- Email: [email protected]
Derivation of hybrid ES cell lines from two different strains of mice
Stem Cell Research 16 (2016) 252–255
Contents lists available at ScienceDirect
Stem Cell Research journal homepage: www.elsevier.com/locate/scr
Lab Resource: Stem Cell Line
Derivation of hybrid ES cell lines from two different strains of mice Ho-Tak Lau a, Lizhi Liu a, Chelsea Ray a, Fong T. Bell a, Xiajun Li a,b,⁎ a b
Black Family Stem Cell Institute, Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA Department of Oncological Sciences, Graduate School of Biological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 31 December 2015 Received in revised form 19 January 2016 Accepted 19 January 2016 Available online 20 January 2016
Parental origin-dependent expression of the imprinted genes is essential for mammalian development. Zfp57 maintains genomic imprinting in mouse embryos and ES cells. To examine the allelic expression patterns of the imprinted genes in ES cells, we obtained multiple hybrid ES clones that were directly derived from the blastocysts generated from the cross between mice on two different genetic backgrounds. The blastocyst-derived ES clones displayed largely intact DNA methylation imprint at the tested imprinted regions. These hybrid ES clones will be useful for future studies to examine the allelic expression of the imprinted genes in ES cells and their differentiated progeny. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Resource table
Name of stem cell construct
Not applicable
Institution Person who created resource Contact person and email Date archived/stock date Origin Type of resource
Icahn School of Medicine at Mount Sinai Ho-Tak Lau, Xiajun Li Xiajun Li, [email protected] December, 2011 Mouse blastocysts Biological reagent: Mouse Embryonic Stem (ES) Cell Lines 129/DBA Hybrid ES cell lines Zfp57 Undifferentiated ES cell morphology confirmed (Fig. 1) http://www.ncbi.nlm.nih.gov/pubmed/18854139
Sub-type Key transcription factors Authentication Link to related literature (direct URL links and full references) Information in public databases
None
Resource details Blastocyst-derived hybrid ES clones displayed typical ES cell characters Parental origin-dependent mono-allelic expression of the imprinted genes is essential for mammalian development. Many imprinted genes ⁎ Corresponding author at: Black Family Stem Cell Institute, Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA. E-mail address: [email protected] (X. Li).
are clustered and co-regulated by a cis-acting imprinting control region that harbors germline-derived differential DNA methylation. Zfp57 is a master regulator in genomic imprinting and it maintains DNA methylation imprint at many imprinted regions in mouse embryos and ES cells (Li et al., 2008; Zuo et al., 2012). To facilitate allelic analysis of imprinting control regions and the imprinted genes, we have successfully established multiple wild-type (Zfp57+/+) and Zfp57 heterozygous (Zfp57−/+) hybrid ES clones from the blastocysts generated from the cross between the wild-type male mice on the DBA/2 genetic background and Zfp57+/− heterozygous female mice on the 129 genetic background. These hybrid ES clones are on the 129/DBA hybrid genetic background containing one 129 allele and one DBA/2 allele at all loci of the entire genome, including approximately 150 known imprinted genes. The colonies of these 129/DBA hybrid ES clones exhibited typical undifferentiated ES cell morphology when they were grown on top of the feeder cells, as exemplified by one Zfp57+/+ hybrid ES clone D1911 shown in Fig. 1A. The cells of these hybrid ES clones formed embryoid bodies (EBs) when grown in suspension on non-adherent Petri dish plates (Fig. 1B). These EBs differentiated into three germ layers based on semi-quantitative RT-PCR analysis (Fig. 1C). Indeed, expression of the markers for endoderm (Foxa2), mesoderm (Mlc2a and Pdgfrα) and ectoderm (Ck18) all appeared to be increased in the EB samples compared with the ES cell samples (Fig. 1C). We also counted the metaphase chromosome numbers in five hybrid ES clones (Table 1), with one metaphase chromosome spread as an example shown in Fig. 1D. At least 30% or more cells in these five hybrid ES clones appeared to be euploid cells with 40 chromosomes (Table 1). We also examined expression of OCT4 and NANOG, two pluripotency markers, in four Zfp57+/+ and four Zfp57−/+ hybrid ES clones (Fig. 2). Based on immunostaining, both OCT4 and NANOG are highly expressed in these hybrid ES clones (Fig. 2).
