Derivation of hybrid ES cell lines from two different strains of mice

Abstract

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.

1. Resource table

Name of stem cell construct	Not applicable
Institution	Icahn School of Medicine at Mount Sinai
Person who created resource	Ho-Tak Lau, Xiajun Li
Contact person and email	Xiajun Li, xiajun.li@mssm.edu
Date archived/stock date	December, 2011
Origin	Mouse blastocysts
Type of resource	Biological reagent: Mouse Embryonic Stem (ES) Cell Lines
Sub-type	129/DBA Hybrid ES cell lines
Key transcription factors	Zfp57
Authentication	Undifferentiated ES cell morphology confirmed (Fig. 1)
Link to related literature (direct URL links and full references)	http://www.ncbi.nlm.nih.gov/pubmed/18854139
Information in public databases	None

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.

2. Resource details

2.1. 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 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).

Table 1.

Chromosome numbers of five hybrid ES clones.

Hybrid ES clone	D1902	D1903	D1904	D1906	D1911
Genotype	Zfp57−/+	Zfp57+/+	Zfp57−/+	Zfp57−/+	Zfp57+/+
# of counted metaphase spreads	20	15	6	10	20
# of spreads with 40 chromosomes	7	7	2	4	10
% of euploid cells	35	46.7	33.3	40	50

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.

2.2. Most hybrid ES clones exhibited intact DNA methylation imprint

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.

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).

3. Materials and methods

3.1. Mouse maintenance

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).

3.2. 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.

3.3. 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 con-fluent, ES cells were split by trypsin digestion and passaged onto new wells with SNL feeder cells. The ES cell growth medium was changed daily to prevent spontaneous differentiation of ES cell colonies.

3.4. 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 non-adherent 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 RT-PCR analysis.

3.5. 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.

3.6. 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.

3.7. Immunostaining for pluripotency markers expressed in hybrid ES clones

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 (sc-376915). DAPI was used to stain the nuclei.

3.8. Bisulphite mutagenesis

The genomic DNA samples were isolated from the hybrid ES clones and subjected to bisulphite mutagenesis with the EZ DNA Methylation-Gold™ Kit (Zymo Research). The bisulphite-treated DNA product was used for COBRA analysis of the DNA methylation imprint at the imprinted regions.

3.9. Combined bisulphite restriction analysis (COBRA)

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.