MOLECULAR AND CELLULAR BIOLOGY, June 2003, p. 3982–3989 Vol.23,No. 11
0270-7306/03/$08.000 DOI: 10.1128/MCB.23.11.3982–3989.2003
Copyright ? 2003, American Society for Microbiology. AllRightsReserved.
Hybrid Embryonic Stem Cell-Derived Tetraploid Mice Show
Apparently Normal Morphological, Physiological, and
Neurological Characteristics
Frieder Schwenk,1 Branko Zevnik,1 Jens Bru¨ning,2 MathiasRo¨hl,2Antje Willuweit,1
Anja Rode,1 Thomas Hennek,1 Gunther Kauselmann,1RudolfJaenisch,3
and Ralf Ku¨hn1*
Artemis Pharmaceuticals GmbH1 and Klinik II und Poliklinikfu¨rInnere Medizin der Universita¨t Ko¨ln and
Center of Molecular Medicine,2 Cologne, Germany, andWhiteheadInstitute for Biomedical Research and
Department of Biology, Massachusetts Institute ofTechnology,Cambridge, Massachusetts3
Received 23 October 2002/Returned for modification 25November2002/Accepted 24 February 2003
ES cell-tetraploid (ES) mice are completely derived fromembryonicstem cells and can be obtained at high
efficiency upon injection of hybrid ES cells intotetraploidblastocysts. This method allows the immediate
generation of targeted mouse mutants from genetically modifiedEScell clones, in contrast to the standard
protocol, which involves the production of chimeras andseveralbreeding steps. To provide a baseline for the
analysis of ES mouse mutants, we performed aphenotypiccharacterization of wild-type B6129S6F1 ES mice
in relation to controls of the same age, sex, and genotyperaisedfrom normal matings. The comparison of 90
morphological, physiological, and behavioral parametersrevealedelevated body weight and hematocrit as the
only major difference of ES mice, which exhibited anotherwisenormal phenotype. We further demonstrate that
ES mouse mutants can be produced from mutant hybrid ES cellsandanalyzed within a period of only 4
months. Thus, ES mouse technology is a valid research toolforrapidly elucidating gene function in vivo.
The standard protocol to derive mouse mutants currently
requires the production of germ line chimeras from heterozygous
targeted embryonic stem (ES) cells, followed by at least
two breeding steps to obtain homozygous mutants (2). Thus,
the production of a mutant strain is a time-intensivetaskexceeding
12 months prior to the analysis of ***** mutants. In
addition, substantial resources are required for the breeding
and genotyping of several hundred mice involved in a typical
knockout project. Besides this classical approach, conditional
gene targeting through Cre/LoxP-mediated recombination is
increasingly used as it allows the spatial and temporal control
of gene inactivation (13, 21). Given that a Cre transgene needs
to be introduced via additional breeding steps, the production
of conditional knockout mice involves four reproductive cycles
requiring at least 16 months before the target gene’s function
can be analyzed in vivo.
Due to these extensive timelines, the impact of targeted
mutants in high-throughput functional genome analysisiscurrently
limited, creating a demand for a time-saving single-step
procedure. Cloning of mice does not provide a viablealternative
because the nuclear transplantation procedure is inefficient
and a variety of abnormalities have been described in
cloned mice which likely result from incompletegenomereprogramming
(8, 17, 25, 30). Alternatively, ES cell-tetraploid
(ES) mice can be produced in a single step throughtheintroduction
of diploid ES cells into tetraploid blastocysts (16). The
latter provide an initial host environment forthedifferentiation
of ES cells but do not contribute to the embryo at later
developmental stages. Although the methodology to produce
ES mice from inbred ES cell lines was described more than a
decade ago, its application was limited due to the extremely
low frequency at which viable ES pups are recovered (16).
Recently, this technology was significantly improved through
the discovery that ES cell lines derived from hybrid mouse
strains support the development of viable ES mice at a 50-fold
higher rate than inbred ES cells (4). Importantly,theproduction
of ES mice is technically not more demanding than the
generation of chimeras (15). Thus, ES mouse technology now
offers the opportunity to efficiently produce targeted mouse
mutants directly from hybrid ES cell clones within a single
mouse generation and without the requirement for further
breeding. With this approach, classical as well as conditional
mouse mutants can be obtained in less than half the time
compared to the current knockout protocol. The technical
feasibility of this novel approach is demonstrated by therecent
report that hybrid ES cell lines tolerate multiple consecutive
gene-targeting cycles without loosing their potencyfortetraploid
blastocyst complementation (5, 23).
The biological characteristics of ES cell-tetraploid mice have
not yet been fully described. This aspect is ofparticularinterest
because faulty expression of imprinted genes and increased
body weight have been documented for both cloned neonates
and ES mice (9). A validation of the biological characteristics
of ES mice is therefore required to provide a baseline for the
phenotyping of ES mouse mutants.
We report here an extensive phenotypic characterization of
***** B6129S6F1 ES mice derived from wild-type ES cells in
relation to controls of the same age, sex, and genotype raised
by normal breeding. The comparison of multiple morphologi-
* Corresponding author. Present address: GSF Research Center,
Institute for Developmental Genetics, IngolstaedterLandstrasse,1,
85764 Neuherberg/Munich, Germany. Phone: 49 89 3187 3674.Fax:49
89 3187 3099. E-mail: ralf.kuehn@gsf.de.
3982
cal, physiological, and neurological parametersrevealedelevated
body weights and hematocrits of ES mice as the only
differences and an otherwise normal phenotype. We further
demonstrate that ES mouse mutants can be produced from
mutant hybrid ES cells and analyzed within a period of only 4
months. Our results indicate that ES mouse technology provides
a useful research tool which expedites the generation and
analysis of designed mouse mutants for functional genome
analysis.