http://dx.doi.org/10.1016/j.scr.2016.01.017 1873-5061/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
H.-T. Lau et al. / Stem Cell Research 16 (2016) 252–255
253
Fig. 1. Blastocyst-derived 129/DBA hybrid ES clones displayed typical properties of ES cells. A, one Zfp57+/+ 129/DBA hybrid ES clone (D1911) was shown as an example that exhibited typical undifferentiated ES cell colonies (red arrows) when grown on the SNL feeder cells. B, the cells of D1911 hybrid ES clone formed embryoid bodies (EBs, arrowheads) when cultured on non-adherent Petri dish plates. C, RT-PCR expression analysis of the marker genes in four hybrid ES clones and the EBs derived from these hybrid ES clones after growing in suspension culture for 6 or 7 or 9 days. Lanes 1–2, negative control without reverse transcription (−RT) of the same total RNA samples in Lanes 4 and 5, respectively. Lanes 3–6, ES cell samples. Lanes 7–10, EB samples. Lane 3, D1903 Zfp57+/+ 129/DBA hybrid ES clone. Lane 4, D1911 Zfp57+/+ 129/DBA hybrid ES clone. Lane 5, D1902 Zfp57−/+ 129/DBA hybrid ES clone. Lane 6, D1904 Zfp57−/+ 129/DBA hybrid ES clone. Lane 7, day 7 EBs of D1911 Zfp57+/+ 129/DBA hybrid ES clone. Lane 8, day 7 EBs of D1902 Zfp57−/+ 129/DBA hybrid ES clone. Lane 9, day 6 EBs of D1904 Zfp57−/+ 129/DBA hybrid ES clone. Lane 10, day 9 EBs of D1911 Zfp57+/+ 129/DBA hybrid ES clone. Mlc2a and Pdgfrα are markers for mesoderm, whereas Foxa2 and Ck18 are the lineage markers for endoderm and ectoderm, respectively. A house-keeping gene Rps17 was similarly amplified as the loading control. Rps17 was subjected to 25 cycles of PCR amplification, whereas 30 PCR cycles were applied to Mlc2a and Pdgfrα. Ck18 was amplified for 25 PCR cycles (Ck18–1) or 30 PCR cycles (Ck18–2). D, an example is shown here for DAPI-stained metaphase chromosome spread of a cell of D1911 Zfp57+/+ 129/DBA hybrid ES clone.
Most hybrid ES clones exhibited intact DNA methylation imprint
Materials and methods
The genotypes of Zfp57 for these hybrid ES clones were confirmed by a PCR-based approach that were used in our previous studies (Li et al., 2008; Takikawa et al., 2013), as exemplified by six hybrid ES clones shown in Fig. 3A. To test if these 129/DBA hybrid ES clones are suitable for allelic expression analysis of imprinted genes in ES cells and their differentiated progeny in future studies, we analyzed DNA methylation imprint of six hybrid ES clones at multiple imprinted regions. We found genomic imprinting was maintained in most 129/DBA hybrid ES clones based on COBRA analysis of DNA methylation at multiple imprinted regions. The COBRA result for the Snrpn imprinted region was provided as an example (Fig. 3B). Except for partial loss of DNA methylation imprint observed in one Zfp57+/+ hybrid ES clone (D1903, Lane 3 of Fig. 3B), DNA methylation imprint at the Snrpn imprinted region was largely intact at two other Zfp57+/+ hybrid ES clones (D1908 and D1911, Lanes 6–7 of Fig. 3B) and three Zfp57−/+ hybrid ES clones (D1902, D1904 and D1906 in lanes 2, 4–5 of Fig. 3B). This indicates that these hybrid ES clones will be useful for allelic analyses in genomic imprinting in future research.
