Heterogeneity in imprinted methylation patterns of pluripotent embryonic
germ cells derived from pre-migratory mouse germ cells
Tanya C. Shovlin⁎, Gabriela Durcova-Hills, Azim Surani, Anne McLaren†
The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK
Received for publication 3 September 2007; revised 25 October 2007; accepted 2 November 2007
Available online 17 November 2007
Pluripotent stem cells, termed embryonic germ (EG) cells, have been generated from both human and mouse primordial germ cells (PGCs). Like
embryonic stem (ES) cells, EG cells have the potential to differentiate into all germ layer derivatives and may also be important for any future
clinical applications. The development of PGCs in vivo is accompanied by major epigenetic changes including DNA demethylation and imprint
erasure. We have investigated the DNA methylation pattern of several imprinted genes and repetitive elements in mouse EG cell lines before and
after differentiation. Analysed cell lines were derived soon after PGC specification, “early”, in comparison with EG cells derived after PGC
colonisation of the genital ridge, “late” and embryonic stem (ES) cell lines, derived from the inner cell mass (ICM). Early EG cell lines showed
strikingly heterogeneous DNA methylation patterns, in contrast to the uniformity of methylation pattern seen in somatic cells (control), late EG cell
and EScell lines. We also observed that allanalysed XX cell lines exhibitedless methylation thanXY.We suggest thatthis heterogeneity may reflect
the changes in DNA methylation taking place in the germ cell lineage soon after specification.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Embryonic germ (EG) cells; Primordial germ cells (PGCs); Embryonic stem (ES) cells; DNA methylation; Germ cell development
Pluripotent stem cells in the mouse can be derived either
from the inner cell mass (ICM)/early epiblast of the blastocyst
(embryonic stem (ES) cells) or from primordial germ cells
(PGCs) (Evans and Kaufman, 1981; Martin, 1981; Matsui et al.,
1992; Resnick et al., 1992).
At first glance, mouse ES and EG cells appear to be similar.
Morphologically, they look identical; both form multi-cellular
colonies positive for tissue non-specific alkaline phosphatase
(TNAP) activity and for Oct-4 and SSEA-1 (Matsui et al.,
1992). On the removal of LIF, both cell types differentiate into
ectoderm, mesoderm and endoderm derivatives (Beddington
and Robertson, 1989; Rohwedel et al., 1996). Injection of ES or
EG cells into the blastocyst produces a high rate of chimerism in
the developing embryo, including the germ cell lineage (Brad-
ley et al., 1984; Labosky et al., 1994; Stewart et al., 1994).
Finally, both ES and EG cells have the ability to reprogramme
somatic cell lineages after cell fusion, resulting in the up-
regulation of pluripotency genes and the down-regulation of
genes involved in differentiation (Tada et al., 1997, 2001).
Despite these similarities, there is a crucial difference
between these pluripotent cell lines. Unlike the ICM, from
which ES cells are derived, PGCs are a highly specified cell
lineage and undergo major epigenetic modifications during their
development, including DNA demethylation and erasure of
parental imprints (Monk et al., 1987; Hajkova et al., 2002; Lee
et al., 2002). DNA demethylation and imprint erasure begin for
some imprinted genes during migration and are completed by
e13.5 (Hajkova et al., 2002). EG cells have been derived from
various stages of germ cell development shortly after
specification of the germ line, “early”, i.e. before imprint
erasure and DNA demethylation has begun (Matsui et al., 1992;
Resnick et al., 1992; Labosky et al., 1994) (e8.0, 8.5), during
migration, “mid” (e9.5) (Durcova-Hills et al., 2001) and after
the germ cells have colonised the genital ridges, “late” (e11.5,
Available online at www.sciencedirect.com
Developmental Biology 313 (2008) 674–681
⁎Corresponding author. Fax: +44 1223 334089.
E-mail address: firstname.lastname@example.org (T.C. Shovlin).