MATERIALS AND METHODS
Cell culture. ES cells were cultured in Dulbecco’s modifiedEagle’smedium
with 15% fetal calf serum containing 2,000 U of leukemiainhibitoryfactors (LIF)
(Chemicon International, Hofheim, Germany) per ml onmitomycinC-treated
embryonic fibroblasts as previously described (27). Fortheestablishment of
wild-type ES lines and adenomatous polyposis colimultipleintestinal neoplasia
(APCMin) ES cell lines, blastocysts were collected 3.5dayspostcoitum from
C57BL/6B6JRj females (Janvier, Le Genest St Isle, France) matedto129S6/
SvEvTac@Bom males (M&B,Ry,Denmark) or from C57BL/6J-APCMin females
(Jackson Laboratories, Bar Harbor, Maine) matedto129S2/SvPasIco-
CrlBR males (Charles River Laboratories, Sulzfeld,Germany),respectively.
Blastocysts were cultured in ES cell medium on a feeder layer;atday 5 the
outgrowth was dissociated by pipetting in trypsin solution, andthecell suspension
was replated on a fresh feeder layer. These plates were screened3days later
for the presence of ES cell colonies. About half of thedissociatedblastocysts
developed into ES cell lines, which were further expanded.
All ES cell lines were controlled for a correct karyotypebychromosome
counts on Giemsa-stained metaphases, and their sex was determinedbyhybridization
of Southern-blotted genomic DNA with a Ychromosome-specificprobe
(1). ES cell lines derived from mice heterozygous for theAPCMinmutation were
further characterized for the presence or absence of theAPCMinallele with a
PCR assay of genomic DNA with primers 5-GCCATCCCTTCACGTTAG-3
(0.02 M), 5-TTCCACTTTGGCATAAGGC-3 (1.0 M), and 5-TTCTGAG
AAAGACAGAAGTTA-3 (3.5 M) and cycling conditions of 94°C for5min,
56°C for 2 min, and 72°C for 3 min for 28 cycles, followed byafinal extension step
at 94°C for 5 min.
Production of ES and control mice. ES mice were producedbytetraploid
embryo complementation as previously described (4, 15).Briefly,embryo culture
was carried out in microdrops on standard bacterial petridishes(Falcon) under
mineral oil (Sigma). Modified CZB medium was used for embryocultureunless
otherwise noted. HEPES-buffered CZB was used for roomtemperatureoperations.
After administration of hormones, superovulated B6D2F1femaleswere
mated with B6D2F1 males (Janvier). Fertilized oocytes wereisolatedfrom the
oviduct, and any remaining cumulus cells were removedwithhyaluronidase.
After overnight culture, two-cell embryos were electrofused withtheCF-150B
cell fusion instrument (BLS Ltd., Budapest, Hungary) toproducetetraploid
embryos. Embryos that had not undergone fusion within 1 hwerediscarded.
Embryos were then cultured in vitro to the blastocyst stage.Formicroinjection,
10 to 20 blastocysts were placed in a drop of Dulbecco’smodifiedEagle’s
medium with 15% fetal calf serum under mineral oil. Aflat-tippedpiezo-actuated
microinjection pipette with an internal diameter of 12 to 15 mwasused to
inject 20 ES cells into each tetraploid blastocyst. Priortoblastocyst injection, ES
cells were trypsinized, resuspended in ES cell medium, andplatedfor 30 min to
remove feeder cells and debris. After recovery, 10injectedblastocysts were
transferred to each uterine horn of pseudopregnant NMRI females2.5days post
coitum. Recipient mothers were sacrificed at day ofembryonicdevelopment 19.5
(E 19.5), and pups were quickly removed and cross-fosteredtolactating NMRI
females.
B6129S6F1 ES mice were generated with the wild-type ES celllineART4/12
derived from a male B6129S6F1 blastocyst. Control males oftheB6129S6F1
genotype were raised from matings of 129S6/SvEvTac@Bom males(M&B)with
C57BL/B6JRj females (Janvier). ES and control mice were born inthesame
week and raised under the same housing conditions. A second groupofnormal
mice (in vitro controls) were raised from B6129S6F1 zygotesthatwere treated
like B6D2F1 tetraploid blastocysts except that electrofusion andEScell injections
were omitted. B6129S6F1 zygotes were cultured to theblastocyststage in
modified CZB medium and transferred into pseudopregnantNMRIfemales.
Pups of the in vitro control group were recovered like ES micebycaesarean
section at E 19.5 and cross-fostered to lactating NMRI females.
B6129S2F1-APCMin ES mice were generated with the ES celllineART/
APCMin-8, established from a male B6129S2F1-APCMinblastocyst.Control
males of the B6129S2F1-APCMin genotype were obtained from matingsof129S2/
SvPasIcoCrlBR males (Charles River Laboratories)withC57BL/6J-APCMin females
(Jackson Laboratories). All mice were typed for the presenceofthe
APCMin allele with a specific PCR assay with tail DNA asdescribedabove. ES
and control mice were born in the same week and raised underthesame housing
conditions. Both groups were maintained on the high-fat diet US17asdescribed
previously (19).
The analysis of glycosylphosphatidylinositol (GPI) isoformswasperformed
exactly as described previously (15). Briefly, tissues from 8-to12-week-old
B6D2F1 control or B6129S6F1 ES mice were homogenized insamplebuffer and
centrifuged. Aliquots of the supernatants were applied toTitan-IIIcelluloseacetate
plates with the Super Z applicator kit and run for 90 min at 300Vin a
zip zone chamber with Supreheme buffer (all reagents fromHelenaLaboratories
Inc., Beaumont, Tex.). Next, the plates were overlaid withanagarose-staining
solution mixture, incubated for 10 min in the dark, and fixedinacetic acidglycerol
before being photographed.