Mouse maintenance
Table 1 Chromosome numbers of five hybrid ES clones. Hybrid ES clone
D1902
Genotype # of counted metaphase spreads # of spreads with 40 chromosomes % of euploid cells
Zfp57−/+ Zfp57+/+ Zfp57−/+ Zfp57−/+ Zfp57+/+ 20 15 6 10 20
D1903
D1904
D1906
D1911
7
7
2
4
10
35
46.7
33.3
40
50
The mouse strains were maintained in the animal facility of Icahn School of Medicine at Mount Sinai in compliance with the animal care protocol approved by IACUC of the institution. The wild-type DBA/2 mice were obtained from the Jackson Laboratories. The Zfp57 heterozygous (Zfp57+/−) mice on the 129 genetic background were generated from our previously published study (Li et al., 2008). Derivation of the 129/DBA hybrid ES clones from blastocysts Mouse blastocytsts were isolated from the timed pregnancy mating between the wild-type male mice on the DBA/2 genetic background and Zfp57+/− heterozygous female mice on the 129 genetic background. Hybrid ES clones were derived from these blastocysts based on the previously published protocol (Meissner et al., 2009). First, blastocysts were added onto the feeder cells to allow outgrowth of the ES colonies. The colonies with ES cell colony-like morphology were picked individually to a well of 24-well plate seeded with feeder cells. Then these clones were passaged to a new well with feeder cells until stable ES cell lines were established. These established ES cell lines are on the 129/DBA hybrid genetic background with one 129 allele and one DBA/2 allele at all loci of the genome including the imprinted regions. ES cell culture The ES cell growth medium was made of high-glucose DMEM medium supplemented with 15% fetal bovine serum (FBS). The ES cells were cultured with the ES cell growth medium on top of the irradiated SNL feeder cells (McMahon and Bradley, 1990). Before they became too confluent, ES cells were split by trypsin digestion and passaged onto new
254
H.-T. Lau et al. / Stem Cell Research 16 (2016) 252–255
Fig. 2. Hybrid ES clones express pluripotent markers OCT4 and NANOG. Immunostaining was performed to analyze the expression of OCT4 (a) and NANOG (b) in four Zfp57+/+ and four Zfp57−/+ hybrid ES clones. D1903, D1905, D1908 and D1911 are Zfp57+/+ wild-type 129/DBA hybrid ES clones, whereas D1902, D1904, D1906 and D1907 are Zfp57−/+ heterozygous 129/ DBA hybrid ES clones generated in this study. Blue signal, DAPI staining. Red signal, OCT4 immunostaining. Green signal, NANOG immunostaining.
wells with SNL feeder cells. The ES cell growth medium was changed daily to prevent spontaneous differentiation of ES cell colonies. Suspension culture for generation of embryoid bodies (EBs) Growing ES cells on a well of 6-well plate seeded with irradiated SNL feeder cells were collected by trypsin digestion and plated onto a nonadherent 10-cm Petri dish plates pre-treated with poly-hema (Sigma). The medium was changed once every 2–3 days without disrupting the aggregated EBs. Floating EBs were harvested at day 6 or day 7 or day 9 for total RNA sample preparation followed by semi-quantitative RTPCR analysis.
