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cells have been reported in a variety of animals, including
mice [12–15], rats [16], rabbits [17], sheep [18], cattle
[19], and pigs [20–22], chimeras derived from EG cells
have only been reported in mice [23,24] and pigs [25,26],but notin goats. To determine if caprine EG cells have the
pluripotency to generate all three germ layers and form
chimeras when injected into host blastocysts, we isolated
EG cells from goat fetuses and injected these cells into
host blastocysts.
2. Materials and methods
2.1. Materials and animals
The sources of materials were: Dulbecco’s modifiedEagle’s medium (DMEM) and knockout serum repla-
cement (KSR; Gibco, Life Technologies, Grand Island,
NY, USA); nonessential amino acids, b-mercaptoetha-
nol, and L-glutamine (Invitrogen Corp., Carlsbad, CA,
USA); dNTPs (Takara. Bio Inc., Otsu, Japan); cDNA II
kit (Ambion Europe Ltd., Huntingdon, Cambridgeshire,
UK); recombinant murine leukemia inhibitory factor
(LIF), human recombinant basic fibroblast growth
factor (bFGF), stem cell factor (SCF), and antibodies
against SSEA-1 and c-kit (Chemicon International Inc.,
Temecula, CA, USA); pregnant mare serum gonado-
tropin (PMSG) and prostaglandin (PGF2a; NingboHormone Co., Ningbo, Zhejiang, China). Unless
otherwise indicated, all other chemicals were purchased
from the Sigma chemical company (St. Louis, MO,
USA).
Local black goats were used to produce host
blastocysts, whereas white Guanzhong goats were used
as a source of EG cells and to serve as surrogates. The
coat color of the local breed was completely black;
however, when these goats were crossed with a white
Guanzhong goat, the coat color of the resulting kids was
brown (no black hair nor white spots).
2.2. Isolation and culture of goat embryonic germ
cells
To isolate PGCs, nine fetuses were collected fromseven white Guanzhong dairy goat fetuses at 28–42 days
of pregnancy (Table 1). The gonadal ridge tissue was
removed, washed three times with PBS plus 0.02%
ethylenediaminetetraacetic acid (EDTA), dissected
manually, and incubated for 30 min at 38.5 8C in a cell
dissociation solution containing 0.25% collagenase IV.
The cell suspension was filtered through sterile gauze
(100 mesh, 149 mm) and washed in PBS once, then
pelleted by centrifugation at 1000 Â g for 5 min. The
suspension of cell mixture of gonadal tissue was co-
cultured with goat embryonic fibroblasts (GEF) that hadbeen inactivated with mitomycin C treatment on gelatin-
coated culture dishes. After 10–12 days of growth, EG-
like cell colonies with 100–200 cells were formed and
then subcultured by picking up individual colonies and
seeding on 35 mm culture dish; this subculture was
considered the first passage of EG cells. Although goat
EG cells can grow well with both GEF and mouse
embryonic fibroblast (MEF), we routinely used MEF as
the feeder cells to culture goat EG cells, since it was
easier to use. For the immunohistochemistry assay, MEF
and EG cells were cultured on cover slides that were laid
on a culture plate. The culture medium was DMEMsupplemented with 15% KSR, 1000 IU/mL LIF, 10 ng/
mL bFGF, 10 ng/mL SCF, 0.1 mM nonessential amino
acids, 0.1 mM b-mercaptoethanol, 2 mM L-glutamine,
100 IU/mL penicillin, and 0.1 mg/mL streptomycin. The
above three factors, LIF, bFGF and SCF, were always
added in media as supplements, even when typical EG
colonies were formed.
The isolated EG cells were cryopreserved in liquid
nitrogen by a method similar to that used for MEF cells.