Phenotype analysis. An experienced veterinarianpathologistperformed the
external examination and necropsy of B6129S6F1 ES and controlmice.For the
preparation of histological sections, mice were perfused intheheart with Bouin’s
solution, and the organs were embedded in paraffin, sectioned,andstained with
hematoxylin-eosin. Brains were stained in addition with acombinedNissl/Luxol
fast blue stain. Images were recorded with a Leica DMEmicroscopeconnected
to a Hitachi HVC20 M camera with the Diskus imaging program(C.Hilgers,
Ko¨nigswinter, Germany). Tumors in the complete small intestineofAPCMin ES
and control mice were counted and also subjected tohistologicalanalysis.
For measurement of hematological parameters, blood-EDTAsampleswere
collected from the retrobulbar venous plexus from each animalfordetermination
of complete blood counts, including differentiation of whitecells.Hematology
parameters were measured from EDTA-blood with anautomaticelectronic cell
counter (CD3500; Abbott Diagnostics, Baar, Switzerland).Forclinical biochemistry
tests, serum was prepared immediately after blood coagulationandanalyzed
in a Cobas Integra 700 instrument (Roche Diagnostics,Rotkreuz,Switzerland)
with Roche reagent kits under the measurement conditionsspecifiedby the
International Federation of Clinical Chemistry at 37°C.Precedingthe hematological
and biochemical measurements, the CD3500 and the Cobas Integra
instruments were tested for accuracy and precision withqualitycontrol EDTAblood
and serum samples, respectively. Data analysis was performedwitha
Mann-Whitney U test, and the level of significance was set atP0.05. All
analyses were performed by Frimorfo Ltd. (Fribourg,Switzerland).Body weights
were measured at the age of 9 to 30 weeks with a standardlaboratoryelectronic
balance; data analysis was performed with a Student’s t test,andthe level of
significance was set at P 0.05.
For the behavioral assessment of 10-week-old ES (n 5) and control(n5)
mice, mice were housed individually per cage and maintained inanincubator
with controlled temperature (21 to 22°C) and a reversedlight-darkcycle (12 h/12
h) with food and water available ad libitum. All experimentswerecarried out by
Neurofit S.A. (Illkirch, France) in accordance withinstitutionalguidelines. The
test battery was based on a modified Irwin screen (10).Allparameters were
scored to provide a quantitative assessment. Aggressivenessandconvulsions
when the animals were handled were recorded. To assessnormalbehavior, each
animal was placed in a glass viewing jar 17 cm in height and 21cmin diameter
for 5 min. On the back of the jar, a sheet of white absorbentpaperwas placed.
The jar was placed in a room with red lights. Without disturbingtheanimal, the
spontaneous activity, respiration rate, and tremors wererecorded,and the
amount of urination or defecation was measured at the end oftheobservation
period.
Afterwards, each animal was transferred from the viewing jar toanopen field
without being handled. The observation was performed in aPlexiglas(52 by 52
by 40 cm) open field divided into nine equal squares, placed inadark room with
red light. During the transfer into the new environment,transferarousal was
noted, and palpebral closure was recorded immediately afterthetransfer. Within
the open field, the locomotor activity, tail elevation,touchescape, and startle
response (90-dB noise) were recorded. Finally, the animalwasremoved from the
open field to record visual placing, grip strength, body tone,andcorneal and
righting reflexes as described previously (10). The skin colorwasrecorded from
the plantar surface and digits of forelimbs. Data analysiswasperformed with the
Mann-Whitney U test. The level of significance was set atP0.05.
Serum insulin and leptin levels were determined byenzyme-linkedimmunosorbent
assay (ELISA) with serum from fed and fasted mice,respectively.
Blood was collected from the tail vein, and plasma was separatedbycentrifugation
at 4°C. The ELISAs were performed according to themanufacturer’sprotocol
(Crystal Chem. Inc.). Glucose and insulin tolerance testswereperformed
VOL. 23, 2003 CHARACTERISTICS OF ES CELL TETRAPLOID MICE 3983
on animals that had been fasted overnight. Blood glucose valuesweredetermined
from tail venous blood with an automatic glucosereader(Glucomen
sensor; A. Menarini Diagnostics). For the glucose tolerancetest,animals were
injected with 2 mg of D-glucose per g of body weight intotheperitoneal cavity.
Blood glucose levels were measured before and 15, 30, 60, and120min after the
administration of glucose. For the insulin tolerance test,animalswere injected
with 1 IU of human insulin (Novo Nordisk Pharma) per kg ofbodyweight. Blood
glucose levels were measured before and 15, 30, and 60 minafterintraperitoneal
administration of insulin. For measurement of white adiposetissuemass, the
peritoneal cavity was opened and epididymal fat pads werecompletelyremoved
and weighed. Data analysis was performed with a Student’s ttest,and the level
of significance was set at P 0.05.
RESULTS
Generation of wild-type ES mice. To generate ES mice from
wild-type ES cells, we established hybrid ES cell lines from
blastocysts of the (C57BL/6 129S6)F1 genotype (B6129S6F1).
Upon injection into tetraploid B6D2F2 blastocysts, ES cell
lines of this genotype generated ES pups at an efficiency of 10
to 15%, comparable to the results obtained with other hybrid
ES lines (4). The ES cell origin of these mice was confirmed by
the analysis of glucose phosphate isomerase (GPI) isoenzymes
(15) in tissue lysates. Figure 1 shows a comparison of samples
from a B6D2F1 control (GPI-a/b) with two mice derived from
tetraploid B6D2F2 blastocysts (GPI a/a, a/b, or b/b) injected
with cells of the B6129S6F1 ES line ART4/12 (GPI-b/c). The
presence of the GPI-c isoform as either the c/c homodimer or
c/b heterodimer in all samples of ES mice confirmed their
origin from ART4/12 ES cells. GPI c/c dimers are unstable and
show a less intense signal than GPI b/b dimers (18, 29). The
GPI a isoform was not detected in lysates from ES mice (Fig.
1), excluding a contribution from GPI a/a or GPI a/b host
blastocysts; only a small fraction (one-eighth) of tetraploid
blastocysts are expected to exhibit only the GPI b isoform. The
same results were obtained from the analysis of six additional
ES mice (R. Ku¨hn, unpublished data).
