BIOLOGY OF REPRODUCTION 69, 48–56 (2003)
Published online before print 5 February 2003.
DOI 10.1095/biolreprod.102.014522
Somatic Cell-Like Features of Cloned Mouse Embryos Prepared with Cultured
Myoblast Nuclei1
Shaorong Gao,3 Young Gie Chung,3 Jean W. Williams,3 Joan Riley,5 Kelle Moley,5 and Keith E. Latham2,3,4
The Fels Institute for Cancer Research and Molecular Biology3 and Department of Biochemistry,4
Temple University School of Medicine, Philadelphia, Pennsylvania 19140
Division of Reproductive Endocrinology and Infertility,5 Washington University School of Medicine,
St. Louis, Missouri 63130
ABSTRACT
Cloning by somatic cell nuclear transfer requires silencing of
the donor cell gene expression program and the initiation of the
embryonic gene expression program (nuclear reprogramming).
Failure to silence the donor cell program could lead to altered
embryonic phenotypes. Cloned mouse embryos produced using
myoblast nuclei fail to thrive in standard embryo culture media
but flourish in somatic cell culture media favored by the donor
myoblasts themselves, forming blastocysts at a significant rate,
with robust morphologies, high total cell number, and a normal
allocation of cells to the inner cell mass in most embryos. Myo-
blast cloned embryos continue expressing the GLUT4 glucose
transporter, which is typically expressed in muscle but not in
preimplantation stage embryos. Myoblast clones also exhibit
precocious enrichment of GLUT1 at the cell surface. Both myo-
blast and cumulus cell cloned embryos exhibit enhanced rates
of glucose uptake. These observations indicate that silencing of
the donor cell genome during cloning either is incomplete or
occurs progressively over the course of preimplantation devel-
opment. As a result, cloned embryos initially exhibit many so-
matic cell-like characteristics. Tetraploid constructs, which pos-
sess a transplanted somatic cell genome plus the oocyte-derived
chromosomes, exhibit a more embryonic-like pattern of gene
expression and culture preference. We conclude that preim-
plantation stage cloned embryos have profoundly altered char-
acteristics that are donor cell type specific and that exposure of
cloned embryos to standard embryo culture conditions may lead
to disruptions in basic homeostasis and inhibition of a range of
essential processes including further nuclear reprogramming,
contributing to cloned embryo demise.
developmental biology, early development, embryo, gene regu-
lation
INTRODUCTION
Cloning by somatic cell nuclear transfer, while possible,
remains highly inefficient [1–7]. A majority of cloned em-
bryos arrest development before implantation, and most re-
maining embryos arrest before or during fetal development
[8]. The reasons for the poor development of cloned em-
bryos have not been elucidated. Defects in DNA methyla-
1
These studies were supported by grants from the NIH to K.E.L.
(HD38381), to K.M. (HD/DK40390), and to the Fels Institute for Cancer
Research (5 T32 CA09214-20); S.G., Y.G.C., and J.W.W. contributed
equally to the study.
2
Correspondence. FAX: 215-707-1454; e-mail: klatham@unix.temple.edu
Received: 19 December 2002.
First decision: 13 January 2003.
Accepted: 3 February 2003.
Q 2003 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
48
tion observed in some cases have prompted the hypothesis
that epigenetic information, including parental genomic im-
printing, may be disrupted in cloned embryos [9–16]. Such
disruptions are unlikely to cause early arrest, as even uni-
parental embryos and embryos deprived of the maternally
inherited DNA methyltransferase Dnmt1o [17] exhibit the
capacity to complete early stages of development.
Successful cloning requires silencing of the donor cell
gene expression program and the initiation of the embry-
onic gene expression program (nuclear reprogramming).
Failure to silence completely the donor cell program could
lead to expression in the early cloned embryo of gene prod-
ucts more typical of the donor cell. Combined with an em-
bryo-specific pattern of protein synthesis directed by ma-
ternal mRNAs, this would create a hybrid gene expression
pattern intermediate between that of normal embryos and
somatic cells. This hybrid gene expression pattern would
cause the cloned embryo to have an altered phenotype man-
ifested in its physiology, metabolism, and culture medium
requirements [18, 19], leading to reduced development of
cloned embryos in culture media optimized for normal fer-
tilized embryos. Our earlier studies revealed that the donor
cell nucleus can affect cloned embryo phenotype, as re-
flected in culture medium preference, even as early as the
one-cell stage [18].
This effect of the original donor cell gene expression
program on cloned embryo phenotype could lead to differ-
ent outcomes in cloning using different donor cell types.
Earlier studies led to the suggestion that cells in the G0
compartment of the cell cycle may be better nuclear donors
than actively proliferating cells, even those in G1 [6]. Clear-
ly, nonproliferating cells differ dramatically from actively
dividing cells on many levels, including gene transcription
and metabolic state. Other studies have concluded that the
differentiated state of the donor cell from which the nucleus
is obtained may affect the efficiency of nuclear reprogram-
ming [7, 20], and cloning with embryonic stem (ES) cells
has been comparatively successful [21, 22]. Although the
effect of differentiated cell state is often discussed in terms
of chromatin structure, the metabolic and physiological
state of the differentiated cell, if imposed on the cloned
embryo, could also have an effect. Stem cells and their
differentiated derivatives often occupy spatially separate
domains within tissues or distinct locales within the em-
bryo. During differentiation, basic cell parameters such as
membrane potential and metabolic parameters can change
dramatically. Additionally, different differentiated cell
types may reside within specialized environments and be
uniquely specialized to function in those environments.
