethics statement The pig experiments were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Veterinary and Quarantine Service and were supervised by Gyeonggido Livestock and Veterinary Service. Each study was approved by the animal ethics committee of Sooam biotech research foundation (license number AEC20081021-0001). Pig ovaries were provided by the regional slaughterhouse (Hyup-Shin, Anyang, Korea). 2.2. In vitro maturation (IVM) Ovaries were collected from prepubescent gilts at the slaughterhouse and transported to the laboratory in 0.9% (w/v) NaCl supplemented with 100 mg/ml streptomycin sulfate (Amresco, Solon, OH) within 1 h at 37 ◦ C. Cumulus–oocyte complexes (COC) were obtained from follicles that were 3 to 6 mm in diameter using 18gauge microneedles. Oocyte with an evenly granulated cytoplasm and a compact surrounding cumulus mass were collected and washed twice with TL–HEPES–PVA medium (Tyrode’s lactate–HEPES medium supplemented with 0.01% polyvinyl alcohol). After washing, 40 to 50 COC were transferred to 500 l of IVM medium (TCM199; Gibco, Carlsbad, CA) supplemented with 10 ng/ml epidermal growth factor (EGF), 1 g/ml insulin (SigmaAldrich, St. Louis, MO), 4 IU/ml eCG (Intervet, Boxmeer, The Netherlands), hCG (Intervet), and 10% (v/v) porcine follicular fluid (pFF). After 22 h of culture, the COC were transferred to an IVM medium without hormones and cultured for an additional 22 h at 38.5 ◦ C in an atmosphere containing 5% CO2 and 100% humidity. 2.3. In vitro fertilization (IVF) Fertilization was performed as described in a previous study (Park et al., 2011). At 42 h of IVM, 15 to 20 denuded MII oocytes were placed in 40 l drops of modified Tris-buffered medium (mTBM) that had been covered
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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with warm mineral oil in a 60-mmdish. Fresh semen ejaculated from a Duroc boar was supplied by DARBY A.I. center (Chungju, South Korea). The semen sample was washed twice by centrifugation at 350 × g for 3 min in phosphate-buffered saline (PBS). The sperm pellet was then re-suspended and adjusted to a concentration of 1 × 106 sperm/ml. The appropriate concentration of sperm was introduced into the oocyte-containing medium drop and the cells were incubated for 6 h at 38.5 ◦ C. After fertilization, excess spermatozoa were removed from oocytes by a repetitive pipetting action, and fertilized oocytes were washed three times in a culture medium (Porcine Zygote Medium-3; PZM3) containing a 1% nonessential amino acid/minimum essential medium solution. 2.4. In vitro culture (IVC) Fertilized or post-activated oocytes (n = 50 to 80) were cultured in 4-well dishes containing 500 l of PZM3 for 168 h. Embryo culture conditions were maintained at 38.5 ◦ C in an atmosphere containing 5% CO2 , 5% O2 , and 100% humidity. 2.5. Porcine ES-like cell culture Hatched blastocysts collected at 7 days were transferred onto mitomycin C-treated mouse embryonic fibroblasts (MEFs), prepared on 0.1% gelatin treated four-well culture dishes, cultured in PES medium consisting of low glucose DMEM (Gibco) and Ham’s F10 medium (Gibco) (50:50 mixture) supplemented with 15% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 2 mM l-glutamine (Gibco), 0.1 mM -mercaptoethanol (Gibco), 1% MEM nonessential amino acids (Gibco), 1% antibiotic-antimycotic (Gibco) containing cytokines 40 ng/ml human recombinant SCF (hrSCF; R&D Systems, Minneapolis, MN), 20 ng/ml human recombinant bFGF (hrbFGF; R&D Systems). When the primary colonies fully expanded at 12 to 14 days after seeding, these colonies were mechanically dissociated into several pieces and transferred onto new feeder cells. The pES cells showing typical molphology were subcultured within 5 to 6 days. 2.6. Alkaline phosphastase (AP) activity analysis The pES cells were fixed with 4% formaldehyde for 15 min at room temperature after washing twice with PBS. Fixed cells were stained by using nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP) stock solution (Roche, Madison, WI) in buffer solution for 30 min at room temperature. 2.7. Karyotyping The ES cells were incubated in medium that was supplemented with 0.02 g/ml colcemide (Sigma-Aldrich) for 1 h at 37 ◦ C in an atmosphere of 5% CO2 in air. After trypsinization and treatment with hypotonic KCl (0.56%) for 20 min, the cells were placed in a hypotonic solution, fixed in a 3:1 (v/v) mixture of methanol and acetic acid and spread on clean microscopic slides by gentle dropping. After staining
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with Giemsa (1:20 dilution; Sigma-Aldrich) for 20 min, the chromosomes were examined at ×1000 magnification. 2.8. In vitro differentiation Embryoid bodies (EB) formation was performed to assess the differentiation ability in vitro of PES cells via immuno-staining. PES colonies were detached from the plates and dissociated into small pieces, and these cells were cultured in suspension condition. Spontaneous EB were grown in PES medium, supplemented with 10 ng of recombinant human BMP4 (R&D Systems), without bFGF and SCF. To confirm the ability of in vitro differentiation, the EB cultured during 6 days were plated onto the 60 mm dishes and cultured 20 days more. After 20 days, cells derived from EB were fixed and used for immunostaining. 2.9. mRNA synthesis and quantitative PCR Embryos that reached the blastocyst stage on Day 7 (cultured for 7 days after fertilization or parthenogenetic activation) with good morphological features were selected and treated with Tyrode’s acid to remove the zona pellucida prior to use for mRNA isolation. The mRNA was immediately isolated from eight pooled embryos and total RNA was extracted from ES cells by using the Dynabeads mRNA Direct Kit (Invitrogen) and easy-BLUETotal RNA Extraction Kit (iNtRON Biotechnology, Korea) respectively according to the manufacturers’ instructions. The concentration and integrity of the mRNA was determined using a NanoDrop ND-1000 (Thermo Scientific, Wilmington, DE). cDNA synthesis was performed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturers’ instructions. Using a final volume of 20 l, synthesis was carried out at 37.5 ◦ C for 60 min and samples were subsequently incubated at 95 ◦ C for 5 min to inactivate reverse transcriptase. Amplification and detection were conducted with the ABI 7300 Real-Time PCR System (Applied Biosystems) using a Power SYBR Green PCR master mix (Applied Biosystems) under the following conditions: 95 ◦ C for 15 min, 40 cycles of denaturation at 95 ◦ C for 15 s, and annealing at 60 ◦ C for 60 s. All of the threshold cycle (CT) values of the tested genes were normalized to amount of mRNA for ACTB, and relative ratios were calculated using the 2−Ct method. The amount and amplification patterns of ACTB were not different between cell lines when 1 l of each cDNA samples was used for the experiment. Three independent experiments were performed with each replicate containing eight pooled blastocysts and ES cells. The replicate CT values showed coefficients of variation more than 2% were rejected. Specificities of all of the designed primers used in this study were confirmed via sequencing analysis (Table 1). 2.10. Immuno-staining For immuno-staining, fixed cells were washed and exposed to 10% goat serum (R&D Systems) and 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 60 min. The cells
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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Table 1 PCR primers used to detect gene expression in porcine blastocysts and ES-like cells. Primers OCT4 NANOG SOX2 H19 IGF2 ACTB
Sequences For 5 -AACGATCAAGCAGTGACTATTCG-3 Rev 5 -GAGTACAGGGTGGTGAAGTGAGG-3 For 5 -AATCTTCACCAATGCCTGAG-3 Rev 5 -GGCTGTCCTGAATAAGCAGA-3 For 5 -CGGCGGCAGGATCGGC-3 Rev 5 -GAGCTCCGCGAGGAAAA-3 For 5 -CTCAAACGACAAGAGATGGT-3 Rev 5 -AGTGTAGTGGCTCCAGAATG-3 For 5 -AAGAGTGCTCTTCCGTAG-3 Rev 5 -TGTCATAGCGGAAGAACTTG-3 For 5 -GTGGACATCAGGAAGGACCTCTA-3 Rev 5 -ATGATCTTGATCTTCATGGTGCT-3
were then incubated with primary antibodies overnight at 4 ◦ C. Primary antibodies used are: OCT4, SOX2, SSEA1, SSEA-4, TRA-1-60, TRA-1-81, cytokeratin 17, desmin, and vimentin (MAB4305, MAB4343, MAB4301, MAB4304, MAB4360, MAB4381, MAB1625, MAB3430, MAB3400, 1:200; Millipore, Temecula, CA), NANOG (ab21624, 1:200; Abcam, Cambridge, UK). After washing with 0.1% Tween20 (Sigma-Aldrich) solution, these cells were incubated with secondary antibody and then nuclei were stained with 10 g/ml Hoechst 33342. Secondary antibodies used are: goat anti-rabbit IgG, goat anti-mouse IgG, goat anti-mouse IgM (A11012, A11001, A21044, 1:500; Molecular Probes, Carlsbad, CA).