†We dedicate this paper to Anne McLaren who died on 7th July 2007.
0012-1606/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
12.5) (Matsui et al., 1992; Labosky et al., 1994; Tada et al.,
Little is known about the differences in methylation status
and developmental potential of early verus late EG cell lines.
Early EG cells derived from pre-migratory PGCs have been
examined for only one imprinted gene (Igf2r), for which they
showed variable imprint erasure (Labosky et al., 1994). In
vivo Igf2r has however been shown to initiate demethylation
earlier than other imprinted genes (Hajkova et al., 2002; Lee
et al., 2002; Sato et al., 2003). Mid and late EG cells do not
reflect the methylation status of the cells from which they
were derived; however, with rare exceptions, they were fully
demethylated for all imprinted genes investigated (Tada et al.,
1998; Durcova-Hills et al., 2001). It was concluded that the
erasure process continued after the PGCs were placed in
culture. Chimeras made from late EG cells showed skeletal
abnormalities while no such abnormalities have yet been
detected with early EG cells, although numbers were low
(Labosky et al., 1994; Stewart et al., 1994; Tada et al.,
In this study, we have investigated the methylation status
early EG cell lines for several imprinted genes (Igf2r,
p57Kip2, lit1, Snrpn, H19 and Igf2) and repetitive elements
in comparison with late EG and ES cell lines. We show that
the early pre-migratory EG cell lines are highly heterogeneous
with respect to the methylation status of their imprinted genes.
Most female EG cells, like female ES cells, have highly
demethylated repetitive sequences. These differences between
the pluripotent stem cell populations may reflect changes in
the epigenetic state from epiblast to newly established PGCs to
Materials and methods
Derivation of EG cell lines
Matings between MF1 females and either C57BL/6-Rosa26, carrying a
LacZ transgene, or 129 males, were used to produce offspring. Noon on the day
of the vaginal plug was taken as e0.5. Embryos from e8.5 pregnant females were
dissected free of extraembryonic tissues. Derivation of the EG cell lines was as
previously described (Durcova-Hills and McLaren, 2006). Further cultures were
maintained in the same way, and subsequent passage onto 35-mm tissue culture
disheswas denotedaspassage1.LateEG andEScelllines wereavailablewithin
Details of the established EG cell lines
Cell lineStageSex Strain PassageRepeats
MF1 X Rosa
MF1 X Rosa
MF1 X Rosa
MF1 X Rosa
MF1 X 129
MF1 X 129
MF1 X Rosa
MF1 X Rosa
MF1 X Rosa
MF1 X 129
MF1 X 129
MF1 X Rosa
MF1 X Rosa
129 X 129
129 X 129
129 X 129
129 X 129
129 X Mix
129 X Mix
129 X 129
129 X 129
129 X 129
aES cell line seen in the Southern blots.
Fig. 1. Characterisation of EG cell lines. All eleven e8.5 EG cell lines express a high level of TNAP activity (A), Oct-4 (B), SSEA-1 (C), GCNA (D), EMA-1 (E) and
were negative for VASA (F).
675 T.C. Shovlin et al. / Developmental Biology 313 (2008) 674–681
Characterisation of EG cell lines
EG cells grown on cover slides were fixed with 4% paraformaldehyde. The
cells were stained for tissue non-specific alkaline phosphatase activity using an
Alkaline Phosphatase Leukocyte kit (Sigma) according to the manufacturer’s
instructions. The expression of Oct-4, SSEA-1, GCNA, EMA-1 and VASAwas
also checked by immunostaining, as previously described (Durcova-Hills and
McLaren, 2006). All the lines were karyotyped (Roberston, 1987), and the sex
of the cell lines was determined by amplifying the UBE1X gene by PCR
(Chuma and Nakatsuji. 2001).