This specialization of donor cells of different developmen-
tal states for different environments or different functions
 SOMATIC FEATURES OF CLONED EMBRYOS
could affect the outcome of cloning by directing uniquely
altered repertoires of gene expression in the early cloned
embryo. Thus, apparent difference in cloning efficiency be-
tween stem cells and differentiated cells, between prolif-
erating and arrested cells, or between donor cells of differ-
ent tissue origins could reflect, in part, differences in donor
cell metabolic or physiological characteristics that persist
in the cloned embryo.
Exploring the effects of the donor cell genome on clon-
ing efficiency will require studies to be undertaken on an
array of cell types. Muscle tissue presents an interesting
opportunity for such cloning studies because it is an abun-
dant tissue that is somewhat accessible and because stem
cells are readily identifiable and can be isolated, cultured,
and enriched in vitro [23, 24]. As undifferentiated, prolif-
erating stem cells, myoblasts could provide a source of nu-
clei that would exhibit a significant ability to support clonal
development. To test the hypothesis that different types of
donor cells can produce cloned embryos with different
characteristics, we compared cloned embryos made with
myoblast nuclei to clones made with cumulus cell nuclei.
We find that cloned embryos made with myoblast donor
cell nuclei differ profoundly from fertilized control embry-
os, exhibiting a clear intolerance for embryo-optimized cul-
ture conditions and a clear preference for the same somatic
cell-like culture conditions preferred by the myoblast donor
cells themselves. The change in culture requirements was
thus much more severe with myoblast cloned embryos than
that observed for cumulus cell cloned embryos [18]. Par-
thenogenetic control embryos resembled fertilized embry-
os, as expected, and tetraploid embryos, prepared in the
same manner as cloned embryos but with the oocyte ge-
nome left in place, did not show characteristics as dramat-
ically altered, indicating that the effects of the donor cell
genome can be ameliorated by the presence of the oocyte-
derived genome. Along with altered culture requirements
in myoblast cloned embryos, we observe an altered pattern
of glucose transporter expression and localization indicative
of persistent execution of the donor cell program. The sig-
nificance of these observations to understanding nuclear re-
programming and cloned embryo biology is discussed.
MATERIALS AND METHODS
Myoblast Cell Culture
Two kinds of myoblasts were employed in these studies. C2C12 myo-
blasts were obtained from the American Type Culture Collection (ATCC,
Manassas, VA). This is an established myoblast cell line with compara-
tively relaxed growth requirements (DMEM supplemented with 10% fetal
bovine serum [FBS]). Myoblasts were also isolated from C57BL/6 3 Mus.
m. castaneus females as described [24]. Muscle tissue (adult hind limb)
was thoroughly minced in PBS and then digested with 10 ml of 0.1%
hyaluronidase (Sigma, St. Louis, MO) and 0.1% collagenase type V (Sig-
ma) in PBS for 20 min at 378C. Before the incubation, and periodically
during the incubation, the tube was vigorously vortexed. The tube was
then centrifuged briefly at ;350 3 g for 1–2 min and the supernatant
discarded. The pelleted tissue was rinsed 23 in 10 ml of PBS and digested
with 0.05% trypsin/EDTA in Ca21- and Mg21-free Hank’s balanced saline
(HBSS, GIBCO/InVitrogen, Carlsbad, CA) for 20 min at 378C with trit-
uration using a 25-ml pipette after 10 min of incubation to facilitate di-
gestion. After sedimenting the tissue pieces, the supernatant was collected,
and 1 ml of FBS was added. The pellet was redigested in the same manner
and resedimented, and the two supernatants were combined. The super-
natants were then filtered through 0.5-mm mesh Nitex nylon membrane,
and the cells were pelleted by centrifugation for 10 min and resuspended
in myoblast enrichment medium [Ham’s F10 (GIBCO), 20% FBS, 5 ng/
ml basic fibroblast growth factor (bFGF, Promega, Madison, WI), 100 U/
ml penicillin (GIBCO), 100 mg/ml streptomycin (GIBCO) sulfate, and
0.25 mg/ml fungizone (GIBCO)] and plated on 100- 3 20-mm collagen
coated dishes.
49
Extensive selection procedures were applied to remove contaminating
cell types as described [23]. Briefly, cells were grown at 378C in 5% CO2
in myoblast enrichment medium, which favors myoblast growth over fi-
broblast growth. After 2 days, the cells were fed with fresh medium. After
5 days, culture medium was replaced with PBS, and the myoblasts were
selectively released by vigorously tapping the plate. The cells were pel-
leted, then exposed to a collagen coated dish for 10–20 min in enrichment
medium, and then the medium and unattached cells were transferred to a
new dish. This selective release of myoblasts was repeated eight more
times as cells reached ;60% confluency. Analysis of myoblast clonal col-
onies revealed that all (35/35) contained differentiated multinucleated
myofibers (.97% myogenic). This indicates that the enrichment proce-
dures were very effective. After enrichment for myoblasts, the cells were
maintained and passaged in myoblast growth medium (1:1 mixture of
Ham’s F10 and DMEM, supplemented with 20% FBS, 5ng/ml bFGF, pen-
icillin, streptomycin, and fungizone as described previously). These myo-
blasts are referred to hereafter as F1 myoblasts.
Nuclear Transfer and Embryo Culture
Metaphase II-arrested oocytes were obtained from superovulated
(B6D2)F1 adult females (8–12 wk of age). Oocyte-cumulus cell complexes
were isolated at 13–14 h post-hCG and cumulus cells removed using hy-
aluronidase as described [18]. Oocytes were cultured in Chatot, Ziomek,
Bavister (CZB) medium [25] supplemented with 5.5. mM glucose
(CZBG). Spindle-chromosome complexes (SCCs) were removed using a
narrow bore pipette attached to a piezo pipette driver as described [5].