For the phenotypic characterization of ES mice, we selected
a group of 10 males derived from the B6129S6F1 ES cell line
ART4/12 through tetraploid blastocyst complementation and
an age-matched control group of the same genotype raised by
normal breeding. To control for potential effects of the embryo
culture and transfer procedure, we raised a third group of mice
from B6129S6F1 zygotes that were cultivated like tetraploid
blastocysts and transferred into pseudopregnant females (in
vitro controls).
Morphological and metabolic analysis of ES and control
mice. The morphology analysis program for B6129S6F1 ES
mice and controls included skeleton radiography,externalexamination,
body weight measurement, macroscopic examination
of body cavities, organs, and tissues (necropsy),andpathological
diagnosis based on histological sections of various
organs. Inspection of all external and internal organs and the
skeleton of five 10-week-old ES mice and five controls revealed
no visible abnormalities in either group. Thehistologicalexamination
of sections prepared from liver, lung, and intestine
(Fig. 2) as well as heart, kidney, and brain also showed no
difference between ES and control mice. These results indicate
that the embryonic and postnatal development of organs and
tissues in ES mice proceeds normally.
As expected from previous studies (4), ***** ES mice and
controls from in vitro-cultured embryos exhibited elevated
body weight relative to controls derived from natural matings
(Fig. 3). The relative weights of ES mice versus normalcontrols
did not increase but were stable over the time measured
(9 to 30 weeks), with a mean elevation of 21%. The same result
was obtained for ES mice derived from an independent
B6129S6F1 ES cell line (R. Ku¨hn, unpublished data). We did
not measure the birth weight of ES mice and controls, but an
earlier study reported 20% elevated birth weight for ES
neonates (4).
In order to characterize whether the elevated weight of ES
mice resulted from the development of obesity, we measured
food intake, white adipose tissue mass, plasma insulin, and
leptin levels of five 11-month-old B6129S6F1 ES mice and five
normal controls. We found no significant differences between
these groups for any of these parameters (Fig. 4A to D). To
further characterize glucose metabolism in ES mice, weperformed
insulin and glucose tolerance tests with five 11-monthold
B6129S6F1 ES mice and five normal controls of the same
age and genotype. These studies revealed that ES mice showed
a normal blood glucose response upon challenge with insulin
or glucose (Fig. 4E and F). We conclude that ES mice, unlike
mice cloned by nuclear transfer (25), have a normal glucose
and lipid metabolism and do not become obese.
Hematological analysis of ES and control mice. To further
assess the health status of ES mice, we performedahematological
analysis and determined levels of metabolites, enzymes,
and electrolytes in the serum of five B6129S6F1 ES and five
control mice at the age of 10 weeks. The concentrations of four
metabolites, three enzymes, and seven electrolytes under study
showed no significant differences between ES and control mice
(Table 1), indicative of a normal metabolism and normal liver
and kidney functions in ES mice. The numbers ofbloodlymphocytes,
monocytes, basophils, eosinophils, and neutrophils
showed no significant differences between the groups (Table
1), suggesting a normal immune cell lineage differentiation in
ES mice. The only differences found in the ES mouse group
were mildly enhanced hematocrit values and erythrocyte numbers.
Behavioral analysis of ES and control mice. To compare the
behavioral and neurological functions of B6129S6F1 ES mice
FIG. 1. Analysis of GPI isoenzymes in tissue lysates of ES and
control mice. Lysates of the indicated tissues of a B6D2F1(GPIa/b)
control mouse and two ES mice (ES#T35, ES#T34) derived from the
ART4/12 ES cell line (GPI b/c) were separated byelectrophoresison
a cellulose-acetate gel. The gel was stained for GPI enzymeactivityand
fixed. The run positions of the GPI homo- and heterodimers are
indicated by arrows. The anode () and cathode () positions are
indicated.
3984 SCHWENK ET AL. MOL. CELL. BIOL.
and normal mated controls, five mice each were assessed
through the behavioral observation profile described by Irwin
(10). As shown in Table 2, ES mice performed like normal
mated controls for all 17 parameters of the test batterywithout
statistically significant differences between the groups.ESmice
exhibited normal responses to environmental stimuli, including
social, exploratory, and avoidance behavior, indicating that
their muscle and motor neuron, spinocerebellar, and sensory
functions were within the normal range. Furthermore,theautonomic
functions and reflexes of the ES mice were indistinguishable
from those of the controls; bizarre behavior and
convulsions were not observed in any of the groups. Upon
mating to wild-type C57BL/6 females, all ES males tested (n
11) proved to be fertile, with an average first litter sizeofsix
pups (range, three to nine), indicating normal mating behavior.
Tumor development in APCMin mutant ES mice. ES mouse
technology allows the assessment of mutant phenotypes within
a short time, as mice can be produced directly from genetically
modified ES cells through tetraploid embryo complementation.
To demonstrate the feasibility of this approach, we generated
ES mice and control mice harboring the Min allele
of the adenomatous polyposis coli (APC) tumor suppressor
gene, an established genetic model for colorectal cancer in
humans (6, 24). Mutant ES mice were produced with a male
ES cell line (ART/APCMin-8), established from a blastocyst
of the (C57BL/6-APCMin 129S2)F1 genotype (B6129S2F1-
APCMin). Tumor development in mice of this genetic background
has been described previously (7).
Upon injection of ART/APCMin-8 ES cells into tetraploid
blastocysts, ES mice were obtained at normal frequency (10%).
A control group of B6129S2F1-APCMin males was raised from
contemporaneous normal matings. At 3 months of age, the
small intestines of three B6129S2F1-APCMin ES mice and two
normal mated controls were analyzed for the presence of tumors.