Chromosome number counting to examine the integrity of hybrid ES clones Metaphase chromosome spread was prepared for five hybrid ES clones by Karyomax (Invitrogen Cat# 15210-040). The dividing ES cells were arrested by addition of Karyomax with a final concentration of Colcemid at 1 μg/ml. The arrested ES cells were collected by trypsin digestion followed by centrifugation. The cell pellets were resuspended and gently mixed with ice-cold 0.56% KCl solution. Then the ES samples were collected by centrifugation and fixed with a mixture solution of acetic acid and methanol (1:3). The resuspended pellets in this fixative solution with DAPI were spotted onto the slides and dried in the air for one hour before microscopy. Photos were taken for well-separated metaphase chromosome spread of these ES clones and the chromosome numbers for each spread were counted afterwards. The results are summarized in Table 1. RT-PCR expression analysis of the markers for three germ layers The ES cells and the collected EBs were lysed in TRIzol reagent (Invitrogen) and total RNA samples were purified according to the manual provided by the manufacturer. Similar amount of total RNA was used for reverse transcription (RT) with Transcriptor First Strand cDNA Synthesis Kit (Roche). RT reaction was primed with the anchored-oligo(dT)18 primer included in the kit, along with two negative control samples without reverse transcriptase (−). 1 μl of RT reaction mixture was used for each PCR amplification. The RT-PCR product was separated on agarose gels before photography. Immunostaining for pluripotency markers expressed in hybrid ES clones
Fig. 3. Most hybrid ES clones exhibited intact DNA methylation imprint at the Snrpn imprinted region. A, PCR-based genotyping of six hybrid ES clones, similar to our previous studies (Li et al., 2008; Takikawa et al., 2013). The positions for the PCR product on the agarose gel are marked for the mutant allele and wild-type (WT) allele of Zfp57, respectively. D1903, D1908 and D1911 are Zfp57+/+ wild-type 129/DBA hybrid ES clones, whereas D1902, D1904 and D1906 are Zfp57−/+ heterozygous 129/DBA hybrid ES clones. B, COBRA analysis was carried out for analyzing DNA methylation imprint at the maternally inherited Snrpn imprinted region. HhaI restriction enzyme digestion was performed for the bisulphite PCR product of six hybrid ES samples and control (Ctrl) wild-type mouse tail DNA sample (Lane 1). U and M, unmethylated (U) and methylated (M) product after HhaI digestion, respectively. Lanes 2–7, six hybrid ES clones (D1902, D1903, D1904, D1906, D1908 and D1911).
The hybrid ES clones were grown a 24-well plate seeded with irradiated SNL feeder cells before they were stained with the antibodies from Santa Cruz Biotechnology against OCT4 (sc-5279) or NANOG (sc376915). DAPI was used to stain the nuclei. Bisulphite mutagenesis The genomic DNA samples were isolated from the hybrid ES clones and subjected to bisulphite mutagenesis with the EZ DNA MethylationGold™ Kit (Zymo Research). The bisulphite-treated DNA product was used for COBRA analysis of the DNA methylation imprint at the imprinted regions.
H.-T. Lau et al. / Stem Cell Research 16 (2016) 252–255
255
Combined bisulphite restriction analysis (COBRA)
Acknowledgments
COBRA was used to examine DNA methylation imprint at the imprinted regions. The bisulphite-treated genomic DNA samples were purified with the columns provided in EZ DNA Methylation-Gold™ Kit before being subjected to PCR amplification with the primers spanning a portion of the imprinted regions (Zuo et al., 2012). The PCR amplified bisulphite-treated product was subjected to restriction enzyme digestion with restriction enzyme sites covering the CpG sites located in the amplified imprinted regions. If the CpG sites were not methylated in the genomic DNA samples, the restriction enzyme sites may be lost after bisulphite mutagenesis. If they were methylated in the genomic DNA samples, the CpG sites were protected from bisulphite mutagenesis and the restriction enzyme sites were retained in the PCR product and could be recognized by the respective restriction enzymes. The restriction enzyme digested PCR product was separated by gel electrophoresis so that we could distinguish the product derived from the DNA with the methylated CpG sites from the product generated from the DNA with the unmethylated CpG sites.