Removal of the SCCs was performed in HEPES buffered CZB medium
[26] supplemented with 5.5 mM glucose as described and 2.5 mg/ml cy-
tochalasin B [18]. Polar bodies were also removed during this operation.
After removal of the SCCs, the oocytes were washed and intact myoblasts
inserted under the zona pellucida. Myoblasts of the smallest size (predom-
inantly G1) were selected manually for injection. Electrofusion of the
myoblasts to the oocytes was accomplished using electrical pulses of 90
V DC in fusion medium (275 mM mannitol, 5 mM CaCl2, 10 mM MgSO4,
0.3% bovine serum albumin [BSA]). If needed, an additional one or two
pulses were given at 30–45-min intervals. Typically, 90% fusion was
achieved with only two pulses. At 30 min after fusion, oocytes were ac-
tivated by 5 h culture in Ca21-free CZB medium supplemented with 10
mM Sr21 as described and 5 mg/ml cytochalasin B [5]. Diploid parthe-
nogenetically activated embryos were obtained using the same activation
protocol without removal of the SCC. The parthenotes were obtained from
the same pools of oocytes used to make cloned embryos and were acti-
vated at the same time. Parthenogenetic embryos resemble normal fertil-
ized embryos with respect to culture requirements and many aspects of
gene expression (with the obvious exception of maternally imprinted
genes) and have the added advantage that they develop in close temporal
synchrony with the activated cloned embryos. Tetraploid embryos were
prepared by introducing somatic cell nuclei into oocytes without removal
of the SCC. These embryos thus undergo all the same procedures as the
diploid clones, with the exception of SCC removal. They provide a means
for evaluating whether the presence of an oocyte-derived genome affects
embryo characteristics [18]. Additional media employed in the study were
Whitten’s medium (WM) [27] and potassium simplex optimized medium
with (KSOMaa) or without (KSOM) amino acid supplementation [28] and
the 1:1 Ham’s F10:DMEM mixture used previously for myoblast culture
without serum or antibiotics but supplemented with 1mM glutamine, 0.27
mM sodium pyruvate, and 0.4% bovine serum albumin. Cloned embryos
prepared with cumulus cell donor nuclei were also made for comparative
purposes as described [18]. It should be noted that the investigators who
participated in making the cloned constructs (YGC and SG) have suc-
cessfully produced live cloned offspring using cumulus cell and/or ES cell
nuclei. All studies adhered to procedures consistent with the National Re-
search Council Guide for the Care and Use of Laboratory Animals.
Blastocyst Analysis
To determine total cell number, embryos were fixed in buffered for-
malin, stained with DAPI, and examined under fluorescent illumination to
view nuclei. Immunosurgery [29] on live blastocysts was used to isolate
inner cell masses from cloned blastocysts in order to facilitate the deter-
mination of the number of cells in the inner cell mass. After immunosur-
gery, the ICMs were fixed, stained with DAPI, and mounted on glass
slides, and the cells were counted under fluorescent illumination.
50 GAO ET AL.
TABLE 1. Development of cloned embryos in WM/KSOMaa medium combination.a
Activated
Medium constructs (Repl.)
C2C12 myoblast donor
WM/KSOMaa 130 (5)
WM/KSOMaa; (DMSO) 46 (2)
Cumulus donor
WM/KSOMaa 155 (6)
a Medium changed from WM to KSOMaa at the eight cell/morula stage.
b vs. c significantly different, P , 0.05.
Analysis of Glucose Uptake
For glucose uptake studies, embryos were cultured overnight in CZB
medium for analysis at either the late one-cell stage or the two-cell stage.
Glucose uptake was assayed by culturing embryos in CZB medium (glu-
cose free) supplemented with 0.5 mCi/ml 2-[1,2 3H]-deoxyglucose. After
incubation, embryos were washed extensively in CZBG and lysed in
groups of 5–10 embryos each. The incorporated label was quantified by
scintillation counting for 1 h per sample. Glucose uptake was expressed
as cpm per embryo per hour.
Analysis of Glucose Transporter Expression
Preimplantation embryos were freed of the zona pellucida using acid-
ified (pH 2.5) Tyrode’s medium and fixed for 10 to 15 min at room tem-
perature in 3.7% formaldehyde in PBS. All solutions for immunofluores-
cence were prepared in PBS, and procedures were performed at room
temperature unless specified otherwise. The fixed cells were blocked for
at least 1 h in blocking buffer (3% BSA, 0.1% Triton X-100).
For the glucose transporter immunohistochemistry, the fixed embryos
were placed on slides, permeabilized with 0.1% Tween, and washed with
phosphate buffered saline containing 2% bovine serum albumin (PBS/
BSA). Embryos were then blocked by incubating for 60 min in 20% don-
key serum in PBS/BSA, followed by 1 h incubation in affinity-purified
primary antibody to mouse GLUT1 or GLUT4 (both rabbit polyclonal
antibodies obtained from Dr. Mike Mueckler, Washington University
School of Medicine) at a dilution of 15 mg/ml. These antibodies have been
used extensively in previous studies, and their specificity is well docu-
mented [30, 31]. The embryos were then washed three times for 10 min
each PBS-BSA and incubated with the secondary antibody, fluorescein
isothiocyanate (FITC)-labeled goat anti-rabbit IgG (Chemicon, Temecula,
CA), at a concentration of 1:80, followed by TO-PRO-3 iodide
(MolecularProbes, Eugene, OR) at a concentration of 4mM for 20 min to
visualize DNA. Finally, the embryos were washed three times for 10 min
each in PBS-BSA and mounted in drops of Vectosheild (Vecto Labs, Bur-
lingame, CA) under a supported coverslip. Fluorescence was detected with
a Bio-Rad MRC-600 laser-scanning confocal microscope. Confocal im-
ages were taken at 633 magnification. Total fluorescence per embryo was
expressed as a number ranging from 3 to 0 as judged by two observers
blinded to the identity of the embryo. The fluorescence values for each
group of embryos were averaged. At least 10 embryos existed in each
group, and each experiment was repeated at least twice.