All mice under study exhibited intestinal tumors typical
of the APCMin mutation (five to nine tumors in ES mice, 7 to
17 tumors in controls). While the small groups do not allow
quantitative comparison of tumorigenesis, the phenotype of
the APCMin ES mice demonstrates that ES mouse mutants are
suitable for assaying tumor suppressor gene function. With the
availability of genetically modified ES cells, we produced and
analyzed ***** mutants within a period of 4 months. Inaddition,
ART/APCMin-8 ES cells provide a useful tool allowing us
to inactivate other putative tumor modifiers in the APCMin
FIG. 2. Histological analysis of ES and control mice. Tissuesof10-week-old B6129S6F1 ES mice and normal mated controls werefixed,paraffin
embedded, sectioned, and stained with either cresyl violet(brain)or hematoxylin and eosin (liver, lung, and colon).Magnification,40.
FIG. 3. Body weights of B6129S6F1 ES and control mice. The body
weights of ES males (solid squares), control mice fromnormalmatings
(open squares), and control mice from in vitro-culturedembryos(open
triangles) were measured at the indicated ages. Resultsareexpressed
as mean values   standard deviations.
VOL. 23, 2003 CHARACTERISTICS OF ES CELL TETRAPLOID MICE 3985
background to assess the phenotype of compound mutants in a
time-saving manner.
DISCUSSION
The recent finding that viable ES mice can be efficiently
produced with hybrid ES cells (4), even after multiple rounds
of gene targeting (5, 23), led us to study the utility of these
mice for biological studies. To assess the phenotype of hybrid
ES mice, we studied a variety of morphological, physiological,
and neurological parameters able to indicate abnormal embryonic
or postnatal development as well as disease states of the
*****. We found that ***** B6129S6F1 ES mice, despite their
full in vitro origin, are apparently normal and healthy. The
elevated body weight of ES mice did not result from obesity or
diabetes. A similar weight increase was found for normalcontrol
mice derived from in vitro-cultured embryos. Thus, weight
increase is not unique to ES mice but likely results from the
common experimental procedure used to derive the ES and
control mice. In particular, the specific pre- and postnatal
nursing conditions of ES mice and in vitro controls may be of
critical importance, since both were raised by outbred (NMRI)
females upon embryo transfer. In contrast, the F1 controls
were derived from normal matings by inbred (C57BL/6) mothers.
It is well known that the offspring’s body weight can be
increased through uterine heterosis, depending on the mother’s
genotype (3, 20, 22).
Our results are consistent with an earlier report showing that
ES pups and neonates derived from in vitro-cultured blastocysts
exhibit 20% elevated birth weight (4). In addition, a
great variability in the expression of imprinted genes that are
frequently involved in fetal and placental growth (8, 31) was
documented in ES cell lines and neonatal ES mice (9). The in
vitro culture of murine preimplantation embryos has also been
shown to cause the altered expression ofgrowth-relatedimprinted
genes (12). Thus, the deregulated expression of such
genes could also contribute, besides uterine heterosis, to the
increased body weight of ES mice and pups derived from in
vitro-cultured embryos. Presently we cannot determine which
of these explanations is the main cause for the observed weight
increase.
The elevated weight of ES mice and control mice may be
distinguished from the neonatal overgrowth found in cloned
FIG. 4. Metabolic parameters of B6129S6F1 ES and control mice.Allassays were performed with groups of five 11-month-old *****miceas
indicated. (A) Food intake. Daily food intake of ES mice (leftbar)and F1 controls (right bar). (B) Insulin levels. Plasmainsulinconcentrations
were determined by ELISA with tail venous blood. (C) Leptinlevels.Plasma leptin concentrations were determined by ELISA withtailvenous
blood of fasted mice. (D) Body fat. White adipose tissue masswasexpressed as a percentage of total body weight. (E)Glucosetolerance test. Blood
glucose levels were measured before and afterintraperitonealadministration of glucose (2 mg/g of body weight).Results areexpressed as mean
glucose levels of ES mice (solid circles) and controls(opensquares)   standard error of the mean. (F) Insulintolerancetest. Animals were injected
with 1.0 IU of human insulin/kg of body weight and analyzedforblood glucose levels at the indicated time points. Resultsareexpressed as a
percentage of the initial glucose level   standard error ofthemean of ES mice (solid circles) and controls (open squares).Glucoseand insulin
tolerance tests were performed on animals that had beenfastedovernight.
3986 SCHWENK ET AL. MOL. CELL. BIOL.
mice as a direct consequence of the cloning procedure. Newborn
mice cloned from ES cell nuclei were reported to exhibit
a 60% weight increase, and placental weights were more than
doubled (4). Cloned mice also exhibit abnormal imprinted
gene expression (9), and severe health impairments such as
reduced life span, frequent pneumonia, and obesityweredescribed
(17, 25, 30). It was recently shown that genome reprogramming
in mice cloned from both nuclei of cultured ES cells
and freshly isolated cumulus cells is associated with a broadly
disturbed expression profile in the placenta, representing at
least 4% of all expressed genes (8). The majority of these
genes were common to both types of clones. Gene expression
changes in the livers of cloned pups were less pronounced than
in the placentas and affected a largely distinct set ofgenes(8).
Even surviving clones may not be normal at birth or laterinlife
as a result of severe placental dysfunction during gestation.In
contrast to clones, the extra-embryonic tissues of ES mice,such
as the placenta, are largely derived from the tetraploid host
blastocysts rather then the donor cells (16). It was further
shown that neither the placenta nor the birth weight of ES
pups exhibited overgrowth and did not differ from those of
neonates derived from in vitro-cultured control embryos (4).
Vice versa, neonatal overgrowth and other abnormalities may
be reduced if cloned mice were derived by tetraploid embryo
complementation with ES cell lines established from cloned
blastocysts.