The work in the author's laboratory is currently supported by the grant from NIH (R01GM093335). XL conceived the study. HTL, LL, CR, FTB and XL performed the experiments. XL wrote the manuscript. References Li, X., Ito, M., Zhou, F., Youngson, N., Zuo, X., Leder, P., Ferguson-Smith, A.C., 2008. A maternal-zygotic effect gene, Zfp57, maintains both maternal and paternal imprints. Dev. Cell 15, 547–557. McMahon, A.P., Bradley, A., 1990. The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain. Cell 62, 1073–1085. Meissner, A., Eminli, S., Jaenisch, R., 2009. Derivation and manipulation of murine embryonic stem cells. Methods Mol. Biol. 482, 3–19. Takikawa, S., Wang, X., Ray, C., Vakulenko, M., Bell, F.T., Li, X., 2013. Human and mouse ZFP57 proteins are functionally interchangeable in maintaining genomic imprinting at multiple imprinted regions in mouse ES cells. Epigenetics 8. Zuo, X., Sheng, J., Lau, H.T., McDonald, C.M., Andrade, M., Cullen, D.E., Bell, F.T., Iacovino, M., Kyba, M., Xu, G., et al., 2012. Zinc finger protein ZFP57 requires its co-factor to recruit DNA methyltransferases and maintains DNA methylation imprint in embryonic stem cells via its transcriptional repression domain. J. Biol. Chem. 287, 2107–2118.
Contents lists available at ScienceDirect
Stem Cell Research journal homepage: www.elsevier.com/locate/scr
Lab Resource: Stem Cell Line
Derivation of hybrid ES cell lines from two different strains of mice Ho-Tak Lau a, Lizhi Liu a, Chelsea Ray a, Fong T. Bell a, Xiajun Li a,b,⁎ a b
Black Family Stem Cell Institute, Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA Department of Oncological Sciences, Graduate School of Biological Sciences, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA
a r t i c l e
i n f o
a b s t r a c t
Article history: Received 31 December 2015 Received in revised form 19 January 2016 Accepted 19 January 2016 Available online 20 January 2016
Parental origin-dependent expression of the imprinted genes is essential for mammalian development. Zfp57 maintains genomic imprinting in mouse embryos and ES cells. To examine the allelic expression patterns of the imprinted genes in ES cells, we obtained multiple hybrid ES clones that were directly derived from the blastocysts generated from the cross between mice on two different genetic backgrounds. The blastocyst-derived ES clones displayed largely intact DNA methylation imprint at the tested imprinted regions. These hybrid ES clones will be useful for future studies to examine the allelic expression of the imprinted genes in ES cells and their differentiated progeny. © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Resource table
Name of stem cell construct
Not applicable
Institution Person who created resource Contact person and email Date archived/stock date Origin Type of resource
Icahn School of Medicine at Mount Sinai Ho-Tak Lau, Xiajun Li Xiajun Li, [email protected] December, 2011 Mouse blastocysts Biological reagent: Mouse Embryonic Stem (ES) Cell Lines 129/DBA Hybrid ES cell lines Zfp57 Undifferentiated ES cell morphology confirmed (Fig. 1) http://www.ncbi.nlm.nih.gov/pubmed/18854139
Sub-type Key transcription factors Authentication Link to related literature (direct URL links and full references) Information in public databases
None
Resource details Blastocyst-derived hybrid ES clones displayed typical ES cell characters Parental origin-dependent mono-allelic expression of the imprinted genes is essential for mammalian development. Many imprinted genes ⁎ Corresponding author at: Black Family Stem Cell Institute, Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, New York, NY 10029, USA. E-mail address: [email protected] (X. Li).
are clustered and co-regulated by a cis-acting imprinting control region that harbors germline-derived differential DNA methylation. Zfp57 is a master regulator in genomic imprinting and it maintains DNA methylation imprint at many imprinted regions in mouse embryos and ES cells (Li et al., 2008; Zuo et al., 2012). To facilitate allelic analysis of imprinting control regions and the imprinted genes, we have successfully established multiple wild-type (Zfp57+/+) and Zfp57 heterozygous (Zfp57−/+) hybrid ES clones from the blastocysts generated from the cross between the wild-type male mice on the DBA/2 genetic background and Zfp57+/− heterozygous female mice on the 129 genetic background. These hybrid ES clones are on the 129/DBA hybrid genetic background containing one 129 allele and one DBA/2 allele at all loci of the entire genome, including approximately 150 known imprinted genes. The colonies of these 129/DBA hybrid ES clones exhibited typical undifferentiated ES cell morphology when they were grown on top of the feeder cells, as exemplified by one Zfp57+/+ hybrid ES clone D1911 shown in Fig. 1A. The cells of these hybrid ES clones formed embryoid bodies (EBs) when grown in suspension on non-adherent Petri dish plates (Fig. 1B). These EBs differentiated into three germ layers based on semi-quantitative RT-PCR analysis (Fig. 1C). Indeed, expression of the markers for endoderm (Foxa2), mesoderm (Mlc2a and Pdgfrα) and ectoderm (Ck18) all appeared to be increased in the EB samples compared with the ES cell samples (Fig. 1C). We also counted the metaphase chromosome numbers in five hybrid ES clones (Table 1), with one metaphase chromosome spread as an example shown in Fig. 1D. At least 30% or more cells in these five hybrid ES clones appeared to be euploid cells with 40 chromosomes (Table 1). We also examined expression of OCT4 and NANOG, two pluripotency markers, in four Zfp57+/+ and four Zfp57−/+ hybrid ES clones (Fig. 2). Based on immunostaining, both OCT4 and NANOG are highly expressed in these hybrid ES clones (Fig. 2).