RESULTS
Alterations in Culture Medium Preference for Myoblast
Cloned Embryos
We previously reported that cloned embryos prepared
with cumulus cell donor nuclei, without DMSO exposure
during the procedure, exhibit altered culture medium pref-
erences relative to normal and other control mouse embryos
[18]. Specifically, cumulus cell cloned embryos developed
poorly in KSOMaa, CZB, or CZBG but developed better
when cultured in WM until the eight-cell/morula stage and
then switched to KSOMaa until blastocyst formation.
Cloned embryos also exhibited an unexpected preference
for glucose containing media. We therefore tested whether
myoblast cloned embryos can likewise develop in the WM/
KSOMaa combination or have different culture medium re-
quirements. Cloned embryos constructed with nuclei from
% Development
Two-cell Four-cell Eight-cell/morula Blastocyst
52 38 35 22b
57 37 28 24b
93 56 45 34c
C2C12 myoblasts developed to blastocysts in this medium
at a reduced rate as compared with cumulus cell cloned
embryos (Table 1). Also unlike cumulus cell cloned em-
bryos, about half the C2C12 myoblast cloned embryos
failed to progress to the two-cell stage. Treatment with
DMSO during oocyte activation, which previously im-
proved cumulus cell cloned embryo development in CZB
medium [18], failed to overcome this early block.
Different results were obtained using F1 myoblasts as
nuclear donors. Clones made with these nuclei developed
to the two-cell stage efficiently in WM but then exhibited
a pronounced arrest in development to the four-cell stage
(Table 2). Similarly poor results were obtained with cloned
embryos made with another established transgenic mouse
myoblast line (data not shown). To test whether this two-
cell arrest was possibly related in any way to the two-cell
arrest exhibited by some strains of fertilized embryos in
some media, we tried different embryo culture medium for-
mulations. The two-cell arrest was also seen in both CZBG
and KSOM. Interestingly, cumulus cell cloned embryos
cultured initially in KSOM and then switched to KSOMaa
formed blastocysts more efficiently under these conditions
than when cultured continuously in KSOMaa ([18] and Ta-
ble 2), similar to a previous study [19]. Parthenogenetic
control embryos developed efficiently to the blastocyst
stage in all these media ([18] and Table 2). Tetraploid em-
bryos also develop well in these media [18]. Thus, the fail-
ure of the F1 myoblast cloned embryos to develop efficient-
ly beyond the two-cell stage was a unique feature of these
cloned embryos rather than a nonspecific effect of the cul-
ture system. These results also distinguish the established
and easily cultivated C2C12 cell line from the F1 myoblasts
with respect to effects of these donor nuclei on cloned em-
bryo phenotype.
Our previous studies with cumulus cell cloned embryos
prompted the hypothesis that the somatic cell genome may
be expressed in the early cloned embryo and thereby alter
its phenotype [18]. Such an effect of the somatic cell ge-
nome could contribute to the overall poor success observed
when culturing the cloned embryos made with F1 myoblasts
in standard embryo culture media by shifting their culture
medium preference toward a more somatic cell-like for-
mulation. We therefore tested whether the somatic cell cul-
ture medium used to prepare growth medium for the F1
myoblasts could support improved cloned embryo devel-
opment. This medium consisted of a 1:1 mixture of Ham’s
F10 and DMEM media, without serum, and was supple-
mented with 0.4% BSA, 1 mM glutamine, and 0.27 mM
pyruvate. The results were striking. The two-cell block was
virtually eliminated, and over 40% of the cloned embryos
developed to the blastocyst stage (Table 2). Interestingly,
DMSO treatment of oocytes during activation did not im-
prove the outcome. However, we found that prolonging
slightly the interval between hCG injection to induce su-
 SOMATIC FEATURES OF CLONED EMBRYOS 51

TABLE 2. Development of myoblast cloned embryosa prepared using primary F1 myoblasts and parthenogenetic control embryos in different media.

% Development
Activated
Medium constructs (Repl.) Two-cell Four-cell Eight-cell/morula Blastocyst
Cloned embryos
WMb 50 (2) 96 14 2 0A
CZBGb 49 (2) 96 20 12 2A
KSOM -. KSOMaab 55 (2) 93 16 7.3 5.5A
KSOM -. KSOMaa (cumulus) 130 (4) 82 50 40 22B
F10:DMEMb 113 (2) 91 79 69 43C
F10:DMEM; DMSObd 131 (2) 94 84 65 38CE
F10:DMEMc 108 (3) 88 68 35 17D
F10:DMEM; DMSOcd 54 (2) 89 65 53 31E
F10:DMEM -. KSOMaabe 98 (3) 83 59 30 18D
F10:DMEM -. WMbe 102 (3) 85 62 34 25DE
Tetraploid embryos
F10:DEM2 34 (2) 97 59 35 0F
CZBG2 49 (2) 100 96 96 61G
Parthenogenetic embryos
WMb 50 (2) 98 96 94 92H
CZBGb 76 (3) 100 99 97 93H
KSOM -. KSOMaab 50 (2) 100 100 100 88H
F10:DMEMb 102 (3) 98 64 14 1I
F10:DMEM; DMSObd 71 (2) 100 33 9.9 0I
F10:DMEM -. KSOMaabe 63 (2) 100 52 16 3.2I
F10:DMEM -. WMbe 68 (2) 96 50 28 19 J
a All data for diploid cloned embryos are from myoblast cloned constructs except cumulus cloned constructs where indicated. Tetraploid embryos were
prepared using myoblast nuclei.
b,c Activated 19.5- and 23.5-h post-hCG injection, respectively. For later time, oocytes isolated at 17-h post-hCG.
d DMSO present at 1% concentration during activation step.
e Medium changed on third day (eight-cell/morula stage) after activation.