Apart from the increased body weight, our results show that
all other studied biological characteristics of B6129S6F1 ES
mice fell into the wild-type range as defined byisogeniccontrols
derived from normal matings. The histological, physiological,
and neurological parameters reported in this work provide
a phenotypic baseline for ***** hybrid ES mice and and
confirm their general suitability for the analysis of mutant
phenotypes. We believe that these results strongly encourage
the future use of this technology for the rapid production of
targeted mouse mutants. It is beyond the scope of a single
study to investigate all biological features of ES mice and
controls. Our results, however, will also stimulatefurthercharacterization
of ES mice in more specialized disciplines.
ES mouse technology offers the benefit of producing *****
mutants directly from genetically modified ES cells without the
requirement for breeding. Mutants are thus availableforanalysis
in less than half the time required by the current methodology
involving the generation of germ line chimeras and multiple
breeding cycles. In an earlier report, we confirmed the
technical feasibility of this approach through the productionof
ES mice from homozygous mutant ES cell clones generated by
gene targeting (23). In a proof-of-principle experiment, withan
ES cell clone harboring a biologically relevant mutant tumor
suppressor gene, we now demonstrate that the phenotypic
analysis of ***** ES mouse mutants, including their production
from ES cells, can be completed within 4 months. In addition,
this ES cell line provides a tool to expedite the mutagenesisof
other tumor modifiers to assess the phenotype of compound
mutants.
Besides the production of classical knockout mice, ES
mouse technology will also allow the direct,time-savingproduction
of conditional mutants with hybrid ES cell lines established
from Cre recombinase transgenic mouse strains. Conditional
alleles may be introduced into such ES cells by two
TABLE 1. Hematology and clinical biochemistry of
ES and control mice
Parameter
Value for:
Controls ES mice Pa
Mean SD Mean SD
Metabolites
Glucose (mmol/liter) 7.84 2.13 9.86 1.78 0.17
Albumin (g/liter) 25.8 1.30 26.2 1.64 0.91
Cholesterin (mmol/liter) 2.4 0.12 2.6 0.19 0.08
Triglycerides (mmol/liter) 1.46 0.39 1.5 0.65 0.75
Enzymes
Alkaline phosphatase (U/liter) 130.2 10.4 122.4 13.2 0.47
Aspartate amino transferase (U/liter) 68.8 2.9 62.8 15.9 0.92
Alanine amino transferase (U/liter) 39.2 16.6 42.8 10.4 0.60
Electrolytes
Ca2 (mmol/liter) 2.4 0.05 2.45 0.07 0.25
Mg (mmol/liter) 1.41 0.09 1.37 0.09 0.21
Fe (mol/liter) 31.6 2.5 33.9 2.1 0.25
Phosphorus (mmol/liter) 2.65 0.19 2.71 0.17 0.56
Na (mmol/liter) 167.6 3.9 16.7 1.6 0.83
K (mmol/liter) 4.78 0.40 4.96 0.59 0.60
Cl (mmol/liter) 122.8 2.8 122.8 2.0 0.83
Erythrocytes
Hematocrit (%) 47.2 1.4 49.6 1.1 0.03
Hemoglobin (g/dl) 15.6 0.5 16.2 0.3 0.02
Erythrocytes (106/l) 9.8 0.39 10.6 0.3 0.01
Mean corpuscular hemoglobin (pg) 1.6 0 15.2 0.5 0.04
Mean corpuscular hemoglobin
content (g/dl)
33.2 0.5 32.8 0.5 0.35
Mean corpuscular volume (fl) 4.8 0.7 46.8 0.8 0.60
Leukocytes
Leukocytes (103/l) 4.1 0.8 5 0.8 0.08
Neutrophils/l 1,410 2,060 70.2 15.8 0.25
Eosinophils/l 9.4 5.2 8.1 3.1 0.34
Basophils/l 24 23 2.8 26
0.99
Monocytes/l 5.6 5.1 10.8 7.9 0.34
Lymphocytes/l 3,424 81.5 4,073 69.0 0.60
a P values were calculated by using the Mann-Whitney U test;thelevel of
significance was set at a P value of 0.05.
TABLE 2. Behavioral analysis of ES and control mice
Behavioral or
physiologic parameter
Response of ES mice
and controls Pa
Aggressivity Normal
0.99
Spontaneous activity Normal
0.99
Respiration rate Normal
0.99
Defecation Normal 0.89
Urination Normal 0.74
Palpebral closure Normal
0.99
Locomotor activity Normal 0.26
Tail elevation Normal 0.12
Touch escape Normal 0.67
Startle response Normal 0.60
Struggle response Normal
0.99
Visual placing Normal
0.99
Grip strength Normal
0.99
Body tone Normal
0.99
Corneal reflex Normal
0.99
Righting reflex Normal
0.99
Skin color Normal
0.99
a P values were calculated by using the Mann-Whitney U test;thelevel of
significance was set at a P value of 0.05.
VOL. 23, 2003 CHARACTERISTICS OF ES CELL TETRAPLOID MICE 3987
sequential gene targeting cycles followed by the removal of
selection markers flanked by FLP recombinase recognition
sites prior to blastocyst injection. As a prerequisite for this
approach, we recently established B6129S6F1 ES cell lines
harboring cell type-specific or inducible Cre transgenes,ableto
derive ES mice at the same frequency as wild-type ES lines (R.
Ku¨hn, F. Schwenk, and B. Zevnik, unpublished data).
Since ES mice are efficiently produced from hybrid but not
inbred ES cell lines, mutants derived from targeted F1 ES cells
necessarily exhibit a hybrid genetic background. Provided that
B6129F1 ES lines are employed (B6129S6F1 [Art4/12] cells in
this study), ES mouse mutants can be studied in a genetic
background that has been frequently used for phenotypeanalysis.
So far, most targeted mutations have been introduced into
129-derived inbred ES cells, and mutant strains have been
established through the cross of chimeras to C57BL/6 females
and subsequent intercrosses resulting in homozygous mouse
mutants in a mixed 129 C57BL/6 background (14). In contrast,
ES mouse technology can deliver homozygous mutants at
a defined F1 (129 C57BL/6) genetic background, since further
intercrossing is not required. However, ES mouse technology
is not suitable for the generation of mutants which
require phenotype analysis on an inbred background.