Briefly, 5 Â 105 cells collected from cultural plates
were pelleted in a 10 mL centrifuge tube, and
W. Jia et al. / Theriogenology 69 (2008) 340–348 341
Table 1
Isolation of PGCs from caprine fetuses
Goat Fetus Gestational
age (days)
Fetus length (cm) EG cells Passage no.a
1 1 28 1.2 EGC#1 P3
2 2a 35 1.7 EGC#2 P3
2b 35 1.8 EGC#3 P123 3 35 2.1 EGC#4 P12
4 4 42 4.5 EGC#5 P0
5 5 35 2.4 EGC#6 P10
6 6 35 2.0 EGC#7 P8
7 7a 37 2.6 EGC#8 P10
7b 37 2.8 EGC#9 P9
a The last passage of EG cells that were cultured in medium.
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resuspended in 1 mL of cryo-storage solution contain-
ing DMEM with 20% fetal bovine serum and 10%
dimethyl sulfoxide. Cells were then transferred to a
storage tube (2 mL) and stored in liquid nitrogen. To
reuse the cryopreserved cells, a tube of cells was
removed from liquid nitrogen and immediately trans-
ferred to a 37 8C water bath to incubate for 1–2 min.The thawed cells were subsequently cultured in the
60 mm plate with MEF feeder cells.
2.3. Immunohistochemistry and cellular assay
To characterize the origin of the isolated cells, we
determined several stem cell specific markers. To
confirm the presence of alkaline phosphotase (AP), EG
cells grown on a cover slide were fixed with 4%
paraformaldehyde for 20 min, washed 10 min with PBS
for three times, and stained with 200 mg/mL of naphtholAS-MX phosphate and 1 mg/mL of Fast Red TR in
0.1 mM Tris buffer (pH 8.2) for 15 min at room
temperature. To detect cell surface markers, cells fixed
with 4% paraformaldehyde were incubated with 3%
H2O2 for 5 min, and blocking solution for 15 min;
antibodies against SSEA-1 (1:100) and c-kit (1:50) were
incubated separately with cells for 12 h at 4 8C. The
secondary goat anti-mouse IgG antibody and chromo-
gen solutions (3,3-diaminobenzidine; DAB), were
applied according to the manufacturer’s instructions
(Beijing ZhongShan Golden Bridge, Beijing, China).Positive cells were detected by red/brown staining and
the control was only incubated with secondary antibody,
followed by staining. In the karyotype assay, EG cells at
the stage of exponential growth were treated with
0.1 mg/mL colcemid for 2 h, and then spread and dried
on the glass slide. The karyotype was determined by
microscopic examination after conventional Giemsa
staining.
2.4. RT-PCR reaction
An RT-PCR was done to detect the Nanog gene thatis specifically expressed in ES cells. The EG cells were
lysed by incubation at 75 8C for 10 min with cell lysate
buffer. Genomic DNA was degraded by incubation with
DNAase I for 15 min at 37 8C; 0.5 mg of total RNAwas
used to synthesize cDNA by MLV reverse transcriptase
and random hexamers using cDNA II kit in 10mL
reaction volume. We subsequently used 5 mL of the
reverse transcription products in the subsequent PCR to
amplify the Nanog gene. The PCR were conducted in a
total volume of 25 mL at 35 cycles of 94 8C for 30 s,
558C for 30 s, and 72
8C for 80 s; PCR products were
separated on 1.0% agarose gel with ethidium bromide.
Reverse transcriptase negative controls were included to
monitor genomic contamination. Primers based on goat
Nanog gene (AY786437) were synthesized to amplify
the 819 bp cDNA fragment. The sequence of primers
was F-Nanog: 50ATGCCTGAAGAAAGTTACGC, and
R-Nanog: 50AGGCTGTATGTTGAGAGGGT.