A–J Significantly different as follows: A vs. B, P , 0.01; A vs. C, C vs. D, A vs. H, F vs. G, H vs. I, H vs. J, I vs. J, P , 0.001; D vs. E, P , 0.05.

perovulation and the oocyte activation step negatively af-

fected the outcome, and DMSO treatment did have a pos-
itive effect in this situation. Not surprisingly, parthenoge-
netic embryos were unable to form blastocysts efficiently
under these conditions. Tetraploid embryos likewise did not
form blastocysts efficiently in this medium but developed
well in CZBG, indicating an effect of the oocyte-derived
chromosomes on embryo phenotype.
The F1 myoblast cloned blastocysts obtained by culture
in the F10:DMEM medium exhibited very robust, well-
expanded morphologies (Fig. 1A) with a greater average
number of cells per blastocyst (59.5 6 12.9 SD, 120 h post-
hCG) than cumulus cell cloned embryos grown under the
optimum WM/KSOMaa combination [18] (Fig. 1, B and
C). In fact, this cell number approached that typically seen
for fertilized embryos (range 68–75 cells/embryo) [18]. Out
of 22 blastocysts subjected to immunosurgery, 14 yielded
visible inner cell masses. Eleven of these ICMs were suc-
cessfully recovered for cell counts and yielded an average
of 18 cells/ICM (SD 7.8). Thus, inner cell masses were, on
average, a significant fraction of the total cells contained in
a majority of the blastocysts.
We next tested whether switching the F1 myoblast cloned
embryos from the F10:DMEM mixture to a standard em-
bryo culture medium at the eight-cell/morula stage might
further enhance development to the blastocyst stage (Table
2). Switching embryos to either KSOMaa or WM signifi-
cantly reduced blastocyst formation in comparison to con-
tinuous culture in F10:DMEM (43% versus 18% and 25%,
respectively). Switching parthenogenetic control embryos
to WM but not KSOMaa had a slightly beneficial effect.

Glucose Uptake in Cloned Embryos FIG. 1. Blastocysts developing from myoblast cloned embryos. A) Bright-
field image of representative blastocysts. B and C) Bright-field and fluo-
We previously found that cumulus cell cloned embryos rescent DAPI stained image of a hatching myoblast cloned blastocyst. The
favored culture media that contained a high concentration mouse embryo is approximately 80 mM in diameter.
52 GAO ET AL.
FIG. 2. Glucose uptake in cloned embryos. A) Cumulus cloned embryos
at the late one-cell stage. B) Cumulus cloned embryos that cleaved during
or just prior to labeling (i.e., early two-cell stage). C) Myoblast cloned
embryos at the late one-cell stage. D) Myoblast cloned embryos at the
mid two-cell stage. Data are expressed as the number of counts per mi-
nute taken up per embryo per hour 6 SEM. Statistically significant dif-
ferences ascertained by t-test are indicated by different letters above bars.
Clone, diploid cloned embryos; Parth, diploid parthenotes; Tetra, tetra-
ploid constructs made by injecting single nuclei into intact oocytes (SCC
not removed); Fert, Fertilized embryos.
of glucose even as early as the one-cell stage [18]. The
preference of F1 myoblast cloned embryos for the F10:
DMEM culture medium suggested that these embryos may
also manifest an alteration in glucose requirement. To test
whether these two types of cloned embryos differed from
control embryos with regard to glucose uptake, we exposed
embryos of each type to tritiated 2-deoxyglucose and then
after extensive washing monitored uptake by scintillation
counting. Cloned embryos of either type exhibited a sig-
nificant increase in glucose uptake at the late one-cell stage
relative to control parthenogenetic embryos (Fig. 2). The
cumulus cell cloned embryos also exhibited enhanced up-
take of glucose relative to tetraploid control embryos and
fertilized control embryos, and this pattern persisted in em-
bryos immediately after cleavage. The tetraploid one-cell
stage embryos prepared with myoblast nuclei exhibited the
same enhanced rate of glucose uptake as the diploid cloned
embryos at the one-cell stage. Glucose uptake was signifi-
cantly elevated in myoblast cloned embryos relative to par-
thenogenetic embryos and tetraploid embryos at the two-
cell stage, when embryo gene transcription has begun (Fig.
2D). In fact, two-cell stage myoblast cloned embryos dis-
played a rate of uptake that was nearly 6-fold greater than
that of parthenogenetic embryos and nearly 3-fold greater
than that of tetraploid embryos. Thus, while the rate of
glucose uptake in diploid cloned embryos remained ele-
vated, the enhanced rate of glucose uptake seen in tetra-
ploid embryos at the one-cell stage was reduced. The rate
of glucose uptake was similar between cloned embryos and
in vivo fertilized embryos (38 h post-hCG) (Fig. 2D). It
should be noted that parthenogenetic and tetraploid embry-
os are activated simultaneously and cultured in parallel with
the cloned embryos, whereas the fertilized embryos may
be slightly more advanced developmentally. These data in-
dicate that during the two-cell stage, the rate of glucose
uptake increases to a level similar to that in fertilized em-
bryos more rapidly in myoblast cloned embryos than in
parthenogenetic or tetraploid controls.