Southern blot hybridisation analysis
EG cells were passaged twice onto gelatine-coated 10-cm tissue culture
dishes to remove any contaminating feeder cells, and the medium was sup-
plemented with twice the amount of LIF to prevent differentiation. When near
confluent, the cultures were rinsed three times in PBS and then lysed in DNA
lysis buffer (10 mM Tris, pH 7.5, 50 mM EDTA and 1% SDS containing
500 μg/ml Proteinase K) overnight at 55 °C followed by phenol:chloroform:
isoamyl alcohol (25:24:1) and ethanol precipitation. The DNA (10–15 μg) was
digested with the appropriate restriction enzymes, separated on 0.8–1.2%
agarosegels andtransferredonto HybondN+ membraneby the alkaline method.
Southern blots were then hybridised with32P dCTP-labelled probes specific for
each imprinted gene tested.
In vitro differentiation
The EG cells were differentiated into embryoid bodies (EBs) as previously
described (Roberston. 1987). DNAwas collected and Southern blot analysis was
performed as before. Immunostaining of all three germ layers was also per-
formed (as above). The antibodies used were as follows: Ectoderm, nestin
(1:100, BD Biosciences), mesoderm, fibronectin (1:40, Chemicon) and endo-
derm, TROMA-1 (1:1, Developmental Studies Hybridoma Bank).
Eleven EG cell lines were derived from e8.5 PGCs, six male
and five female (Table 1). All the EG cell lines contained a
normal diploid set of 40 chromosomes. All showed high levels
TNAP activity (Fig. 1A), expressed Oct-4, SSEA-1, GCNA and
EMA-1 and were negative for the germ cell marker VASA
(Figs. 1B–F). ES cells cultured to high passage numbers have
Fig. 2. DNA methylation analysis of Igf2r, Lit1, p57Kip2, Snrpn, H19 and Igf2. Igf2r: DNA was digested with PvuII and the methylation-sensitive MluI and was
hybridised with a probe for region 2 of the Igf2r gene (Stoger et al., 1993). The methylated DNA fragment is detected at 3 kb and the unmethylated at 2 kb. Lit1: DNA
was digested with PvuII and the methylation-sensitive EagI and was hybridised with a probe specific to Lit1 (Smilinich et al., 1999). The methylated fragment is
detected at 4 kb and the unmethylated at 3 kb. p57Kip2: The DNA was digested as for Lit1 and was hybridised with a probe specific for p57Kip2. The methylated
fragment is detected at 1.1 kb and the unmethylated at 0.8 kb. Snrpn: DNA was digested with XbaI and the methylation-sensitive HhaI and was hybridised with a
probe specific for the differentially methylated region 1 (DMR1) of the Snrpn gene (Shemer et al., 1997). The methylated DNA fragment is detected at 2.2 kb and the
unmethylated at 0.9 kb. H19 5′ promoter region: DNAwas digested with ApaI and the methylation-sensitive HpaII and was hybridised with a probe located at the 5′
end of the promoter region (Ferguson-Smith et al., 1993). The methylated fragment is detected at 1 kb and the unmethylated at 0.8 kb. Igf2 DMR1: DNAwas digested
with EcoRI and the methylation-sensitive HpaII and was hybridised with a probe specific to the DMR1 (Sasaki et al., 1992). The methylated fragments are detected
from 1.5 kb to 2.2 kb and the unmethylated from 1.3 kb and below.
676 T.C. Shovlin et al. / Developmental Biology 313 (2008) 674–681
been shown to exhibit epigenetic changes, so only early passage
EG cells were used (5–12) during these experiments.
EG cells derived from migrating PGCs and from PGCs that
have already colonised the genital ridges have a quite uniform
imprint erasure for most imprinted genes tested (Tada et al.,
1998; Durcova-Hills et al., 2001). However, Labosky et al.