2.11. Genome methylation assay Genomic DNA was isolated from each 10 IVF and parthenogenetic blastocysts and pES cells with the Gspin Genomic DNA extraction kit for Cell/Tissue (iNtRON Biotechnology) according to the manufacturer’s instruction. The genomic DNA was digested with EcoRI (New England Bolabs, Germany). The bisulfite treatment of DNA was performed as described (Clark et al., 2006). The bisulfite-treated DNA was purified using the PCR quickspin PCR product purification kit (iNtRON Biotechnology) and desulfonated with 0.3 M NaOH and 10 g Escherichia coli tRNA (Sigma Aldrich) for 20 min at 37 ◦ C. The DNA was purified again and then re-suspended in distilled water. Nested PCR amplifications of bisulfite-treated DNA were performed using the primers for H19 DMR3 region described in our previous study (Park et al., 2009). Tworound PCR was conducted with a 2X PCR master mix solution (iNtRON Biotechnology) containing 1 pmol of the primers. The PCR product was gel purified with the MEGAspin agarose gel extraction kit (iNtRON Biotechnology) and the purified products were cloned into the pGEMT-Easy vector (Promega, USA) followed by transformation into E. coli cells (Novagen, USA). More than 10 insert-positive plasmid clones were sequenced by an ABI PRISM 3730 automated sequencer (Applied Biosystems, Carlsbad, CA). The methylation patterns were analyzed in sequences derived from clones with ≥98% cytosine conversions only and all experiments were repeated at least three times for blastocysts and pES cells, respectively.
Genbank accession number
Size (bp)
AF074419
153
DQ447201
141
EU519824
113
AY044827
122
NM213883
156
U07786
137
2.12. Statistical analysis The data obtained in this study were analyzed using the GraphPad Prism statistical program (GraphPad Software, San Diego, CA). Data are presented as the mean ± S.E.M. and were examined using analysis of variance (ANOVA). A probability of P < 0.05 was considered statistically significant. 3. Results 3.1. Establishment of parthenogenetic embryonic stem-like cell lines Primary outgrowths were formed 5 to 7 days after transferring hatched PG blastocysts onto mouse embryonic fibroblasts (MEFs; Fig. 1A). As the outgrowths expanded, these were divided into 5 to 10 pieces by manual dissection using a glass capillary tube until Day 14, at which time the outgrowths were moved onto fresh feeder layers. A total of 99 blastocysts were seeded onto feeder layers, however, only four primary outgrowths maintained long term culture up to passage 40; one failed to be maintained. Although the derivation efficiency was very low, three PG ES lines (pESP1, pESP2, pESP3) were successfully cryopreserved, and survived after thawing. The ES colonies were morphologically similar to porcine-induced pluripotent stem (iPS) cells and grew in monolayer. Alkaline phosphatase (AP) was highly expressed in the current PG ES lines (Fig. 1B). Karyotype analysis revealed that these three cell lines contained a normal number of 38 XX chromosomes at mid-passages (20–30) (Fig. 1E). Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that three PG ES cell lines and an in vitro fertilization (IVF) ES cell line expressed pluripotent genes including OCT4, SOX2, and NANOG (Fig. 1C, pESP3 data not shown). Furthermore, the expressions of OCT-4, NANOG, SOX2, SSEA1, TRA1-60, and TRA1-81 genes in these three PG ES lines were confirmed by immunocytochemistry both at early (9–12) and late (40–42) passages (Fig. 1D). 3.2. Differentiation of parthenogenetic porcine embryonic stem cells To identify the differentiation capacity of PG ES cells, the embryoid body (EB) formation method was utilized.
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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Fig. 1. Characterization of porcine PG ES cells. (A) A primary outgrowth derived from a PG blastocyst at Day 8 after seeding. (B) Alkaline phophatase activity of PG ES cells (pESP1). (C) Expression of pluripotent genes in PG ES cells by RT-PCR. RT(−) lane shows product of PCR conducted without template cDNA. (D) Immunoflourescence staining of pluripotent markers (pESP2 at passage 42). (E) Karyotype of PG ES cells (pESP2 at passage 22) by G banding analysis (Scale bar: 200 m).
The PG ES cells were dissected into small clumps of cells and then cultured in hanging drops for 3 days. The cells that aggregated in the hanging drops were transferred to a nonadherent plate and cultured for an additional 3 days. After 6 days of suspension culture, cystic EB were formed from the three cell lines (Fig. 2A) and the OCT4 gene expression
in the cells rapidly decreased compared to undifferentiated cells (data not shown). To induce the spontaneous differentiation of PG ES cells into three germ layers, EB formed in the suspension condition for 6 days were placed directly onto the tissue culture plate. After 20 days, these EB differentiated into cells showing several different morphologies.
Fig. 2. In vitro differentiation of PG ES cells of pigs. (A) Embryoid bodies derived by suspension culture under differentiation condition during 7 days are shown. (B) Spontaneous differentiation of PG ES cells (pESP1) in vitro into three germ layers (DESMIN, mesoderm; CYTOKERATIN 17, endoderm; VIMENTIN, ectoderm). Cell nuclei were stained with HOECHST 33342 (Scale bar: 50 m).
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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Immunocytochemistry analysis revealed that PG ES cells were positive for DESMIN (mesoderm), CYTOKERATIN 17 (endoderm), and VIMENTIN (ectoderm), indicating that PG ES cells of pigs can differentiate into three embryonic germ layers in an in vitro environment. The IVF ES cell line (pESF6) also contains general properties of ES cells and details including pluripotent marker expression, karyotype, and differentiation ability are described in a previous report (Park et al., 2013). 3.3. Expression patterns of H19 and Igf2 in blastocysts and porcine embryonic stem cells To verify whether the expression of imprinted genes is stable in PG ES cells of pigs, the mRNA patterns of H19 and IGF2genes were initially examined in 7 day blastocysts, the progenitors of PG ES cells. The mRNA of H19, a paternally imprinted gene, in PG blastocysts was 16-fold greater compared to IVF blastocysts (Fig. 3A). Although the amount of mRNA forH19of PG ES cells remained greater than IVF ES cells at passage 10, the difference in transcript abundance decreased relative to blastocysts (Fig. 3B). However, one PG ES line (pESP1) had less mRNA for the H19 gene compared to IVF ES cells, indicating that the H19 gene expression of PG
ES cells is not stable in an in vitro culture environment. In contrast to H19, the amount of mRNA of IGF2 gene, a maternally imprinted gene, was two-fold less in PG blastocysts than in IVF blastocysts, as expected, and this gene expression tendency was maintained in PG ES cells, except for the pESP1 line (Fig. 3B). While the amount of IGF2 mRNA for the pESP2 and pESP3 lines was two-fold less than the pESF6 line of cells, there was no significant difference in mRNA between pESP1 and pESF6 (Fig. 3B). These data suggest that in vitro culture can re-program the expression of H19 and IGF2 genes in PG ES cells after isolation from blastocysts. 3.4. Methylation status of IGF2/H19 DMR3 in blastocysts and porcine embryonic stem cells The H19 and IGF2 genes share enhancers that reside downstream of the H19 gene and the reciprocal expression of both genes is regulated by DNA methylation at the IGF2/H19 DMR. To understand the altered expression patterns of the H19 and IGF2 genes in PG ES cells, a methylation analysis was conducted on the IGF2/H19 DMR3 region in blastocysts and ES cells using a bisulfite sequencing method. Prior to the examination of blastocysts and ES cells, the adult liver of pigs was analyzed to validate
Fig. 3. Relative amounts of mRNA for the H19 and IGF2 genes in porcine (A) IVF (n = 24)/PG (n = 24) blastocysts and (B) IVF (pESF6)/PG (pESP1, pESP2, pESP3) ES cells. The relative amounts of mRNA were quantified using qRT-PCR and then calculated with the 2−Ct method. Three replicate samples were examined for each class. ACTB served as internal controls. Three replicate samples were examined for each class. Bars indicate mean ± SEM (n = 3).* P < 0.05 compared with IVF counterparts (control).