http://dx.doi.org/10.1016/j.scr.2016.01.017 1873-5061/© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
H.-T. Lau et al. / Stem Cell Research 16 (2016) 252–255
253
Fig. 1. Blastocyst-derived 129/DBA hybrid ES clones displayed typical properties of ES cells. A, one Zfp57+/+ 129/DBA hybrid ES clone (D1911) was shown as an example that exhibited typical undifferentiated ES cell colonies (red arrows) when grown on the SNL feeder cells. B, the cells of D1911 hybrid ES clone formed embryoid bodies (EBs, arrowheads) when cultured on non-adherent Petri dish plates. C, RT-PCR expression analysis of the marker genes in four hybrid ES clones and the EBs derived from these hybrid ES clones after growing in suspension culture for 6 or 7 or 9 days. Lanes 1–2, negative control without reverse transcription (−RT) of the same total RNA samples in Lanes 4 and 5, respectively. Lanes 3–6, ES cell samples. Lanes 7–10, EB samples. Lane 3, D1903 Zfp57+/+ 129/DBA hybrid ES clone. Lane 4, D1911 Zfp57+/+ 129/DBA hybrid ES clone. Lane 5, D1902 Zfp57−/+ 129/DBA hybrid ES clone. Lane 6, D1904 Zfp57−/+ 129/DBA hybrid ES clone. Lane 7, day 7 EBs of D1911 Zfp57+/+ 129/DBA hybrid ES clone. Lane 8, day 7 EBs of D1902 Zfp57−/+ 129/DBA hybrid ES clone. Lane 9, day 6 EBs of D1904 Zfp57−/+ 129/DBA hybrid ES clone. Lane 10, day 9 EBs of D1911 Zfp57+/+ 129/DBA hybrid ES clone. Mlc2a and Pdgfrα are markers for mesoderm, whereas Foxa2 and Ck18 are the lineage markers for endoderm and ectoderm, respectively. A house-keeping gene Rps17 was similarly amplified as the loading control. Rps17 was subjected to 25 cycles of PCR amplification, whereas 30 PCR cycles were applied to Mlc2a and Pdgfrα. Ck18 was amplified for 25 PCR cycles (Ck18–1) or 30 PCR cycles (Ck18–2). D, an example is shown here for DAPI-stained metaphase chromosome spread of a cell of D1911 Zfp57+/+ 129/DBA hybrid ES clone.
Most hybrid ES clones exhibited intact DNA methylation imprint
Materials and methods
The genotypes of Zfp57 for these hybrid ES clones were confirmed by a PCR-based approach that were used in our previous studies (Li et al., 2008; Takikawa et al., 2013), as exemplified by six hybrid ES clones shown in Fig. 3A. To test if these 129/DBA hybrid ES clones are suitable for allelic expression analysis of imprinted genes in ES cells and their differentiated progeny in future studies, we analyzed DNA methylation imprint of six hybrid ES clones at multiple imprinted regions. We found genomic imprinting was maintained in most 129/DBA hybrid ES clones based on COBRA analysis of DNA methylation at multiple imprinted regions. The COBRA result for the Snrpn imprinted region was provided as an example (Fig. 3B). Except for partial loss of DNA methylation imprint observed in one Zfp57+/+ hybrid ES clone (D1903, Lane 3 of Fig. 3B), DNA methylation imprint at the Snrpn imprinted region was largely intact at two other Zfp57+/+ hybrid ES clones (D1908 and D1911, Lanes 6–7 of Fig. 3B) and three Zfp57−/+ hybrid ES clones (D1902, D1904 and D1906 in lanes 2, 4–5 of Fig. 3B). This indicates that these hybrid ES clones will be useful for allelic analyses in genomic imprinting in future research.