2.5. Generation of chimera
The EG cells were cultured for three or four passages
and incubated for 30 min in 0.05% trypsin and 0.02%
EDTA. After neutralization by culture medium, cells
formed small clusters (which contained 10–20 cells). By
using a microinjector (Leica, DM IRB, Wetzlar,
Germany) with a protocol similar to that described by
Schoonjans [17], an EG cell cluster was delivered into a
blastocyst that was isolated from a naturally mated black goat and cultured for 7 days in vitro (Fig. 1A). The
injected blastocysts were incubated in a CO2 incubator
for 30 min, and then immediately transferred into white
recipient goats on Day6 (estrus = Day 0). To synchronize
estrus, recipient goats were given an intravaginal
progestagen-impregnated sponge (Ova-Gest; Bioniche
Australasia, Armidale, NSW, Australia) for 10 days;
goats were injected with 200 IU PMSG 7 days after
sponge insertion, followed by 0.2 mg PGF2a 24 and 12 h,
respectively, before sponge removal. Goats were
observed frequently for estrous activity for 48 h (starting24 h after the second PGF2a treatment).
2.6. Characterization of chimeric kids
For characterization of chimeric kids, DNA samples
were isolated from EG cells and tissues including blood
and skin (from the head) with whitehair or with black hair
from both male and female kids, and from various tissues
(includingliver,placenta, lung,heart, spleen, muscle, and
brain) from the dead fetus. The DNA samples were also
isolated from ear skin of black Dam #1 and Sire #1 of the
host blastocyst and surrogatewhite Dam #2. We used a Y-chromosome-specific gene SRY (Z30646) to determine
if the injected EG cells had differentiated into various
tissues and organs. To detect this gene, two pairs of
primers were synthesized according to a published
sequence [27]. The primer sequences were:
SRY, forward: 50ATGAATAGAACGGTGCAATCG 30
SRY, reverse: 50GAAGAGGTTTTCCCAAAGGC30
Aml-X, forward: 50CAGTAGCTCCAGCTCCAG
CT30
Aml-X, reverse: 50
GTGCATCCCTTCATTGGC30
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The 116 bp fragment of Y-chromosome-specific
gene SRY and 300 bp fragment of internal control
gene Aml-X (AF215887) were amplified by PCR,
performed in 20 mL of reaction mixture that contained
20 ng DNA, 200 mM of dNTP, 1.5 mM MgCl2, 10 pmol
of forward and reverse primers, 0.4 U Taq DNA
polymerase, and carried out for 36 cycles of 94 8C
for 30 s, 58 8C for 30 s, and 72 8C for 45 s. The PCR
products were separated by 12% polyacrylamide gel
electrophoresis and visualized by silver staining.
2.6.1. Microsatellite assay
Ten microsatellite markers, including BMS1004,
BMS1290, BM203, BMS875, BMS574, SR-CRSP1,
SR-CRSP5, SR-CRSP24, OarAE101, and OarFCB11
were selected to characterize the chimerism of the
newborn kids. The PCR were conducted with 20 ng
DNA template isolated from skin and blood samples,with 32 cycles of 94 8C for 30 s, 55 8C for 30 s, and
72 8C for 45 s. The amplified DNA fragments were
separated on 15% polyacrylamide gel and visualized by
silver staining.
3. Results
3.1. Derivation and characterization of goat EG
cell lines
The PGCs isolated from fetal gonads were round oroval, and had a large nucleus and a high nucleus-to-
plasma ratio. When subcultured on feeder layers for 3–5
days, PGCs started to form clusters with morphology
typical of ES cells (Fig. 2A). We isolated eight EG-like
cells from nine fetuses; it was easier to successfully
isolate EG cells from fetal gonads at 35–37 days,
compared to those at 28 or 42 days. Two isolated EG
cells, EGC#3 and EGC#9, had a normal male karyotype
and were used for blastocyst injections (Table 1). The
isolated EG cells were cultured up to 12 passages
without loss of AP activity (Fig. 2B). The EG cellcolonies also had positive staining for SSEA-1 and c-kit
(Fig. 2C and D). At passage 12, the proportion of
isolated EG cells in the entire culture was low, due to
spontaneous differentiation. However, the remaining
EG cells still had AP activity and were positive for
SSEA-1 and c-kit. To determine if the EG cells
contained the ES cell-specific transcription factor, RT-
PCR was done to monitor Nanog mRNA expression;
both PGCs and EG cells expressed the Nanog gene
(Fig. 3). Based on all of these assessments, the isolated
cells were EG cells and had stem cell features.