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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Fig. 4. Methylation status of IGF2/H19 DMR3 in blastocysts and PG ES cells of pigs. Liver was analyzed to validate this methylation assay. Circles are CpG sites within the region analyzed: 䊉, methylated cytosines; , un-methylated cytosines.
the methylation assay. A hemi-methylation of 43.75% indicated that there were no potential artifacts or bias. At Day 8, IVF blastocysts displayed a hemi-methylated (50%) status while a pattern of hypomethylation (22%) was observed in PG blastocysts at the same stage (Fig. 4). The DMR3 region of pESF6 was hemi-methylated (60%) in a manner similar to IVF blastocysts, however, all of the PG ES cells examined displayed an un-methylated status (pESP1: 5.6%; pESP2: 3.5%; pESP3: 3.5%) indicating a reduction in methylation of over 16% relative to PG blastocysts (Fig. 4). 4. Discussion The H19 and Igf2 genes are the most widely studied elements of a well-known cluster of imprinted genes. The H19 gene is expressed by the maternal allele while the Igf2 gene is transcribed from the paternal allele (FergusonSmith et al., 1993). The current findings indicate that the H19 gene expression is much greater in PG blastocysts than IVF blastocysts. However, the expression of the H19 gene is dramatically decreased in one PG ES line (pESP1) and a significantly lesser amount of transcripts were detected relative to IVF ES cells after the ES cells were isolated from PG blastocysts and cultured on MEFs. Interestingly, the pESP1 line that underwent the most remarkable transcriptional change for the H19 gene had a similar IGF2 gene expression to the IVF ES cells whereas the relative amount of gene expression in the other two PG ES lines remained minimal. This indicates that PG ES cells underwent an alteration in the expressions of the H19 and IGF2 genes during ES cell isolation process when cultured in vitro, which is consistent with previous findings that successfully produced live mice pups directly from PG ES cells through tetraploid embryo complementation (Chen et al., 2009). The PG ES cells in the previous study contributed to all organs in chimeras and were indistinguishable in most
aspects from the ES cells derived from fertilized embryos. The subsequent analysis of imprinted genes demonstrated that the extent of transcription of maternally expressed genes, including Igf2r and H19, in PG ES cells decreased similar to the degree observed in IVF ES cells. Moreover, this tendency can be found in paternally expressed genes such as Igf2, Snrpn, Peg1, and U2af1-rs1 which do not significantly differ between PG ES and IVF ES cells (Li et al., 2009). Thus, the present findings support the notion that the expression of imprinted genes in embryonic cells can be changed by in vitro culture (Dean et al., 1998; Humpherys et al., 2001). Results of the present study clearly showed that the majority of CpG sites in PG ES cell lines were almost completely un-methylated, whereas the CpGs showed some methylation in PG blastocysts. Previous study showed that the partial methylation pattern in this region of in vitroderived embryos may be responsible for in vitro culture environments (Park et al., 2011). However, such partial methylation was not observed in PG ES cells, it may be possible that poor quality embryos with epigenetic defects were excluded by the derivation process. Although it is well accepted that the DMRs of H19 and Igf2 genes regulate the reciprocal gene expression in a methylation-sensitive manner according to boundary and chromatin models (Leighton et al., 1995; Kaffer et al., 2001; Davies et al., 2002), the gene expression and methylation results of the pESP1 line are not likely to be explained by this theory. The pESP1 line had a lesser transcript abundance of the H19 than for the pESF6 line, but the PG ES cell line was in hypo-methylation, while a methylation of 60% was observed for the pESF6 line. Furthermore, despite a greater de-methylated status in pESP2 and pESP3 lines than in PG blastocysts, the H19 gene expression of pESP cells decreased. Several studies have shown to be hypomethylated pattern of Igf2/H19 DMRs in the mouse PG ES cells (Horii et al., 2008; Chen et al., 2009; Li et al., 2009).
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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A previous study showed that methylation status of the DMRs is not correlated with the activation and expression of H19 and Igf2 genes (Li et al., 2009). In this previous study, the decreased methylation of Snrpn, Peg1, and U2af1rs1 genes was highly correlated with the increased gene expression, suggesting that the H19 and Igf2 gene expressions are regulated by another mechanism. In addition, the IGF2 gene is expressed from both alleles even though the H19 DMR is indeed differentially methylated in the adult pig liver (Braunschweig et al., 2011). It has been proposed that allele-specific histone acetylation may involve a regulation of the DMRs for the Igf2/H19 gene (Wylie et al., 2000; Carr et al., 2007). In the present study, three putative PG ES cell lines were derived from PG blastocysts produced by electrical activation. These cells were maintained in an undifferentiated state for more than 40 passages, and the cell colony was flattened and grew in a monolayer shape similar to human ES cells rather than mouse ES cells. These cells exhibited normal karyotypes and the pluripotent marker OCT4, NANOG, and SOX2 genes were highly expressed when cultured for more than 20 passages. In addition, the capacity to differentiate into three germ layers was confirmed by EB formation and was followed by the induction of spontaneous differentiation in vitro. The amounts of OCT4 and NANOG gene transcripts in the pESF6 cell line were less compared to PG ES cells but the pluripotent marker gene expression at the level of proteins has been previously confirmed as well as the tissue differentiation capacity (Park et al., 2013). According to the present findings, the pESF6 line used in this study for the comparison of epigenetic status in PG ES cells possesses epiblast stem cell (EpiSC) features even though the cell line did not form teratoma. Moreover, as the PG ES cells were generated and cultured in the same system and had similar characteristics to that of the pESF6 line, these cells are also considered to possess EpiSC markers. Unfortunately, the PG ES cells established in the present study lack the in vivo capacity for differentiation into three germ layers, and the molecular pathways supporting self-renewal and pluripotency might be controversial. Nevertheless, these cells might be very useful tools for understanding genomic imprinting because each cell line originated from individual blastocysts. Analysis of genomic imprinting on individual embryos is necessary to avoid variations that can appear in pooled embryos and advance understanding of this process. Moreover, optimal porcine ES cell derivation and culture systems that can support the maintenance of authentic ES cells are required. If so, pig ES cells hold tremendous potential for studying the epigenetic dynamics of ES cells and aiding our understanding of genomic imprinting at pre- and peri-implantation stages. Conflict of interest The authors have no conflicts of interest that biased or influenced this work. Acknowledgments This research was supported by the grant of Bioindustry Technology Development Program of iPET (Korea
Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries, grant no. 111160-04) funded by the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. This work was also supported by the Next BioGreen 21 program (grant no. PJ009493), Rural Development Administration. References Allen, N.D., Barton, S.C., Hilton, K., Norris, M.L., Surani, M.A., 1994. A functional analysis of imprinting in parthenogenetic embryonic stem cells. Development 120, 1473–1482. Barton, S.C., Surani, M.A., Norris, M.L., 1984. Role of paternal and maternal genomes in mouse development. Nature 311, 374–376. Bischoff, S.R., Tsai, S., Hardison, N., Motsinger-Reif, A.A., Freking, B.A., Nonneman, D., Rohrer, G., Piedrahita, J.A., 2009. Characterization of conserved and nonconserved imprinted genes in swine. Biol. Reprod. 81, 906–920. Braunschweig, M.H., Owczarek-Lipska, M., Stahlberger-Saitbekova, N., 2011. Relationship of porcine IGF2 imprinting status to DNA methylation at the H19 DMD and the IGF2 DMRs 1 and 2. BMC Genet. 12, 47. Brevini, T.A., Pennarossa, G., Attanasio, L., Vanelli, A., Gasparrini, B., Gandolfi, F., 2010. Culture conditions and signalling networks promoting the establishment of cell lines from parthenogenetic and biparental pig embryos. Stem Cell Rev. Rep. 6, 484–495. Carr, M.S., Yevtodiyenko, A., Schmidt, C.L., Schmidt, J.V., 2007. Allelespecific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain. Genomics 89, 280–290. Chen, Z., Liu, Z., Huang, J., Amano, T., Li, C., Cao, S., Wu, C., Liu, B., Zhou, L., Carter, M.G., Keefe, D.L., Yang, X., Liu, L., 2009. Birth of parthenote mice directly from parthenogenetic embryonic stem cells. Stem Cells 27, 2136–2145. Cibelli, J.B., Cunniff, K., Vrana, K.E., 2006. Embryonic stem cells from parthenotes. Methods Enzymol. 418, 117–135. Cibelli, J.B., Grant, K.A., Chapman, K.B., Cunniff, K., Worst, T., Green, H.L., Walker, S.J., Gutin, P.H., Vilner, L., Tabar, V., Dominko, T., Kane, J., Wettstein, P.J., Lanza, R.P., Studer, L., Vrana, K.E., West, M.D., 2002. Parthenogenetic stem cells in nonhuman primates. Science 295, 819. Clark, S.J., Statham, A., Stirzaker, C., Molloy, P.L., Frommer, M., 2006. DNA methylation: bisulphite modification and analysis. Nat. Protoc. 1, 2353–2364. Davies, K., Bowden, L., Smith, P., Dean, W., Hill, D., Furuumi, H., Sasaki, H., Cattanach, B., Reik, W., 2002. Disruption of mesodermal enhancers for Igf2 in the minute mutant. Development 129, 1657–1668. Dean, W., Bowden, L., Aitchison, A., Klose, J., Moore, T., Meneses, J.J., Reik, W., Feil, R., 1998. Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes. Development 125, 2273–2282. Dindot, S.V., Kent, K.C., Evers, B., Loskutoff, N., Womack, J., Piedrahita, J.A., 2004. Conservation of genomic imprinting at the XIST, IGF2, and GTL2 loci in the bovine. Mamm. Genome 15, 966–974. Fang, Z.F., Gai, H., Huang, Y.Z., Li, S.G., Chen, X.J., Shi, J.J., Wu, L., Liu, A., Xu, P., Sheng, H.Z., 2006. Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos. Exp. Cell Res. 312, 3669–3682. Ferguson-Smith, A.C., Sasaki, H., Cattanach, B.M., Surani, M.A., 1993. Parental-origin-specific epigenetic modification of the mouse H19 gene. Nature 362, 751–755. Horii, T., Kimura, M., Morita, S., Nagao, Y., Hatada, I., 2008. Loss of genomic imprinting in mouse parthenogenetic embryonic stem cells. Stem Cells 26, 79–88. Humpherys, D., Eggan, K., Akutsu, H., Hochedlinger, K., Rideout, W.M., 3rd, Biniszkiewicz, D., Yanagimachi, R., Jaenisch, R., 2001. Epigenetic instability in ES cells and cloned mice. Science 293, 95–97. Jiang, H., Sun, B., Wang, W., Zhang, Z., Gao, F., Shi, G., Cui, B., Kong, X., He, Z., Ding, X., Kuang, Y., Fei, J., Sun, Y.J., Feng, Y., Jin, Y., 2007. Activation of paternally expressed imprinted genes in newly derived germlinecompetent mouse parthenogenetic embryonic stem cell lines. Cell Res. 17, 792–803. Kaffer, C.R., Grinberg, A., Pfeifer, K., 2001. Regulatory mechanisms at the mouse Igf2/H19 locus. Mol. Cell. Biol. 21, 8189–8196. Leighton, P.A., Saam, J.R., Ingram, R.S., Stewart, C.L., Tilghman, S.M., 1995. An enhancer deletion affects both H19 and Igf2 expression. Genes Dev. 9, 2079–2089. Li, C., Chen, Z., Liu, Z., Huang, J., Zhang, W., Zhou, L., Keefe, D.L., Liu, L., 2009. Correlation of expression and methylation of imprinted genes
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Sritanaudomchai, H., Pavasuthipaisit, K., Kitiyanant, Y., Kupradinun, P., Mitalipov, S., Kusamran, T., 2007. Characterization and multilineage differentiation of embryonic stem cells derived from a buffalo parthenogenetic embryo. Mol. Reprod. Dev. 74, 1295–1302. Surani, M.A., Barton, S.C., Norris, M.L., 1984. Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 308, 548–550. Vrana, K.E., Hipp, J.D., Goss, A.M., McCool, B.A., Riddle, D.R., Walker, S.J., Wettstein, P.J., Studer, L.P., Tabar, V., Cunniff, K., Chapman, K., Vilner, L., West, M.D., Grant, K.A., Cibelli, J.B., 2003. Nonhuman primate parthenogenetic stem cells. Proc. Nat. Acad. Sci. U.S.A. 100 (Suppl. 1), 11911–11916. Wylie, A.A., Murphy, S.K., Orton, T.C., Jirtle, R.L., 2000. Novel imprinted DLK1/GTL2 domain on human chromosome 14 contains motifs that mimic those implicated in IGF2/H19 regulation. Genome Res. 10, 1711–1718.
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Contents lists available at ScienceDirect
Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs Kyung-Jun Uh a,b , Chi-Hun Park b , Kwang-Hwan Choi a , Jin-Kyu Park a , Yeon-Woo Jeong b , Sangho Roh c , Sang-Hwan Hyun d , Taeyoung Shin b , Chang-Kyu Lee a,∗∗ , Woo Suk Hwang b,∗ a Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute for Agriculture and Life Science, Seoul National University, Seoul, Republic of Korea b Sooam Biotech Research Foundation, Seoul, Republic of Korea c Dental Research Institute and CLS21, Seoul National University School of Dentistry, Seoul, Republic of Korea d College of Veterinary Medicine, Chungbuk National University, Cheongju, Republic of Korea
a r t i c l e
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Article history: Received 11 June 2013 Received in revised form 25 March 2014 Accepted 28 March 2014 Available online xxx
Keywords: Imprinted gene Embryonic stem cell Parthenogenesis DNA methylation
a b s t r a c t To determine whether the genomic imprinting can be maintained during the process of embryonic stem (ES) cell derivation from pig blastocysts, mRNA and DNA methylation at the IGF2/H19 imprinting control region in putative ES cells derived from in vitro fertilized (IVF) and parthenogenetic (PG) embryos were investigated. In the present study, one IVFand three PG ES-like cell lines were established and analyzed for cellular characteristics such as pluripotent marker expression and differentiation capacity. The results showed that these putative ES cells derived from pig blastocysts fulfilled the general “stemness” criteria. The expression of the H19 gene was significantly greater in PG blastocysts than IVF blastocysts, but there were greater amounts of IGF2 in IVF than PG blastocysts. Of these putative ES cell lines, one PG line had less H19 gene expression than a IVF ES cell line while the other two PG lines had much greater expression of the H19 gene than the IVF line. In contrast, the IGF2 gene was upregulated in the same PG cell line relative to the other two PG cell lines and transcript abundance was similar to IVF ES-like cells. Despite the variable amounts of mRNA among the PG cell lines, the IGF2/H19 gene had a differentially methylated region (DMR) 3 was typically un-methylated in all PG cells, and hemi-methylated in the IVF cells. These findings indicated that the mRNA of H19 and IGF2 genes is susceptible to in vitro environments during the process of ES cell derivation from blastocysts but DNA methylation status at this region was well maintained. These altered gene expressions may not be associated with the methylation of the imprinting control region at this locus. Therefore, with their uni-parental genotype, the pluripotent differentiation potentials of PG ES cells could be a valuable tool for understanding genomic imprinting in embryonic development. © 2014 Elsevier B.V. All rights reserved.