Expression of Glucose Transporters
Given the increased rate of glucose uptake in cloned
embryos relative to control embryos, we wished to ascer-
tain whether cloned embryos display altered glucose trans-
porter expression, indicative of continued expression of do-
nor cell type genes. Preimplantation stage mouse embryos
express GLUT1 at all stages [32], and this transporter is
also expressed in many somatic cell types. We therefore
examined the expression of GLUT1 in late one-cell stage
cumulus and myoblast cloned embryos by immunofluores-
cence staining. Cumulus cell cloned embryos exhibited a
modest increase in GLUT1 expression relative to parthe-
nogenetic control embryos (Fig. 3A). Tetraploid embryos
made with cumulus cell donor nuclei exhibited an inter-
mediate level of staining. Myoblast cloned embryos did not
exhibit such an increase in GLUT 1 expression at the late
one-cell stage or any later stage (Fig. 3B) but at the two-
cell stage exhibited enhanced plasma membrane localiza-
tion of GLUT1, which normally does not occur until the
eight-cell stage and which was not seen in either parthe-
nogenetic or control embryos (Fig. 4A). Cloned embryos
treated with a-amanitin to inhibit transcription showed
much reduced expression of GLUT1, indicating that the
expression observed in untreated cloned embryos was tran-
scription dependent (Fig. 3B). At the blastocyst stage, the
localization of GLUT1 to the plasma membrane appeared
to be reduced in myoblast cloned embryos in comparison
to parthenogenetic and fertilized embryos (Fig. 4C).
Because glucose transporter type 4 (GLUT4) is ex-
pressed in muscle but not in preimplantation stage embryos
[32], we examined the expression of this transporter in
myoblast cloned embryos and controls. The myoblast
cloned embryos at the late one-cell stage expressed signif-
icant quantities of GLUT4, well above the low level of
background fluorescence observed in the parthenogenetic
and fertilized control embryos (Figs. 3C and 4B). Diploid
myoblast cloned embryos treated with a-amanitin showed
greatly reduced signals for GLUT4 at the two-cell stage,
indicating that the GLUT4 expression seen in untreated dip-
loid cloned embryos was transcription dependent and not
merely a result of protein or mRNA carryover from the
donor cell. Tetraploid embryos expressed slightly elevated
quantities of GLUT4 at the one-cell stage and two-cell
stage, but this was less than that seen in the diploid cloned
embryos (Figs. 3C and 4B). Expression declined progres-
sively in tetraploid embryos with development to the blas-
tocyst stage. Continued increased staining was seen in dip-
loid myoblast cloned embryos at later stages for GLUT4,
relative to parthenogenetic, tetraploid, and flushed fertilized
embryos (Figs. 3C and 4D). Thus, diploid myoblast cloned
embryos showed aberrant, high expression of GLUT4
throughout preimplantation development, distinguishing
them from all three types of control embryos. Although
enrichment of GLUT4 at the plasma membrane was not
seen, such enhanced localization is often insulin dependent,
and cycling of a fraction of the GLUT4 protein between
cytoplasm and plasma membrane can nevertheless occur
and contribute to glucose uptake.
DISCUSSION
Our results reveal that the initial cellular phenotype of
the donor cell, even a proliferating stem cell, plays a pivotal
role in determining the phenotype of the cloned embryo.
Myoblast cloned embryos can develop to the blastocyst
stage at a significant rate and form blastocysts with robust
 SOMATIC FEATURES OF CLONED EMBRYOS
morphologies that possess an expected total number of cells
and a normal allocation to the inner cell mass. Such de-
velopment, however, requires somatic cell-like culture con-
ditions, which differ dramatically from those required by
normal or parthenogenetic control embryos and even
cloned embryos made with other donor cell type nuclei.
Moreover, we have shown that an established or immortal-
ized cell line (C2C12) can produce dramatically relaxed
culture requirements in cloned embryos as compared with
primary cells or established cell lines with more typical
culture requirements. We have also documented aberrant
expression of GLUT4 (a myoblast expressed protein), pre-
cocious localization of GLUT1 to the plasma membrane,
and enhanced glucose uptake in cloned embryos. Thus, the
shift in cloned embryo phenotype toward more somatic-like
characteristics is manifested at the molecular level as well
as in the response of the cloned embryos to the culture
environment. These results reveal that nuclear reprogram-
ming is slow or inefficient in cloned embryos and that the
donor genome dramatically modifies embryo phenotype.
Based on our experience with myoblasts as donor cells,
it appears that those extracellular conditions that are fa-
vored by the donor cell may serve as a guide for identifying
favorable conditions for the cloned embryo. The degree to
which this rule applies is likely to be influenced by vari-
ables such as the degree of potency (mono-, multi-, or plu-
ripotential) and the degree of disparity between donor cell
environment and the normal oocyte/embryo environment.