3.2. Production of chimera
Twenty-nine injected blastocysts, of which ten used
cryopreserved EG cells, were implanted in nine white
surrogate goats. One surrogate goat, Dam #2, main-
tained pregnancy to full term, and gave birth to three
kids, including a dead malformed fetus of undetermined
gender with three legs and failure of the abdominal wall
to close Table 2. One kid, a black male, was designated
GB1 and the other, a black female with a large white
spot on her forehead (Fig. 1B), was designated GBW1.
W. Jia et al. / Theriogenology 69 (2008) 340–348 343
Fig. 1. Blastocyst injection and chimeric goats. The blastocyst from
the black femalehost was injected with approximately 10–20 EG cells
from a white male goat fetus;injectedblastocystswere transferred into
a white recipient goat. (A) EG cells were injected into the blastocyst;
(B) two kids a few minutes after birth; (C) dead, malformed fetus
(gender unknown; all three fetuses were littermates). The arrows
indicated the white hair in both the female kid and the dead fetus
(they were chimeras).
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The malformed fetus (GBW2) also had white hair that
covered a much larger area of the head than that in
GBW1 (Fig. 1C). Although we had implanted only two
injected blastocysts into Dam #2, that she gave birth to
three kids (in conjunction with other evidence), we
inferred that both female kid GBW1 and malformed
fetus GBW2 were monozygotic twins.
3.3. Molecular analysis of newborn kids
To determine if the newborn kids were chimeras, the
SRY gene was analyzed by PCR. The Aml-X gene (X
amelogenin) co-amplified in PCR assay was used as an
internal control to detect the X-chromosome marker.
We injected EG cells from a white male goat into the
blastocyst of a black goat; if the injected blastocysts
developed to form a fetus, the chimera was expected to
retain two features, white hair and SRY gene positive,
especially in a female offspring. Kid GBW1 not only
had white hair (Fig. 1B), it also had the SRY gene, even
W. Jia et al. / Theriogenology 69 (2008) 340–348344
Fig. 2. Isolation and characterization of goat EG cells. The isolated goat EG cells formed a colony with typical morphology of pluripotent stem cells(A: 200Â); the EG cell colony was positive for alkaline phosphatase activity (B: 200Â), SSEA-1 (C: 200Â), and c-kit (D: 400Â).
Fig. 3. The RT-PCR reaction analysis of Nanog gene in isolated EG
cells. The RT-PCR reactions were done with Nanog gene primers and
the primary culture of goat PGCs (column 1) or EG cells in passages 3
or 4 (column 2). Column 3 was the negative control and M was the
1500 bp DNA ladder.
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though it was a female goat (Fig. 4, column 3). The dead
fetus (GBW2) also had white hair on its head and the
SRY gene; therefore, both GBW1 and GBW2 were
chimeric goats (Fig. 4, column 2).
To further characterize the degree of chimerism, 10
microsatellite markers were analyzed on DNA samples
isolated from GB1, GBW1, and GBW2, the parents of
the host blastocyst (Dam #1 and Sire #1) as well as EGcells and Dam #2. Among the ten markers, there were
three markers (BMS574, BMS875, and BMS1004) that
demonstrated the variation between chimera and normal
goats. The microsatellite assay (Fig. 5a) was based on
marker BMS574; both GBW1 and GBW2 had an
identical DNA pattern, which was significantly different
from the pattern in GB1. On the gel, there were two
groups of DNA bands in GBW1 and GBW2. One group
of bands in the 200 bp area consisted of the combined
bands from both EG cells and parent goat DNA. The
second group of bands in the 300 bp area had two bands,one from the EG cell and the other from Sire #1;
however, the bands in the second group were not present
in samples from Dam #1 and Dam #2. Additionally,
GB1 had a similar DNA pattern to that of Sire #1, but
completely different from that of the EG cells,
indicating that GB1 was not a chimera (Fig. 5a). Blood
samples from GB1 and GBW1 were also analyzed by
marker BMS574; these samples had DNA fingerprint-
ing similar to that from skin samples, suggesting that the
EG cells can differentiate into blood cells in a chimeric
goat (Fig. 5b).