∗ Corresponding authors at: Sooam Biotech Research Foundation, 64, Gyeongin-ro, Guro-gu, Seoul 108-105, Republic of Korea. Tel.: +82 2 2616 5658; fax: +82 2 2616 5672. ∗∗ Corresponding authors at: Department of Agricultural Biotechnology, Seoul National University, 1, Gwanak-ro, Gwanak-gu, Seoul 151-015, Republic of Korea. Tel.: +82 2 880 4805; fax: +82 2 873 4805. E-mail addresses: [email protected] (C.-K. Lee), [email protected] (W.S. Hwang). http://dx.doi.org/10.1016/j.anireprosci.2014.03.020 0378-4320/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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1. Introduction Genomic imprinting is an epigenetic mechanism that results in the expression of certain genes from only one of the two parental chromosomes. The exclusive expression of imprinted genes is dependent on whether these genes originate from either the maternal or paternal allele. To date, more than 80 imprinted genes have been identified in mice and humans and many of these genes are highly conserved across mammalian species, including pigs and cows (Dindot et al., 2004; Bischoff et al., 2009). Parental genomic imprints are deleted and re-established during germ cell development and, once established, the epigenetic imprints are maintained throughout development. Imprinted genes have important roles in early development and growth and influence postnatal behavior. DNA methylation is an important epigenetic marker that regulates parental imprinting for allele-specific gene expression (Reik and Walter, 2001). In mammals, PG embryos with two sets of maternal genomes fail early in development due to a lack of paternal-specific signals for the development of extraembryonic tissues (Barton et al., 1984; Surani et al., 1984). Although these developmental consequences occur within uniparental embryos, embryonic stem (ES) cells derived from these embryos in mice fulfill the criteria of biparental ES cells (Allen et al., 1994). PG ES cells have been successfully achieved in numerous mammalian species including rabbits (Fang et al., 2006), buffalo (Sritanaudomchai et al., 2007), non-human primates (Cibelli et al., 2002; Vrana et al., 2003), and humans (Mai et al., 2007). The PG ES cells provide a valuable source of cells or tissues for cell therapy as a substitute for fertilized ES cells due to their histocompatibility with the oocyte donor (Cibelli et al., 2006). The application of PG ES cells in regenerative medicine would circumvent the immune rejection problem as well as ethical concerns related to destroying fertilized embryos. Moreover, uniparental ES cell lines established from PG or androgenetic (AG) embryos have been a useful tool for understanding genomic imprinting due to their genotype. The comparative study of imprinting among PG, AG, and normal ES cells offers a great opportunity to understand the properties of parental imprinting. Genomic imprinting of uniparental ES cells has been vigorously investigated in mice and humans, however, little is known concerning other species. In the case of pigs, the establishment of putative PG ES cell lines has been reported (Brevini et al., 2010) but an epigenetic analysis has yet to be conducted. Numerous studies have demonstrated that the expression and methylation pattern of imprinted genes in ES cells are susceptible to alterations in in vitro culture (Dean et al., 1998; Humpherys et al., 2001). In vitro culture changes the epigenetic status of imprinted genes during the isolation of PG ES cells from their progenitor embryos and the changes in the expression of several genes, including U2af1rs1, Snrpn, and Igf2r, are correlated with the pluripotency of PG ES cells (Li et al., 2009). Several paternally expressed imprinted genes are activated in PG ES cells, and this phenomenon is associated with a change in the methylation pattern of related differentially methylated regions (DMRs)
(Jiang et al., 2007). It is, however, unclear whether such uniparental pluripotent cells can be used as a model system for studying the mechanisms of epigenetic imprinting events that occur during embryonic development. Therefore, in the present study an allele-specific methylation pattern is maintained in ES cells of pigs and there were investigations as to whether the ES derivation process influences the stability of an epigenetic imprint. For this experiment, one IVF and three porcine PG ES cell lines were established and evaluated for pluripotency following passage number 10. In previous studies, the amount of mRNA and DNA methylation patterns of DMRs at the H19/IGF2 locus have been determined in fertilized and uniparental pre-implantation pig embryos (Park et al., 2009, 2011). Based on these findings, amounts of mRNA and the DNA methylation of DMR3 at the H19/IGF2 locus between PG and IVF ES cell lines were compared. 2. Materials and methods 2.1. Ethics statement The pig experiments were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Veterinary and Quarantine Service and were supervised by Gyeonggido Livestock and Veterinary Service. Each study was approved by the animal ethics committee of Sooam biotech research foundation (license number AEC20081021-0001). Pig ovaries were provided by the regional slaughterhouse (Hyup-Shin, Anyang, Korea). 2.2. In vitro maturation (IVM) Ovaries were collected from prepubescent gilts at the slaughterhouse and transported to the laboratory in 0.9% (w/v) NaCl supplemented with 100 mg/ml streptomycin sulfate (Amresco, Solon, OH) within 1 h at 37 ◦ C. Cumulus–oocyte complexes (COC) were obtained from follicles that were 3 to 6 mm in diameter using 18gauge microneedles. Oocyte with an evenly granulated cytoplasm and a compact surrounding cumulus mass were collected and washed twice with TL–HEPES–PVA medium (Tyrode’s lactate–HEPES medium supplemented with 0.01% polyvinyl alcohol). After washing, 40 to 50 COC were transferred to 500 l of IVM medium (TCM199; Gibco, Carlsbad, CA) supplemented with 10 ng/ml epidermal growth factor (EGF), 1 g/ml insulin (SigmaAldrich, St. Louis, MO), 4 IU/ml eCG (Intervet, Boxmeer, The Netherlands), hCG (Intervet), and 10% (v/v) porcine follicular fluid (pFF). After 22 h of culture, the COC were transferred to an IVM medium without hormones and cultured for an additional 22 h at 38.5 ◦ C in an atmosphere containing 5% CO2 and 100% humidity. 2.3. In vitro fertilization (IVF) Fertilization was performed as described in a previous study (Park et al., 2011). At 42 h of IVM, 15 to 20 denuded MII oocytes were placed in 40 l drops of modified Tris-buffered medium (mTBM) that had been covered
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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with warm mineral oil in a 60-mmdish. Fresh semen ejaculated from a Duroc boar was supplied by DARBY A.I. center (Chungju, South Korea). The semen sample was washed twice by centrifugation at 350 × g for 3 min in phosphate-buffered saline (PBS). The sperm pellet was then re-suspended and adjusted to a concentration of 1 × 106 sperm/ml. The appropriate concentration of sperm was introduced into the oocyte-containing medium drop and the cells were incubated for 6 h at 38.5 ◦ C. After fertilization, excess spermatozoa were removed from oocytes by a repetitive pipetting action, and fertilized oocytes were washed three times in a culture medium (Porcine Zygote Medium-3; PZM3) containing a 1% nonessential amino acid/minimum essential medium solution. 2.4. In vitro culture (IVC) Fertilized or post-activated oocytes (n = 50 to 80) were cultured in 4-well dishes containing 500 l of PZM3 for 168 h. Embryo culture conditions were maintained at 38.5 ◦ C in an atmosphere containing 5% CO2 , 5% O2 , and 100% humidity. 2.5. Porcine ES-like cell culture Hatched blastocysts collected at 7 days were transferred onto mitomycin C-treated mouse embryonic fibroblasts (MEFs), prepared on 0.1% gelatin treated four-well culture dishes, cultured in PES medium consisting of low glucose DMEM (Gibco) and Ham’s F10 medium (Gibco) (50:50 mixture) supplemented with 15% fetal bovine serum (FBS; Hyclone, Logan, UT, USA), 2 mM l-glutamine (Gibco), 0.1 mM -mercaptoethanol (Gibco), 1% MEM nonessential amino acids (Gibco), 1% antibiotic-antimycotic (Gibco) containing cytokines 40 ng/ml human recombinant SCF (hrSCF; R&D Systems, Minneapolis, MN), 20 ng/ml human recombinant bFGF (hrbFGF; R&D Systems). When the primary colonies fully expanded at 12 to 14 days after seeding, these colonies were mechanically dissociated into several pieces and transferred onto new feeder cells. The pES cells showing typical molphology were subcultured within 5 to 6 days. 2.6. Alkaline phosphastase (AP) activity analysis The pES cells were fixed with 4% formaldehyde for 15 min at room temperature after washing twice with PBS. Fixed cells were stained by using nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP) stock solution (Roche, Madison, WI) in buffer solution for 30 min at room temperature. 2.7. Karyotyping The ES cells were incubated in medium that was supplemented with 0.02 g/ml colcemide (Sigma-Aldrich) for 1 h at 37 ◦ C in an atmosphere of 5% CO2 in air. After trypsinization and treatment with hypotonic KCl (0.56%) for 20 min, the cells were placed in a hypotonic solution, fixed in a 3:1 (v/v) mixture of methanol and acetic acid and spread on clean microscopic slides by gentle dropping. After staining