The production of homozygous mutant ES mice requires
two sequential transfection rounds to target both copies of an
autosomal gene in hybrid cells, e.g., B6129F1 ES cells. Thus,F1
ES cells must maintain their pluripotency during prolonged in
vitro culture, and gene targeting vectors should recombine
efficiently with the C57BL/6- and 129-derived allele of the
target gene. In earlier reports we have shown that B6129F1 ES
cells (including line Art4/12) tolerate up to three consecutive
gene targeting cycles without losing the ability to complement
tetraploid blastocysts (5, 23). We have also reported that both
alleles of an autosomal gene (Rosa 26) can betargetedefficiently
in B6129F1 ES cells by using gene targeting vectors with
identical, 129-derived homology arms (23). In our experience,
about 75% of the targeting vectors that we havetestedrecombined
at comparable efficiency with C57BL/6- and 129-derived
alleles with a single set of homology regions derived from one
of these strains (R. Ku¨hn and F. Schwenk, unpublishedresults).
Specific genes, such as the retinoblastoma gene, which exhibit
high sequence diversity in different mouse strains can be
targeted in inbred ES cells only with isogenic homology arms
derived from the same inbred strain (26). The targeting of such
genes in B6129F1 ES cells would require the use oftwoindependent
gene targeting vectors, one derived from C57BL/6
genomic DNA and the other from the respective 129 substrain.
However, with the availability of the complete sequences of the
C57BL/6 genome and several 129 strains (11, 28), it is now
possible to predict beforehand from the sequence diversity of
a given gene whether a single set of homology arms issufficient
to target both alleles in B6129F1 ES cells.
Taken together, our results indicate that ES mouse technology
provides a useful research tool to expedite the generation
and analysis of mouse mutants in a hybrid background. Since
this approach is simple and technically no more demanding
than the current gene targeting protocols, we expect ittobecome
a widely used tool in reverse mouse genetics.
ACKNOWLEDGMENTS
We thank I. Falkner, A. Hortz, D. Schulz, and D. Thielforexcellent
technical assistance, J. Lo¨hler for comments, and G. StottandL.
Jackson-Grusby for critically reading the manuscript.
This work was supported by Artemis Pharmaceuticals GmbH and
the German Ministry for Education and Science (BMBF, grants
0311956 and ZMMKTV2).
F. Schwenk and B. Zevnik contributed equally to this work.
REFERENCES
1. Bishop, C. E., and D. Hatat. 1987. Molecular cloning andsequenceanalysis
of a mouse Y chromosome RNA transcript expressed in thetestis.Nucleic
Acids Res. 15:2959–2969.
2. Capecchi, M. R. 1989. The new mouse genetics: altering thegenomeby gene
targeting. Trends Genet. 5:70–76.
3. Cowley, D. E., D. Pomp, W. R. Atchley, E. J. Eisen, andD.Hawkins-Brown.
1989. The impact of maternal uterine genotype on postnatalgrowthand
***** body size in mice. Genetics 122:193–203.
4. Eggan, K., H. Akutsu, J. Loring, L. Jackson-Grusby, M. Klemm,W.M.
Rideout III, R. Yanagimachi, and R. Jaenisch. 2001. Hybridvigor,fetal
overgrowth, and viability of mice derived by nuclear cloningandtetraploid
embryo complementation. Proc. Natl. Acad. Sci. USA98:6209–6214.
5. Eggan, K., A. Rode, I. Jentsch, C. Samuel, T. Hennek, H.Tintrup,B. Zevnik,
J. Erwin, J. Loring, L. Jackson-Grusby, M. R. Speicher, R.Kuehn,and R.
Jaenisch. 2002. Male and female mice derived from thesameembryonic
stem cell clone by tetraploid embryo complementation.Nat.Biotechnol.
20:455–459.
6. Fodde, R., and R. Smits. 2001. Disease model:familialadenomatous polyposis.
Trends Mol. Med. 7:369–373.
7. Gould, K. A., C. Luongo, A. R. Moser, M. K. McNeley,N.Borenstein, A.
Shedlovsky, W. F. Dove, K. Hong, W. F. Dietrich, and E. S.Lander.1996.
Genetic evaluation of candidate genes for the Mom1 modifierofintestinal
neoplasia in mice. Genetics 144:1777–1785.
8. Humpherys, D., K. Eggan, H. Akutsu, A. Friedman, K.Hochedlinger,R.
Yanagimachi, E. S. Lander, T. R. Golub, and R. Jaenisch.2002.Abnormal
gene expression in cloned mice derived from embryonic stem cellandcumulus
cell nuclei. Proc. Natl. Acad. Sci. USA 99:12889–12894.
9. Humpherys, D., K. Eggan, H. Akutsu, K. Hochedlinger, W.M.Rideout III,
D. Biniszkiewicz, R. Yanagimachi, and R. Jaenisch. 2001.Epigeneticinstability
in ES cells and cloned mice. Science 293:95–97.
10. Irwin, S. 1968. Comprehensive observational assessment. Ia.Asystematic,
quantitative procedure for assessing the behavioral andphysiologicstate of
the mouse. Psychopharmacologia 13:222–257.
11. Kerlavage, A., V. Bonazzi, M. di Tommaso, C. Lawrence, P. Li,F.Mayberry,
R. Mural, M. Nodell, M. Yandell, J. Zhang, and P. Thomas.2002.The
Celera discovery system. Nucleic Acids Res. 30:129–136.
12. Khosla, S., W. Dean, D. Brown, W. Reik, and R. Feil.2001.Culture of
preimplantation mouse embryos affects fetal development andtheexpression
of imprinted genes. Biol. Reprod. 64:918–926.