The degree of physiological or metabolic specializations of
53
FIG. 3. Semiquantitative analysis of
GLUT1 and GLUT4 expression in cloned
embryos. Total fluorescence per embryo
was expressed as a number ranging from 3
to 0 as judged by two observers blinded to
the identity of the embryo. The fluores-
cence values for each group of embryos
were averaged. At least 10 embryos exist-
ed in each group, and each experiment
was repeated at least two times. A) Data
for GLUT1 staining in cumulus cell cloned
embryos and controls at the one-cell
stage. B and C) Data for GLUT1 and
GLUT4 staining, respectively, in myoblast
cloned embryos and controls at the stages
indicated. No expression of GLUT4 was
seen in a-amanitin treated embryos, as in-
dicated by the *. m, Parthenotes; u, Tetra-
ploids; g, Diploid Clones; t, Flushed fer-
tilized embryos; □, a-amanitin treated
embryos. D) Representative photographs
show, from top to bottom, embryos as-
signed grades 0 through 3 of staining. The
mouse embryos are approximately 80 mM
in diameter.
the donor cell may also be important. For example, cumulus
cell nuclei promote a preference for glucose containing me-
dia, and cumulus cells themselves are specialized for en-
hanced glucose metabolism [18]. By analogy, other cellular
specialization may have other effects. For example, elec-
trically excitable cells such as differentiated neurons, car-
diomyocytes, or multinucleated skeletal muscle fibers may
manifest intracellular ionic properties distinct from those of
nonexcitable cells, which, through continuation of the so-
matic cell gene expression program, could then shift the
intracellular ionic properties of the cloned embryo. Such a
change could be accompanied by a shift in extracellular
culture requirements. It will indeed be interesting to ascer-
tain whether cloned embryos might share other features
with their corresponding nuclear donors, such as growth
factor or cytokine responsiveness, and whether such char-
acteristics can synergize with or interfere with the functions
of proteins normally expressed in developing embryos.
The significant reduction in blastocyst formation ob-
served when myoblast cloned embryos are returned to stan-
dard embryo culture media at the morula stage and the
continued expression of GLUT4 through the blastocyst
stage indicate that, despite an otherwise robust morphology,
the cellular phenotypes in these embryos remain skewed
toward that of the donor cells. This indicates that repro-
gramming likely requires a prolonged period extending
even beyond the preimplantation period. Previously ob-
served mosaicism in the expression of other genes such as
Oct4 [33] may thus be explicable on this basis, as very
54 GAO ET AL.

FIG. 4. Immunofluorescent staining for

glucose transporters in myoblast clone em-
bryos and controls. A and C) Embryos
stained with antibody to GLUT1 at the 2-
cell and blastocyst stages, respectively. B
and D) Embryos stained with antibody to
GLUT4 at the 2-cell and blastocyst stages,
respectively. P, parthenote; T, tetraploid
construct, C, diploid cloned embryo; F, in
vivo fertilized embryo; Am, a-amanitin
treated cloned embryo. Note: Blastocyst
stage embryos have been compressed un-
der coverslips to permit visualization of
cell borders. The mouse embryos are ap-
proximately 80 mM in diameter.

slow reprogramming over the course of six or more cell
cycles could lead to such heterogeneity between blasto-
meres. Formation of the inner cell mass and the definitive
embryonic cell lineage may thus be a highly selective pro-
cess that recruits only those cells that have undergone the
greatest degree of nuclear reprogramming. Physiological
and metabolic properties may constitute important param-
eters on which this selection operates. Recruitment of cells
to this lineage may thus be inhibited not only by delayed
or inadequate expression of regulatory proteins like Oct4
[33], which must be reactivated during clonal development,
but also by persistent expression of other somatic cell-type
proteins in some cells, which would make those cells un-
able to flourish within the specialized compartment even-
tually occupied by the ICM and its derivatives.
The apparently leisurely pace of nuclear reprogramming
likely has important consequences for determining when
cloned embryos should be returned to a reproductive tract
environment and how they will fare when placed in that
environment. Because the oviduct provides an environment
that is highly optimized for normal embryos, this same en-
vironment could constitute a poor environment for myo-
blast cloned embryos during the first several cell cycles.
Because the myoblast cloned embryos were only partially
more tolerant of standard embryo culture media at the mor-
ula stage, this negative effect of the oviduct environment
may prevail even beyond the morula stage. Thus, the over-
all pace of reprogramming combined with the initial degree
of disparity between the preferred culture environment and
the reproductive environment in situ will likely affect when
cloned embryos made with various donor cells can be trans-
ferred to foster mothers. Because the period during which
embryos may be transferred is limited, donor cell types for
which the pace of reprogramming is unusually slow or for
which the disparity between environmental requirements is
largest may simply be unable to yield viable progeny at a
significant rate, unless the pace of reprogramming can be
improved by altering the culture environment.
Our results also reveal that procedural factors can affect
the viability of cloned embryos and that these effects may
be cell type specific. For example, the presence of DMSO
during the activation step was able to overcome culture
medium preferences in cloned embryos made with cumulus
cell nuclei [18] but was unable to overcome the detrimental
effects of standard embryo culture media on myoblast
cloned embryos. The length of time between ovulation (re-
lated to the timing of hCG injection) and oocyte activation
significantly affects blastocyst formation rates among myo-
blast cloned embryos, but such an effect was not readily
apparent with cumulus cell cloned embryos ([5] and Y.G.
Chung, unpublished results). The effect of using piezo-me-
diated nuclear injection versus electrofusion is significant
with myoblast nuclei, possibly because of the greater size
 SOMATIC FEATURES OF CLONED EMBRYOS
of the myoblast nucleus as compared with, for example, ES
cell or cumulus cell nuclei.