Mouse maintenance
Table 1 Chromosome numbers of five hybrid ES clones. Hybrid ES clone
D1902
Genotype # of counted metaphase spreads # of spreads with 40 chromosomes % of euploid cells
Zfp57−/+ Zfp57+/+ Zfp57−/+ Zfp57−/+ Zfp57+/+ 20 15 6 10 20
D1903
D1904
D1906
D1911
7
7
2
4
10
35
46.7
33.3
40
50
The mouse strains were maintained in the animal facility of Icahn School of Medicine at Mount Sinai in compliance with the animal care protocol approved by IACUC of the institution. The wild-type DBA/2 mice were obtained from the Jackson Laboratories. The Zfp57 heterozygous (Zfp57+/−) mice on the 129 genetic background were generated from our previously published study (Li et al., 2008). Derivation of the 129/DBA hybrid ES clones from blastocysts Mouse blastocytsts were isolated from the timed pregnancy mating between the wild-type male mice on the DBA/2 genetic background and Zfp57+/− heterozygous female mice on the 129 genetic background. Hybrid ES clones were derived from these blastocysts based on the previously published protocol (Meissner et al., 2009). First, blastocysts were added onto the feeder cells to allow outgrowth of the ES colonies. The colonies with ES cell colony-like morphology were picked individually to a well of 24-well plate seeded with feeder cells. Then these clones were passaged to a new well with feeder cells until stable ES cell lines were established. These established ES cell lines are on the 129/DBA hybrid genetic background with one 129 allele and one DBA/2 allele at all loci of the genome including the imprinted regions. ES cell culture The ES cell growth medium was made of high-glucose DMEM medium supplemented with 15% fetal bovine serum (FBS). The ES cells were cultured with the ES cell growth medium on top of the irradiated SNL feeder cells (McMahon and Bradley, 1990). Before they became too confluent, ES cells were split by trypsin digestion and passaged onto new
254
H.-T. Lau et al. / Stem Cell Research 16 (2016) 252–255
Fig. 2. Hybrid ES clones express pluripotent markers OCT4 and NANOG. Immunostaining was performed to analyze the expression of OCT4 (a) and NANOG (b) in four Zfp57+/+ and four Zfp57−/+ hybrid ES clones. D1903, D1905, D1908 and D1911 are Zfp57+/+ wild-type 129/DBA hybrid ES clones, whereas D1902, D1904, D1906 and D1907 are Zfp57−/+ heterozygous 129/ DBA hybrid ES clones generated in this study. Blue signal, DAPI staining. Red signal, OCT4 immunostaining. Green signal, NANOG immunostaining.
wells with SNL feeder cells. The ES cell growth medium was changed daily to prevent spontaneous differentiation of ES cell colonies. Suspension culture for generation of embryoid bodies (EBs) Growing ES cells on a well of 6-well plate seeded with irradiated SNL feeder cells were collected by trypsin digestion and plated onto a nonadherent 10-cm Petri dish plates pre-treated with poly-hema (Sigma). The medium was changed once every 2–3 days without disrupting the aggregated EBs. Floating EBs were harvested at day 6 or day 7 or day 9 for total RNA sample preparation followed by semi-quantitative RTPCR analysis.