(1994) had shown that the methylation pattern at Igf2r was
markedly heterogeneous in EG cells derived from pre-migratory
PGCs. To obtain a more comprehensive overview of the epi-
genetic status at this earlier time, genomic DNA was extracted
from the 11 early EG cell lines, and the methylation status at
differentially methylated sites of six imprinted genes loci (Igf2r,
p57Kip2, Lit1, Snrpn, H19 and Igf2) was analysed by Southern
blotting, along with late EG cell lines, ES cell lines and a so-
matic cell line, cultured mouse embryonic fibroblasts (MEFs) as
a control (Table 1).
Methylation patterns of imprinted genes
The two late EG cell lines were uniformly hypomethylated at
all differentially methylated sites tested with the exception of
sites in H19, confirming the findings of Tada et al. (1998) and
Durcova-Hills et al. (2001) for H19, see also Durcova-Hills et
al. (2006) (Durcova-Hills et al., 2006). In striking contrast to the
situation in ES cells and late EG lines, the different cell lines of
early EG cells were markedly heterogeneous. There was an
overall tendency for male EG cell lines to show more methyl-
ation than female lines, but Lit1, maternally methylated and
paternally expressed, was completely unmethylated in all the
EG cell lines (Fig. 2). The preferential expression of the ma-
ternal Igf2r depends on upon the differentially methylated
region (region 2), located in the second intron of the gene, and
the presence of the Air antisence transcript (Stoger et al., 1993).
In all the female lines examined, the locus was completely un-
methylated, but three out of the six male cell lines exhibited a
very low level or partially methylated fragment (Fig. 2), similar
to what had been previously seen (Labosky et al., 1994).
p57Kip2, paternally methylated and maternally expressed
(Hatada and Mukai, 1995), retained some level of methylation
in all of the lines, although it did not appear to be specific to the
sex of the cell line (Fig. 2). Half of the male cell lines were also
able to retain the methylation status of their Snrpn gene, which
is maternally methylated and paternally expressed (Fig. 2). The
5′ promoter of H19, a paternally methylated and maternally
expressed gene, was more methylated in EG cells derived from
male embryos when compared to female-derived cells (Fig. 2).
The imprinting control region had also been analysed and we
observed similar results (data not shown). The related gene,
Igf2, also retained a degree of methylation with the male lines
also being more methylated (Fig. 2). For those EG cell lines for
which repeat Southern analysis had been carried out (see Table
1), the methylation patterns were entirely consistent (data not
shown), suggesting that the heterogeneity was real and not
technical. Of the male EG cell lines, 6M showed the most me-
thylation and1M the least. For the female lines, 2F was the most
methylated and 1F the least. Similar to female EG cell lines,
female ES cells were also demethylated when compared to their
male counterparts, which showed a very uniform methylation
pattern. This confirms and reiterates previous findings (Zvet-
kova et al., 2005).
in the EG cell lines, Southern blot analysis was also done for
some repetitive element sequences. Repetitive elements are
the mouse genome and are highly methylated. During normal
embryo development, these sequences are usually exempt, to a
degree, from the demethylation process (Lees-Murdock et al.,
Fig. 3. Methylation status of IAP. DNAwas cleaved with the methylation-sensitive HpaII and probed with an IAP probe that comes from the IΔI element inserted at
the fused locus (Walsh et al., 1998). The MEFs were also digested with MspI to show the expected pattern that would be seen if the DNA is demethylated.
677 T.C. Shovlin et al. / Developmental Biology 313 (2008) 674–681
2003). When analysing the DNA methylation levels, we ob-
served that female EG cell lines, late and early, were hypo-
methylated at the repetitive elements IAP and minor satellite
repeats (Fig. 3 and data not shown). As expected the female ES
cell line was also hypomethylated (Zvetkova et al., 2005).
The question of heterogeneity
To answer the question of whether the level of heterogeneity
was between the lines or within them single cells of the EG cell
line, 4M were seeded to form clones of the cell line. Those cells
that formed a single colony were further propagated; DNAwas
isolated and Southern blots performed for the following genes;
Igf2r, H19 and the repetitive element IAP. The southerns
confirmed that the heterogeneity seen was not within the cell
line as all the clones showed a similar methylation pattern to the
original cell line (Fig. 4 and data not shown).