Our analysis of glucose transporter gene expression in
cloned embryos indicates that the altered culture medium
preference is associated with continued expression of the
somatic cell program. The cumulus cell cloned embryos
exhibited an early increase in GLUT1 expression. The myo-
blast cloned embryos exhibit precocious GLUT1 localiza-
tion to the plasma membrane and continue to express
GLUT4 at all stages of preimplantation development ex-
amined. GLUT4 is typically expressed in muscle cells but
not in preimplantation stage mouse embryos [30, 32]. This
augmentation of glucose transporter expression is reminis-
cent of the enhanced preference for glucose containing me-
dia exhibited by cumulus cell cloned embryos [18]. We also
observed increased uptake of 29-deoxy glucose by both
myoblast and cumulus cell cloned embryos, indicating that
the increased transporter expression is functionally signifi-
cant.
The precocious enrichment of GLUT1 at the cell surface
in two-cell stage cloned embryos and the reduced plasma
membrane localization of GLUT1 in cloned blastocysts re-
veal alterations in the normal temporal pattern of protein
localization. Thus, the donor cell nucleus clearly alters a
variety of basic cellular functions in the cloned embryo,
including gene expression, protein localization, and glucose
uptake. Such alterations are likely to be extensive and re-
sponsible for the unique phenotype observed for the cloned
embryos.
It is clear that an authentic embryonic genome can exert
a potent regulative effect that is noticeably absent or greatly
reduced in the cloned embryo. Others have reported effects
of maternal oocyte-derived chromosomes on DNA meth-
ylation [13]. We observed effects of oocyte-derived chro-
mosomes on the expression of GLUT1 and GLUT4. Tet-
raploid embryos do not exhibit precocious localization of
GLUT1. GLUT4 expression is lower in tetraploids than in
diploid cloned embryos. Tetraploids also failed to form
blastocysts efficiently in the F10:DMEM medium, prefer-
ring instead standard embryo culture conditions. Using cu-
mulus cell donor nuclei, the rate of glucose uptake and
expression of GLUT1 are lower with tetraploid embryos
than with diploid cloned embryos. The silencing of donor
cell genes thus may be a complex process, involving a com-
bination of functions provided by maternally inherited fac-
tors in the oocyte and embryonically expressed factors.
These observations suggest two interesting possibilities that
need not be mutually exclusive. One is that the transplanted
donor cell nuclei fail to express key transcriptional regu-
lators, which may be uniquely expressed by the maternal
chromosomes, or perhaps more simply by an authentic em-
bryonic genome. Alternatively, the maternally derived
chromosomes may have a unique ability to express factors
involved in nuclear reprogramming. With respect to normal
embryos, the oocyte-derived chromosomes may thus direct
a critical part of the transcriptional programming of the
embryonic genome. With respect to cloned embryos, this
transcriptional programming function may be seriously
compromised by removal of the maternal chromosomes.
This creates the interesting possibility that retransfer of a
somatic cell genome after it has been exposed to ooplasm
together with a maternal set of chromosomes, or retransfer
of the cloned embryo genome to enucleated blastomere cy-
toplasm, might result in an improved efficiency of the clon-
ing process.
Overall, our data indicate that nuclear reprogramming
55
during mammalian cloning by somatic cell nuclear transfer
most likely occurs over a protracted period of time. As a
result, many genes expressed in the somatic cell donor nu-
cleus are likely transcribed in the cloned embryos as early
as the one-cell stage. Additionally, posttranscriptional gene
regulation appears to be disrupted in cloned embryos.
These effects confer altered phenotypes and culture require-
ments on the cloned embryos that are donor cell type spe-
cific. Given the inefficient silencing of the donor cell ge-
nome and the effect of this on cloned embryo phenotype,
improving the efficiency of donor cell genome silencing
may constitute a critical threshold that must be overcome
before the overall efficiency of cloning can be improved.
Providing a culture environment that is optimized for each
donor cell type will likely be critical for maintaining cloned
embryo homeostasis and supporting the reprogramming
process. Obviously, identifying culture conditions that sup-
port early cleavage stage development in cloned embryos
is essential to the process. Additional studies seeking to
refine further those conditions that support early develop-
ment may help to improve long-term developmental poten-
tial. The development of multistep culture systems that al-
low the culture environment to change in concert with the
progress of reprogramming may provide the greatest im-
provements to cloned embryo development. Further studies
to characterize in detail the specific alterations in gene ex-
pression, metabolism, and physiology of cloned embryos
made with a variety of donor cell types will be needed in
order to approach the development of optimized culture
systems through rationale design. Such studies should also
further our understanding of the basic biology of normal
fertilized embryos.
Until the effect of continued expression of the donor cell
gene expression program on cloned embryo phenotype is
thoroughly understood and accounted for experimentally,
conclusions related to the relative developmental capacities
of different types of donor cell nuclei or their ability to be
reprogrammed must be viewed with caution. We show here,
for example, that nuclei from differentiated cumulus cell nu-
clei support a greater degree of development under standard
embryo culture conditions than nuclei from undifferentiated
myoblasts, and yet the latter can manifest a much greater
degree of developmental competence to the blastocyst stage
when a different culture system is employed. Thus, it appears
that reprogramming may be equally limited with either dif-
ferentiated or undifferentiated cell nuclei and that the pre-
dominant effect of donor cell type on developmental capac-
ity is the degree to which the persistent donor cell gene
expression program shifts the cloned embryo phenotype
away from that which is typical of normal embryos. It will
be challenging to dissect inherent limitations on reprogram-
ming that may exist within the donor cell nuclei, limitations
related to donor cell type-specific effects on culture require-
ments, and their associated secondary effects on the pace of
reprogramming. The use of developmental milestones will
be inadequate for this purpose, making detailed assays that
evaluate reprogramming at the level of specific gene ex-
pression while minimizing the impact of culture essential.
ACKNOWLEDGMENT
We thank Bela Patel for technical assistance in this work.
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