Based on microsatellite assays, EG cells in GBW1
developed into skin and blood. To determine if EG cells
were able to differentiate into other tissues and organs,
we collected GBW2 tissues that covered all three germ
layers, including skin areas with white and black hair,
liver, placenta, lung, heart, spleen, muscle, and brain,
and did microsatellite assays with BMS574 marker. All
tissues tested had the EG cell signal, indicating that EGcells in the injected blastocyst differentiated into all
three germ layers (Fig. 5c). In addition to using
BMS574 marker, similar experiments were done with
W. Jia et al. / Theriogenology 69 (2008) 340–348 345
Table 2
Caprine chimeras produced by microinjection of EG cells into blastocysts
Blastocysts injected Recipients Term pregnancies Kids Chimeras
EGC#3a 7 4 1 3 2c
Cryo-EGC#3b 10 2 0 0 0
EGC#9a 12 3 0 0 0
a EGC#3 was at passage 4 and EGC#9 was at passage 3.b EGC#3 were cryopreserved in liquid nitrogen, thawed and used.c One of the chimeras was a dead, malformed fetus.
Fig. 4. The PCR assay for SRY and Aml-X genes. The SRY gene
fragment (116 bp) and Aml-X gene fragment (300 bp) were amplified
from EG cells (column 1), the malformed fetus (column 2), female kid
(column 3), male kid (column 4), Dam #1 (column 5), Sire #1 (column
6), and the negative control (column 7). M was a 100 bp DNA ladder.
Fig. 5. Microsatellite assays with BMS574 marker; PCR products
were separated with a 15% polyacrylamide gel. (a) DNA from skin
tissue or EG cells was used for the assay; (b) assay of blood samples
from two newborn kids; (c) assay of nine tissues from the malformed
dead fetus. M , 100 bp DNA marker; FL, Dam #1; ML, Sire #1; EG,
embryonic germ cell; RP, Recipient Dam #2; GB1, male kid; GBW1,
female kid; GBW2, malformed dead fetus; WS, skin from white hair
area; LV, liver; BS, skin from black hair area; CL, placenta; LG, lung;
HT, heart; SP, spleen; MS, muscle; BR, brain.
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markers BMS875 and BMS1004. Both GBW1 and
GBW2 had identical DNA bands (Fig. 6a and b) whichwere completely different from that of GB1. In
summary, we concluded that GB1 was a normal kid
without heterozygosis, and GBW1 and GBW2 were
chimeric kids and monozygotic twins.