Chromosome number counting to examine the integrity of hybrid ES clones Metaphase chromosome spread was prepared for five hybrid ES clones by Karyomax (Invitrogen Cat# 15210-040). The dividing ES cells were arrested by addition of Karyomax with a final concentration of Colcemid at 1 μg/ml. The arrested ES cells were collected by trypsin digestion followed by centrifugation. The cell pellets were resuspended and gently mixed with ice-cold 0.56% KCl solution. Then the ES samples were collected by centrifugation and fixed with a mixture solution of acetic acid and methanol (1:3). The resuspended pellets in this fixative solution with DAPI were spotted onto the slides and dried in the air for one hour before microscopy. Photos were taken for well-separated metaphase chromosome spread of these ES clones and the chromosome numbers for each spread were counted afterwards. The results are summarized in Table 1. RT-PCR expression analysis of the markers for three germ layers The ES cells and the collected EBs were lysed in TRIzol reagent (Invitrogen) and total RNA samples were purified according to the manual provided by the manufacturer. Similar amount of total RNA was used for reverse transcription (RT) with Transcriptor First Strand cDNA Synthesis Kit (Roche). RT reaction was primed with the anchored-oligo(dT)18 primer included in the kit, along with two negative control samples without reverse transcriptase (−). 1 μl of RT reaction mixture was used for each PCR amplification. The RT-PCR product was separated on agarose gels before photography. Immunostaining for pluripotency markers expressed in hybrid ES clones
Fig. 3. Most hybrid ES clones exhibited intact DNA methylation imprint at the Snrpn imprinted region. A, PCR-based genotyping of six hybrid ES clones, similar to our previous studies (Li et al., 2008; Takikawa et al., 2013). The positions for the PCR product on the agarose gel are marked for the mutant allele and wild-type (WT) allele of Zfp57, respectively. D1903, D1908 and D1911 are Zfp57+/+ wild-type 129/DBA hybrid ES clones, whereas D1902, D1904 and D1906 are Zfp57−/+ heterozygous 129/DBA hybrid ES clones. B, COBRA analysis was carried out for analyzing DNA methylation imprint at the maternally inherited Snrpn imprinted region. HhaI restriction enzyme digestion was performed for the bisulphite PCR product of six hybrid ES samples and control (Ctrl) wild-type mouse tail DNA sample (Lane 1). U and M, unmethylated (U) and methylated (M) product after HhaI digestion, respectively. Lanes 2–7, six hybrid ES clones (D1902, D1903, D1904, D1906, D1908 and D1911).
The hybrid ES clones were grown a 24-well plate seeded with irradiated SNL feeder cells before they were stained with the antibodies from Santa Cruz Biotechnology against OCT4 (sc-5279) or NANOG (sc376915). DAPI was used to stain the nuclei. Bisulphite mutagenesis The genomic DNA samples were isolated from the hybrid ES clones and subjected to bisulphite mutagenesis with the EZ DNA MethylationGold™ Kit (Zymo Research). The bisulphite-treated DNA product was used for COBRA analysis of the DNA methylation imprint at the imprinted regions.
H.-T. Lau et al. / Stem Cell Research 16 (2016) 252–255
255
Combined bisulphite restriction analysis (COBRA)
Acknowledgments
COBRA was used to examine DNA methylation imprint at the imprinted regions. The bisulphite-treated genomic DNA samples were purified with the columns provided in EZ DNA Methylation-Gold™ Kit before being subjected to PCR amplification with the primers spanning a portion of the imprinted regions (Zuo et al., 2012). The PCR amplified bisulphite-treated product was subjected to restriction enzyme digestion with restriction enzyme sites covering the CpG sites located in the amplified imprinted regions. If the CpG sites were not methylated in the genomic DNA samples, the restriction enzyme sites may be lost after bisulphite mutagenesis. If they were methylated in the genomic DNA samples, the CpG sites were protected from bisulphite mutagenesis and the restriction enzyme sites were retained in the PCR product and could be recognized by the respective restriction enzymes. The restriction enzyme digested PCR product was separated by gel electrophoresis so that we could distinguish the product derived from the DNA with the methylated CpG sites from the product generated from the DNA with the unmethylated CpG sites.
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