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with Giemsa (1:20 dilution; Sigma-Aldrich) for 20 min, the chromosomes were examined at ×1000 magnification. 2.8. In vitro differentiation Embryoid bodies (EB) formation was performed to assess the differentiation ability in vitro of PES cells via immuno-staining. PES colonies were detached from the plates and dissociated into small pieces, and these cells were cultured in suspension condition. Spontaneous EB were grown in PES medium, supplemented with 10 ng of recombinant human BMP4 (R&D Systems), without bFGF and SCF. To confirm the ability of in vitro differentiation, the EB cultured during 6 days were plated onto the 60 mm dishes and cultured 20 days more. After 20 days, cells derived from EB were fixed and used for immunostaining. 2.9. mRNA synthesis and quantitative PCR Embryos that reached the blastocyst stage on Day 7 (cultured for 7 days after fertilization or parthenogenetic activation) with good morphological features were selected and treated with Tyrode’s acid to remove the zona pellucida prior to use for mRNA isolation. The mRNA was immediately isolated from eight pooled embryos and total RNA was extracted from ES cells by using the Dynabeads mRNA Direct Kit (Invitrogen) and easy-BLUETotal RNA Extraction Kit (iNtRON Biotechnology, Korea) respectively according to the manufacturers’ instructions. The concentration and integrity of the mRNA was determined using a NanoDrop ND-1000 (Thermo Scientific, Wilmington, DE). cDNA synthesis was performed with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturers’ instructions. Using a final volume of 20 l, synthesis was carried out at 37.5 ◦ C for 60 min and samples were subsequently incubated at 95 ◦ C for 5 min to inactivate reverse transcriptase. Amplification and detection were conducted with the ABI 7300 Real-Time PCR System (Applied Biosystems) using a Power SYBR Green PCR master mix (Applied Biosystems) under the following conditions: 95 ◦ C for 15 min, 40 cycles of denaturation at 95 ◦ C for 15 s, and annealing at 60 ◦ C for 60 s. All of the threshold cycle (CT) values of the tested genes were normalized to amount of mRNA for ACTB, and relative ratios were calculated using the 2−Ct method. The amount and amplification patterns of ACTB were not different between cell lines when 1 l of each cDNA samples was used for the experiment. Three independent experiments were performed with each replicate containing eight pooled blastocysts and ES cells. The replicate CT values showed coefficients of variation more than 2% were rejected. Specificities of all of the designed primers used in this study were confirmed via sequencing analysis (Table 1). 2.10. Immuno-staining For immuno-staining, fixed cells were washed and exposed to 10% goat serum (R&D Systems) and 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 60 min. The cells
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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Table 1 PCR primers used to detect gene expression in porcine blastocysts and ES-like cells. Primers OCT4 NANOG SOX2 H19 IGF2 ACTB
Sequences For 5 -AACGATCAAGCAGTGACTATTCG-3 Rev 5 -GAGTACAGGGTGGTGAAGTGAGG-3 For 5 -AATCTTCACCAATGCCTGAG-3 Rev 5 -GGCTGTCCTGAATAAGCAGA-3 For 5 -CGGCGGCAGGATCGGC-3 Rev 5 -GAGCTCCGCGAGGAAAA-3 For 5 -CTCAAACGACAAGAGATGGT-3 Rev 5 -AGTGTAGTGGCTCCAGAATG-3 For 5 -AAGAGTGCTCTTCCGTAG-3 Rev 5 -TGTCATAGCGGAAGAACTTG-3 For 5 -GTGGACATCAGGAAGGACCTCTA-3 Rev 5 -ATGATCTTGATCTTCATGGTGCT-3
were then incubated with primary antibodies overnight at 4 ◦ C. Primary antibodies used are: OCT4, SOX2, SSEA1, SSEA-4, TRA-1-60, TRA-1-81, cytokeratin 17, desmin, and vimentin (MAB4305, MAB4343, MAB4301, MAB4304, MAB4360, MAB4381, MAB1625, MAB3430, MAB3400, 1:200; Millipore, Temecula, CA), NANOG (ab21624, 1:200; Abcam, Cambridge, UK). After washing with 0.1% Tween20 (Sigma-Aldrich) solution, these cells were incubated with secondary antibody and then nuclei were stained with 10 g/ml Hoechst 33342. Secondary antibodies used are: goat anti-rabbit IgG, goat anti-mouse IgG, goat anti-mouse IgM (A11012, A11001, A21044, 1:500; Molecular Probes, Carlsbad, CA).
2.11. Genome methylation assay Genomic DNA was isolated from each 10 IVF and parthenogenetic blastocysts and pES cells with the Gspin Genomic DNA extraction kit for Cell/Tissue (iNtRON Biotechnology) according to the manufacturer’s instruction. The genomic DNA was digested with EcoRI (New England Bolabs, Germany). The bisulfite treatment of DNA was performed as described (Clark et al., 2006). The bisulfite-treated DNA was purified using the PCR quickspin PCR product purification kit (iNtRON Biotechnology) and desulfonated with 0.3 M NaOH and 10 g Escherichia coli tRNA (Sigma Aldrich) for 20 min at 37 ◦ C. The DNA was purified again and then re-suspended in distilled water. Nested PCR amplifications of bisulfite-treated DNA were performed using the primers for H19 DMR3 region described in our previous study (Park et al., 2009). Tworound PCR was conducted with a 2X PCR master mix solution (iNtRON Biotechnology) containing 1 pmol of the primers. The PCR product was gel purified with the MEGAspin agarose gel extraction kit (iNtRON Biotechnology) and the purified products were cloned into the pGEMT-Easy vector (Promega, USA) followed by transformation into E. coli cells (Novagen, USA). More than 10 insert-positive plasmid clones were sequenced by an ABI PRISM 3730 automated sequencer (Applied Biosystems, Carlsbad, CA). The methylation patterns were analyzed in sequences derived from clones with ≥98% cytosine conversions only and all experiments were repeated at least three times for blastocysts and pES cells, respectively.
Genbank accession number
Size (bp)
AF074419
153
DQ447201
141
EU519824
113
AY044827
122
NM213883
156
U07786
137
2.12. Statistical analysis The data obtained in this study were analyzed using the GraphPad Prism statistical program (GraphPad Software, San Diego, CA). Data are presented as the mean ± S.E.M. and were examined using analysis of variance (ANOVA). A probability of P < 0.05 was considered statistically significant. 3. Results 3.1. Establishment of parthenogenetic embryonic stem-like cell lines Primary outgrowths were formed 5 to 7 days after transferring hatched PG blastocysts onto mouse embryonic fibroblasts (MEFs; Fig. 1A). As the outgrowths expanded, these were divided into 5 to 10 pieces by manual dissection using a glass capillary tube until Day 14, at which time the outgrowths were moved onto fresh feeder layers. A total of 99 blastocysts were seeded onto feeder layers, however, only four primary outgrowths maintained long term culture up to passage 40; one failed to be maintained. Although the derivation efficiency was very low, three PG ES lines (pESP1, pESP2, pESP3) were successfully cryopreserved, and survived after thawing. The ES colonies were morphologically similar to porcine-induced pluripotent stem (iPS) cells and grew in monolayer. Alkaline phosphatase (AP) was highly expressed in the current PG ES lines (Fig. 1B). Karyotype analysis revealed that these three cell lines contained a normal number of 38 XX chromosomes at mid-passages (20–30) (Fig. 1E). Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that three PG ES cell lines and an in vitro fertilization (IVF) ES cell line expressed pluripotent genes including OCT4, SOX2, and NANOG (Fig. 1C, pESP3 data not shown). Furthermore, the expressions of OCT-4, NANOG, SOX2, SSEA1, TRA1-60, and TRA1-81 genes in these three PG ES lines were confirmed by immunocytochemistry both at early (9–12) and late (40–42) passages (Fig. 1D). 3.2. Differentiation of parthenogenetic porcine embryonic stem cells To identify the differentiation capacity of PG ES cells, the embryoid body (EB) formation method was utilized.