4. Discussion
The EG cell lines were derived from cultured PGCs
in media containing LIF, FGF2, and SCF. Compared
with isolation of embryonic cells from the inner cell
mass (ICM) of embryos, the advantage of using PGCs to
purify stem cells was that an aborted fetus was easier to
obtain [28]. However, the primary PGCs were difficult
to subculture and passage; cell-to-cell bonds among
PGCs were tighter than those of embryonic cells derived
from the ICM [7]. To isolate goat PGCs, antibodies
against SSEA-1 and EMA-1 were applied on a flow
cytometer to collect marker-specific PGCs [10]. In theearly 1980’s, Felice et al. established three approaches
to isolate murine PGCs, including collagenase treat-
ment and mechanical procedures, with or without prior
EDTA treatment [23]. To improve isolation efficiency,
we incubated genital ridge segments with 0.02% EDTA
for 20 min, and then incubated them with 0.25%
collagenase IV for 25–30 min. Using this approach,
PGCs were isolated and continually cultured in vitro for
up to 12 passages. The EG cells derived from PGCs of
Guanzhong goats had embryonic stem cell features and
could differentiate into multiple cell types [32].The proliferation and differentiation of goat EG cells
have been reported previously, although the focus was
mainly on the isolation of goat PGCs and culture of EG
cells [7–10]. Lee et al. reported that the AP-positive
PGCs were isolated from a Day 25 goat fetus; these
isolated EG cells were only maintained for four
passages in culture medium [7,9]. Until now, only
Tillmann’s group reported that goat ES cells could be
cultured for prolonged intervals in vitro [11]. There was
no previous report describing a chimeric goat with EG
cells. In our study, we not only successfully isolated andcultured goat PGCs, but also purified EG cells up to 12
passages and document their pluripotency, demonstrat-
ing that they could differentiate into three germ layers in
vivo. However, it is difficult to construct a goat EG cell
line; we still face the difficulty of culturing isolated EG
cells for prolonged intervals without spontaneous
differentiation.
Ten microsatellite markers were used to determine
whether the progeny were chimeras; however, only
three markers, BMS574, BMS875, and BMS1004,
detected differences among EG cells, the parents, and
the newborn kids. Similar observations were reported inpig chimeras that were created by delivering porcine EG
cells into recipient blastocysts [25,26]. On marker
BMS574 assays, GBW1 had chimeric skin and blood
that the only tissues were tested, whereas in GBW2, all
nine tissues and organs tested were chimeric. Therefore,
these EG cells were capable of differentiating into three
germ layers without affecting tissue functions; GBW1
was healthy at birth and has already lived 11 months. In
contrast, Shim et al. reported that stated that in a porcine
chimera, chimerism only occurred in blood, brain,
pancreas and muscle, but not in heart, spleen, liver, or
W. Jia et al. / Theriogenology 69 (2008) 340–348346
Fig. 6. Microsatellite assays of DNA samples, isolated from skin of
littermate goat kids, conducted with markers BMS875 (a) and
BMS1004(b). PCRproducts were separatedon a 15% polyacrylamide
gel. GB1, newborn male kid; GBW1, newborn female kid; GBW2,
malformed dead fetus.
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lung [25]. There are many factors affecting the
developmental capability of EG cells after they are
delivered into a blastocoelic cavity, including viability
of the EG cells, the number of cells injected, and the
interaction between EG cells and ICM, etc. Soon after
EG cells were involved in blood tissue formation, they
were also able to proliferate and differentiate into othertissues and organs, as well as obtain immune tolerance
[29–31].
To further characterize goat chimeras, the SRY gene,
a male gender-specific gene, was analyzed by PCR. The
experiment was designed so that the injected EG cells
contained a Y-chromosome, and if a newborn female
kid had an SRY gene, the animal would be a chimera.
Both GBW1 and GBW3 retained the SRY gene, similar
to that of the injected EG cells. How male cells were
chimerically present in the female tissue and whether
the XY EG cells can develop into the genital tissue, isyet to be determined. However, we plan to raise GBW1
and to determine if it has normal estrous cycles,
ovulation, and fertilization.
This study is the first report of a chimeric goat derived
by injecting caprine embryonic germ (EG) cells into a
host blastocyst. Furthermore, it was noteworthy that the
EG cells differentiated into all three germ layers in vivo.
Acknowledgments
We thank: Dr Hong Chen for discussion; JingzhuangFan and Xiaoliang Han for technical support; Dr Rachel
Maier for comments on the manuscript. This work was
supported in part by grants from the National High-Tech
Research and Development Program of China
(2005AA21905) and the Key Program of Shaanxi
Province (4130253301).
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