Methylation status on differentiation or prolonged culture
To check the stability of the methylation pattern seen in the
EG cell lines, we induced four of the lines, 2M, 1F, 3M, 7M and
an ES cell line to differentiate in vitro by the removal of LIF.
Embryoid bodies (EBs) were grown in droplets for 2 days. After
a further 5 days in suspension culture, the EBs were allowed to
attach and spontaneously differentiate on tissue-culture-grade
dishes for 15 days. To ensure that the cells had differentiated,
immunofluorescencewas performed withantibodies specific for
the three germ layers, ectoderm, mesoderm and endoderm.
Derivatives of all three germ layers were detected (Fig. 5A).
DNA was extracted from the differentiated cells and southern
blot analysis was performed as before. Of the four EG cell lines
tested, only 2M reverted to the somatic methylation upon dif-
ferentiation, for Igf2r, Lit1 and Snrpn (Fig. 5B). All the differ-
entiated cell lines tested increased the level of methylation at
H19, including the ES cell line (Fig. 5B). Also upon differ-
Since ES cells have been reported to show a gain in methyl-
ation at H19 and Igf2 after prolonged culture (Dean et al., 1998),
we cultured to a late passage number (between 13 and 34) the same three EG cell lines used previously to differentiate in vitro
in order to see whether EG cells resembled ES cells in showing
an increase in methylation, and whether other imprinted genes
would show a similar increase. The partial methylation seen at
Igf2r was maintained throughout the passages and still persisted
However, H19 and Igf2 were affected by the continued
passaging of the cells, showing a gradual gain in methylation
(Fig. 6), as seen in ES cells. When analysing the methylation
status at the repetitive element IAP, an increase in methylation
was also observed for the female EG cell line (Fig. 6).
In mammals, DNA methylation is a major epigenetic modifi-
cation that plays a key role in the transcriptional repression of a
Fig. 4. Methylation status of Igf2r, region 2 and H19 5′ promoter, for each clone
derived from single cells of the EG cell line 4M.
Fig. 5. In vitro differentiation of EG cells. (A) Differentiated EG cells were
stained with antibodies (green) specific for each of the germ layers: ectoderm
(Nestin), mesoderm (Fibronectin) and endoderm (TROMA-1). TOTO-3 was
used to counterstain the nuclei (blue). (B) DNA methylation status of the
imprinted genes Igf2r, Lit1, Snrpn and H19 and the repetitive element IAP.
678T.C. Shovlin et al. / Developmental Biology 313 (2008) 674–681
number of sequence classes, including imprinted genes (Li et
al., 1993) and repetitive elements (Walsh et al., 1998). Genomic
imprinting leads to the differential expression of a subset of
genes that is dependent upon their parent of origin, resulting in
monoallelic expression. It is crucial that these epigenetic
imprints be erased in the germ cell lineages in order for new
sex-specific imprints to be established. Since the number of
PGCs is limited, particularly in the early stages of development,
EG cells have provided a unique tool for the study of epigenetic
modification in the germ line and in particular the methylation
status of imprinted genes. However, the initial hope that the
methylation status of EG cells would reflect that of the PGCs
from which they were derived proved ill-founded, at least for
late EG cell lines (Durcova-Hills et al., 2001).
In the present study, we have examined the differences in
methylation at several imprinted genes and repetitive elements
in early EG cell lines compared to late EG cell lines and ES
cells, both before and after differentiation. Our data indicate that
the methylation status of imprinted genes in our early EG cells
is different from ES cells. It is more similar to that of late EG
cells, which have almost all of their site-specific methylation
erased, suggesting that the level of site-specific methylation for
early EG cells is already being down-regulated.