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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Fig. 1. Characterization of porcine PG ES cells. (A) A primary outgrowth derived from a PG blastocyst at Day 8 after seeding. (B) Alkaline phophatase activity of PG ES cells (pESP1). (C) Expression of pluripotent genes in PG ES cells by RT-PCR. RT(−) lane shows product of PCR conducted without template cDNA. (D) Immunoflourescence staining of pluripotent markers (pESP2 at passage 42). (E) Karyotype of PG ES cells (pESP2 at passage 22) by G banding analysis (Scale bar: 200 m).
The PG ES cells were dissected into small clumps of cells and then cultured in hanging drops for 3 days. The cells that aggregated in the hanging drops were transferred to a nonadherent plate and cultured for an additional 3 days. After 6 days of suspension culture, cystic EB were formed from the three cell lines (Fig. 2A) and the OCT4 gene expression
in the cells rapidly decreased compared to undifferentiated cells (data not shown). To induce the spontaneous differentiation of PG ES cells into three germ layers, EB formed in the suspension condition for 6 days were placed directly onto the tissue culture plate. After 20 days, these EB differentiated into cells showing several different morphologies.
Fig. 2. In vitro differentiation of PG ES cells of pigs. (A) Embryoid bodies derived by suspension culture under differentiation condition during 7 days are shown. (B) Spontaneous differentiation of PG ES cells (pESP1) in vitro into three germ layers (DESMIN, mesoderm; CYTOKERATIN 17, endoderm; VIMENTIN, ectoderm). Cell nuclei were stained with HOECHST 33342 (Scale bar: 50 m).
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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Immunocytochemistry analysis revealed that PG ES cells were positive for DESMIN (mesoderm), CYTOKERATIN 17 (endoderm), and VIMENTIN (ectoderm), indicating that PG ES cells of pigs can differentiate into three embryonic germ layers in an in vitro environment. The IVF ES cell line (pESF6) also contains general properties of ES cells and details including pluripotent marker expression, karyotype, and differentiation ability are described in a previous report (Park et al., 2013). 3.3. Expression patterns of H19 and Igf2 in blastocysts and porcine embryonic stem cells To verify whether the expression of imprinted genes is stable in PG ES cells of pigs, the mRNA patterns of H19 and IGF2genes were initially examined in 7 day blastocysts, the progenitors of PG ES cells. The mRNA of H19, a paternally imprinted gene, in PG blastocysts was 16-fold greater compared to IVF blastocysts (Fig. 3A). Although the amount of mRNA forH19of PG ES cells remained greater than IVF ES cells at passage 10, the difference in transcript abundance decreased relative to blastocysts (Fig. 3B). However, one PG ES line (pESP1) had less mRNA for the H19 gene compared to IVF ES cells, indicating that the H19 gene expression of PG
ES cells is not stable in an in vitro culture environment. In contrast to H19, the amount of mRNA of IGF2 gene, a maternally imprinted gene, was two-fold less in PG blastocysts than in IVF blastocysts, as expected, and this gene expression tendency was maintained in PG ES cells, except for the pESP1 line (Fig. 3B). While the amount of IGF2 mRNA for the pESP2 and pESP3 lines was two-fold less than the pESF6 line of cells, there was no significant difference in mRNA between pESP1 and pESF6 (Fig. 3B). These data suggest that in vitro culture can re-program the expression of H19 and IGF2 genes in PG ES cells after isolation from blastocysts. 3.4. Methylation status of IGF2/H19 DMR3 in blastocysts and porcine embryonic stem cells The H19 and IGF2 genes share enhancers that reside downstream of the H19 gene and the reciprocal expression of both genes is regulated by DNA methylation at the IGF2/H19 DMR. To understand the altered expression patterns of the H19 and IGF2 genes in PG ES cells, a methylation analysis was conducted on the IGF2/H19 DMR3 region in blastocysts and ES cells using a bisulfite sequencing method. Prior to the examination of blastocysts and ES cells, the adult liver of pigs was analyzed to validate
Fig. 3. Relative amounts of mRNA for the H19 and IGF2 genes in porcine (A) IVF (n = 24)/PG (n = 24) blastocysts and (B) IVF (pESF6)/PG (pESP1, pESP2, pESP3) ES cells. The relative amounts of mRNA were quantified using qRT-PCR and then calculated with the 2−Ct method. Three replicate samples were examined for each class. ACTB served as internal controls. Three replicate samples were examined for each class. Bars indicate mean ± SEM (n = 3).* P < 0.05 compared with IVF counterparts (control).
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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Fig. 4. Methylation status of IGF2/H19 DMR3 in blastocysts and PG ES cells of pigs. Liver was analyzed to validate this methylation assay. Circles are CpG sites within the region analyzed: 䊉, methylated cytosines; , un-methylated cytosines.
the methylation assay. A hemi-methylation of 43.75% indicated that there were no potential artifacts or bias. At Day 8, IVF blastocysts displayed a hemi-methylated (50%) status while a pattern of hypomethylation (22%) was observed in PG blastocysts at the same stage (Fig. 4). The DMR3 region of pESF6 was hemi-methylated (60%) in a manner similar to IVF blastocysts, however, all of the PG ES cells examined displayed an un-methylated status (pESP1: 5.6%; pESP2: 3.5%; pESP3: 3.5%) indicating a reduction in methylation of over 16% relative to PG blastocysts (Fig. 4). 4. Discussion The H19 and Igf2 genes are the most widely studied elements of a well-known cluster of imprinted genes. The H19 gene is expressed by the maternal allele while the Igf2 gene is transcribed from the paternal allele (FergusonSmith et al., 1993). The current findings indicate that the H19 gene expression is much greater in PG blastocysts than IVF blastocysts. However, the expression of the H19 gene is dramatically decreased in one PG ES line (pESP1) and a significantly lesser amount of transcripts were detected relative to IVF ES cells after the ES cells were isolated from PG blastocysts and cultured on MEFs. Interestingly, the pESP1 line that underwent the most remarkable transcriptional change for the H19 gene had a similar IGF2 gene expression to the IVF ES cells whereas the relative amount of gene expression in the other two PG ES lines remained minimal. This indicates that PG ES cells underwent an alteration in the expressions of the H19 and IGF2 genes during ES cell isolation process when cultured in vitro, which is consistent with previous findings that successfully produced live mice pups directly from PG ES cells through tetraploid embryo complementation (Chen et al., 2009). The PG ES cells in the previous study contributed to all organs in chimeras and were indistinguishable in most
aspects from the ES cells derived from fertilized embryos. The subsequent analysis of imprinted genes demonstrated that the extent of transcription of maternally expressed genes, including Igf2r and H19, in PG ES cells decreased similar to the degree observed in IVF ES cells. Moreover, this tendency can be found in paternally expressed genes such as Igf2, Snrpn, Peg1, and U2af1-rs1 which do not significantly differ between PG ES and IVF ES cells (Li et al., 2009). Thus, the present findings support the notion that the expression of imprinted genes in embryonic cells can be changed by in vitro culture (Dean et al., 1998; Humpherys et al., 2001). Results of the present study clearly showed that the majority of CpG sites in PG ES cell lines were almost completely un-methylated, whereas the CpGs showed some methylation in PG blastocysts. Previous study showed that the partial methylation pattern in this region of in vitroderived embryos may be responsible for in vitro culture environments (Park et al., 2011). However, such partial methylation was not observed in PG ES cells, it may be possible that poor quality embryos with epigenetic defects were excluded by the derivation process. Although it is well accepted that the DMRs of H19 and Igf2 genes regulate the reciprocal gene expression in a methylation-sensitive manner according to boundary and chromatin models (Leighton et al., 1995; Kaffer et al., 2001; Davies et al., 2002), the gene expression and methylation results of the pESP1 line are not likely to be explained by this theory. The pESP1 line had a lesser transcript abundance of the H19 than for the pESF6 line, but the PG ES cell line was in hypo-methylation, while a methylation of 60% was observed for the pESF6 line. Furthermore, despite a greater de-methylated status in pESP2 and pESP3 lines than in PG blastocysts, the H19 gene expression of pESP cells decreased. Several studies have shown to be hypomethylated pattern of Igf2/H19 DMRs in the mouse PG ES cells (Horii et al., 2008; Chen et al., 2009; Li et al., 2009).
Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020
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A previous study showed that methylation status of the DMRs is not correlated with the activation and expression of H19 and Igf2 genes (Li et al., 2009). In this previous study, the decreased methylation of Snrpn, Peg1, and U2af1rs1 genes was highly correlated with the increased gene expression, suggesting that the H19 and Igf2 gene expressions are regulated by another mechanism. In addition, the IGF2 gene is expressed from both alleles even though the H19 DMR is indeed differentially methylated in the adult pig liver (Braunschweig et al., 2011). It has been proposed that allele-specific histone acetylation may involve a regulation of the DMRs for the Igf2/H19 gene (Wylie et al., 2000; Carr et al., 2007). In the present study, three putative PG ES cell lines were derived from PG blastocysts produced by electrical activation. These cells were maintained in an undifferentiated state for more than 40 passages, and the cell colony was flattened and grew in a monolayer shape similar to human ES cells rather than mouse ES cells. These cells exhibited normal karyotypes and the pluripotent marker OCT4, NANOG, and SOX2 genes were highly expressed when cultured for more than 20 passages. In addition, the capacity to differentiate into three germ layers was confirmed by EB formation and was followed by the induction of spontaneous differentiation in vitro. The amounts of OCT4 and NANOG gene transcripts in the pESF6 cell line were less compared to PG ES cells but the pluripotent marker gene expression at the level of proteins has been previously confirmed as well as the tissue differentiation capacity (Park et al., 2013). According to the present findings, the pESF6 line used in this study for the comparison of epigenetic status in PG ES cells possesses epiblast stem cell (EpiSC) features even though the cell line did not form teratoma. Moreover, as the PG ES cells were generated and cultured in the same system and had similar characteristics to that of the pESF6 line, these cells are also considered to possess EpiSC markers. Unfortunately, the PG ES cells established in the present study lack the in vivo capacity for differentiation into three germ layers, and the molecular pathways supporting self-renewal and pluripotency might be controversial. Nevertheless, these cells might be very useful tools for understanding genomic imprinting because each cell line originated from individual blastocysts. Analysis of genomic imprinting on individual embryos is necessary to avoid variations that can appear in pooled embryos and advance understanding of this process. Moreover, optimal porcine ES cell derivation and culture systems that can support the maintenance of authentic ES cells are required. If so, pig ES cells hold tremendous potential for studying the epigenetic dynamics of ES cells and aiding our understanding of genomic imprinting at pre- and peri-implantation stages. Conflict of interest The authors have no conflicts of interest that biased or influenced this work. Acknowledgments This research was supported by the grant of Bioindustry Technology Development Program of iPET (Korea
Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries, grant no. 111160-04) funded by the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea. This work was also supported by the Next BioGreen 21 program (grant no. PJ009493), Rural Development Administration. References Allen, N.D., Barton, S.C., Hilton, K., Norris, M.L., Surani, M.A., 1994. A functional analysis of imprinting in parthenogenetic embryonic stem cells. Development 120, 1473–1482. Barton, S.C., Surani, M.A., Norris, M.L., 1984. Role of paternal and maternal genomes in mouse development. Nature 311, 374–376. Bischoff, S.R., Tsai, S., Hardison, N., Motsinger-Reif, A.A., Freking, B.A., Nonneman, D., Rohrer, G., Piedrahita, J.A., 2009. Characterization of conserved and nonconserved imprinted genes in swine. Biol. Reprod. 81, 906–920. Braunschweig, M.H., Owczarek-Lipska, M., Stahlberger-Saitbekova, N., 2011. Relationship of porcine IGF2 imprinting status to DNA methylation at the H19 DMD and the IGF2 DMRs 1 and 2. BMC Genet. 12, 47. Brevini, T.A., Pennarossa, G., Attanasio, L., Vanelli, A., Gasparrini, B., Gandolfi, F., 2010. Culture conditions and signalling networks promoting the establishment of cell lines from parthenogenetic and biparental pig embryos. Stem Cell Rev. Rep. 6, 484–495. Carr, M.S., Yevtodiyenko, A., Schmidt, C.L., Schmidt, J.V., 2007. Allelespecific histone modifications regulate expression of the Dlk1-Gtl2 imprinted domain. Genomics 89, 280–290. Chen, Z., Liu, Z., Huang, J., Amano, T., Li, C., Cao, S., Wu, C., Liu, B., Zhou, L., Carter, M.G., Keefe, D.L., Yang, X., Liu, L., 2009. Birth of parthenote mice directly from parthenogenetic embryonic stem cells. Stem Cells 27, 2136–2145. Cibelli, J.B., Cunniff, K., Vrana, K.E., 2006. Embryonic stem cells from parthenotes. Methods Enzymol. 418, 117–135. Cibelli, J.B., Grant, K.A., Chapman, K.B., Cunniff, K., Worst, T., Green, H.L., Walker, S.J., Gutin, P.H., Vilner, L., Tabar, V., Dominko, T., Kane, J., Wettstein, P.J., Lanza, R.P., Studer, L., Vrana, K.E., West, M.D., 2002. Parthenogenetic stem cells in nonhuman primates. Science 295, 819. Clark, S.J., Statham, A., Stirzaker, C., Molloy, P.L., Frommer, M., 2006. DNA methylation: bisulphite modification and analysis. Nat. Protoc. 1, 2353–2364. Davies, K., Bowden, L., Smith, P., Dean, W., Hill, D., Furuumi, H., Sasaki, H., Cattanach, B., Reik, W., 2002. Disruption of mesodermal enhancers for Igf2 in the minute mutant. Development 129, 1657–1668. Dean, W., Bowden, L., Aitchison, A., Klose, J., Moore, T., Meneses, J.J., Reik, W., Feil, R., 1998. Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes. Development 125, 2273–2282. Dindot, S.V., Kent, K.C., Evers, B., Loskutoff, N., Womack, J., Piedrahita, J.A., 2004. Conservation of genomic imprinting at the XIST, IGF2, and GTL2 loci in the bovine. Mamm. Genome 15, 966–974. Fang, Z.F., Gai, H., Huang, Y.Z., Li, S.G., Chen, X.J., Shi, J.J., Wu, L., Liu, A., Xu, P., Sheng, H.Z., 2006. Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos. Exp. Cell Res. 312, 3669–3682. Ferguson-Smith, A.C., Sasaki, H., Cattanach, B.M., Surani, M.A., 1993. Parental-origin-specific epigenetic modification of the mouse H19 gene. Nature 362, 751–755. Horii, T., Kimura, M., Morita, S., Nagao, Y., Hatada, I., 2008. Loss of genomic imprinting in mouse parthenogenetic embryonic stem cells. Stem Cells 26, 79–88. Humpherys, D., Eggan, K., Akutsu, H., Hochedlinger, K., Rideout, W.M., 3rd, Biniszkiewicz, D., Yanagimachi, R., Jaenisch, R., 2001. Epigenetic instability in ES cells and cloned mice. Science 293, 95–97. Jiang, H., Sun, B., Wang, W., Zhang, Z., Gao, F., Shi, G., Cui, B., Kong, X., He, Z., Ding, X., Kuang, Y., Fei, J., Sun, Y.J., Feng, Y., Jin, Y., 2007. Activation of paternally expressed imprinted genes in newly derived germlinecompetent mouse parthenogenetic embryonic stem cell lines. Cell Res. 17, 792–803. Kaffer, C.R., Grinberg, A., Pfeifer, K., 2001. Regulatory mechanisms at the mouse Igf2/H19 locus. Mol. Cell. Biol. 21, 8189–8196. Leighton, P.A., Saam, J.R., Ingram, R.S., Stewart, C.L., Tilghman, S.M., 1995. An enhancer deletion affects both H19 and Igf2 expression. Genes Dev. 9, 2079–2089. Li, C., Chen, Z., Liu, Z., Huang, J., Zhang, W., Zhou, L., Keefe, D.L., Liu, L., 2009. Correlation of expression and methylation of imprinted genes
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Please cite this article in press as: Uh, K.-J., et al., Analysis of imprinted IGF2/H19 gene methylation and expression in normal fertilized and parthenogenetic embryonic stem cells of pigs. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.03.020