We have demonstrated that our early EG cell lines are hete-
rogeneous with regard to differential methylation, for the ma-
jority of the imprinted genes tested. This could be because the
PGCs from which the EG cells are derived are also hetero-
geneous with regard to differential methylation. PGCs may
develop according to some internal cell-autonomous clock for
reprogramming, including demethylation, but environmental
cues probably also play a role. Certainly e8.0–8.5 is a time
when striking epigenetic changes are taking place in PGCs, as
they leave their “niche” location at the base of the allantois and
embark on their migration in the endoderm of the hind gut. Seki
etr al. (2005) determined the level of DNA methyl–cytosine in
single PGCs at e8.0, separately from those that were still at the
base of the allantois and those that had already entered the
endoderm. The endodermal PGCs showed a dramatic drop in
the level of DNA methylation while those that had not migrated
into the endoderm retained a DNA methylation level similar to
the surrounding somatic cells.
The early EG cell lines (e8.5) listed in Table 1 were made
from embryos recovered at about midday. Since embryonic
development is by no means synchronous, it is quite possible
that some of our more highly methylated lines (e.g. 2M) were
derived mainly from PGCs at the base of the allantois, while the
Fig. 6. Methylation status after prolonged culture of e8.5 EG cell lines: 2M, 1F and 3M. DNA was obtained after passage numbers 13, 18, 23, 29 and 34 and was
analysed for site-specific differential methylation of Igf2r, Lit1, H19, Igf2 and the repetitive element IAP. p=passage number of the cells.
679 T.C. Shovlin et al. / Developmental Biology 313 (2008) 674–681
PGCs that gave rise to the more hypomethylated lines (e.g. 1F)
had already entered the hindgut endoderm. We therefore made
EG cell lines, from Stella×129, at about 6 a.m., when the PGCs
were observed to be located at the base of the allantois (1 EG
cell line) and a further 2 lines at 6 p.m. from the same source,
when the PGCs had begun to migrate. The 3 cell lines showed
similar methylation (data not shown), suggesting that asyn-
chronous of development was not the explanation of the
heterogeneity that we observed.
For several of the imprinted genes that we investigated, the
male early EG cell lines showed more methylation than the
female. In late EG cells, H19 and Igf2 are the only imprinted
genes to show methylation at differentially methylated sites in
male EG cells (Tada et al., 1998), probably reflecting the early
establishment of the new methylation imprint on these genes
(Davis et al., 2000). For H19, new methylation has appeared
already in the male germ cells at e15.5 and e16.5 (Durcova-Hills
et al., 2006). For both the e15.5, e16.5 germ cells and the late
EG cells, we have shown, using sex-reversed mice, that XX
male germ cells and EG cells were significantly less methylated
than their XY controls. The effect was particularly striking in
EG cells, where there is no tissue environment to influence the
extent of methylation of imprinted genes (Durcova-Hills et al.,
2004, 2006). Both X chromosomes are active in germ cells at
this stage, and also in EG cells as in other pluripotent stem cell
lines. The reduced level of methylation observed in XX cells
resembles that reported in XX compared with XY or XO ES
cells, suggesting that the X chromosome encodes some modifier
locus that represses de novo methyltransferases (Okano et al.,
1999), with over-expression of the locus by the two active X
chromosomes (Zvetkova et al., 2005). After prolonged culture,
the increase in the level of methylation at the repetitive element
IAP could be due to the loss of one of the active X
chromosomes as reported in ES cells (Zvetkova et al., 2005).
The level of demethylation in XX EG cells at the IAP repe-
titive element and the extent of its heterogeneity suggest a level
of instability, which could militate against clinical applications.
We would like to thank Masahiro Kaneda and Colum Walsh
for probes and Peter Beverly for the TG-1 (SSEA-1) antibody.
This work was supported by grants from the BBSRC (to AML)
and the Wellcome Trust (to AS). GD-H is a recipient of a MRC
joint collaborative career development fellowship funded by the
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