13. Kwan, K. M. 2002. Conditional alleles in mice:Practicalconsiderations for
tissue-specific knockouts. Genesis 32:49–62.
14. Mak, T. W. 1998. The gene knockout facts book. AcademicPress,London,
UK.
15. Nagy, A. 2000. Production and analysis of ES cellaggregationchimaeras, p.
177–206. In A. L. Joyner (ed.), Gene targeting: apracticalapproach, 2nd ed.
Oxford University Press, Oxford, United Kingdom.
16. Nagy, A., E. Gocza, E. M. Diaz, V. R. Prideaux, E. Ivanyi,M.Markkula, and
J. Rossant. 1990. Embryonic stem cells alone are able tosupportfetal
development in the mouse. Development 110:815–821.
17. Ogonuki, N., K. Inoue, Y. Yamamoto, Y. Noguchi, K. Tanemura,O.Suzuki,
H. Nakayama, K. Doi, Y. Ohtomo, M. Satoh, A. Nishida, and A.Ogura.2002.
Early death of mice cloned from somatic cells. Nat.Genet.30:253–254.
18. Padua, R. A., G. Bulfield, and J. Peters. 1978.Biochemicalgenetics of a new
glucosephosphate isomerase allele (Gpi-1c) from wildmice.Biochem.
Genet. 16:127–143.
19. Petrik, M. B., M. F. McEntee, B. T. Johnson, M. G.Obukowicz,and J.
Whelan. 2000. Highly unsaturated (n-3) fatty acids, butnotalpha-linolenic,
conjugated linoleic or gamma-linolenic acids, reducetumorigenesisin Apc-
(Min/) mice. J. Nutr. 130:2434–2443.
20. Pomp, D., D. E. Cowley, E. J. Eisen, W. R. Atchley, andD.Hawkins-Brown.
1989. Donor and recipient genotype and heterosis effects onsurvivaland
prenatal growth of transferred mouse embryos. J. Reprod.Fertil.86:493–
500.
21. Rajewsky, K., H. Gu, R. Kuhn, U. A. Betz, W. Muller, J.Roes,and F.
Schwenk. 1996. Conditional gene targeting. J. Clin.Investig.98:600–603.
22. Rhees, B. K., C. A. Ernst, C. H. Miao, and W. R. Atchley.1999.Uterine and
postnatal maternal effects in mice selected for differential rateofearly
development. Genetics 153:905–917.
23. Seibler, S., B. Zevnik, B. Ku¨ter-Luks, S. Andreas, H. Kern,T.Hennek, A.
Rode, C. Heimann, N. Faust, R. Jaenisch, K. Rajewsky, R. Ku¨hn,andF.
3988 SCHWENK ET AL. MOL. CELL. BIOL.
Schwenk. 2003. Rapid generation of inducible mouse mutants.NucleicAcids
Res. in press.
24. Sieber, O. M., I. P. Tomlinson, and H. Lamlum. 2000.Theadenomatous
polyposis coli (APC) tumour suppressor—genetics, functionanddisease.
Mol. Med. Today 6:462–469.
25. Tamashiro, K. L., T. Wakayama, H. Akutsu, Y. Yamazaki, J.L.Lachey,
M. D. Wortman, R. J. Seeley, D. A. D’Alessio, S. C. Woods,R.Yanagimachi,
and R. R. Sakai. 2002. Cloned mice have an obese phenotypenottransmitted
to their offspring. Nat. Med. 8:262–267.
26. te Riele, H., E. R. Maandag, and A. Berns. 1992.Highlyefficient gene
targeting in embryonic stem cells through homologousrecombinationwith
isogenic DNA constructs. Proc. Natl. Acad. Sci.USA89:5128–5132.
27. Torres, R. M., and R. Ku¨hn. 1997. Laboratory protocolsforconditional gene
targeting. Oxford University Press, Oxford, UK.
28. Waterston, R. H., K. Lindblad-Toh, E. Birney, J. Rogers, J.F.Abril, P.
Agarwal, R. Agarwala, R. Ainscough, M. Alexandersson, P. An, S.E.Antonarakis,
J. Attwood, R. Baertsch, J. Bailey, K. Barlow, S. Beck,E.Berry,
B. Birren, T. Bloom, P. Bork, M. Botcherby, N. Bray, M. R. Brent,D.G.
Brown, S. D. Brown, C. Bult, J. Burton, J. Butler, R. D.Campbell,P.
Carninci, S. Cawley, F. Chiaromonte, A. T. Chinwalla, D. M.Church,M.
Clamp, C. Clee, F. S. Collins, L. L. Cook, R. R. Copley, A.Coulson,O.
Couronne, J. Cuff, V. Curwen, T. Cutts, M. Daly, R. David,J.Davies, K. D.
Delehaunty, J. Deri, E. T. Dermitzakis, C. Dewey, N. J.Dickens,M.
Diekhans, S. Dodge, I. Dubchak, D. M. Dunn, S. R. Eddy, L.Elnitski,R. D.
Emes, P. Eswara, E. Eyras, A. Felsenfeld, G. A. Fewell, P.Flicek,K. Foley,
W. N. Frankel, L. A. Fulton, R. S. Fulton, T. S. Furey, D. Gage,R.A. Gibbs,
G. Glusman, S. Gnerre, N. Goldman, L. Goodstadt, D. Grafham,T.A.
Graves, E. D. Green, S. Gregory, R. Guigo, M. Guyer, R. C.Hardison,D.
Haussler, Y. Hayashizaki, L. W. Hillier, A. Hinrichs, W. Hlavina,T.Holzer,
F. Hsu, A. Hua, T. Hubbard, A. Hunt, I. Jackson, D. B. Jaffe, L.S.Johnson,
M. Jones, T. A. Jones, A. Joy, M. Kamal, E. K. Karlsson, etal.2002. Initial
sequencing and comparative analysis of the mouse genome.Nature420:520–
562.
29. West, J. D., and J. H. Flockhart. 1989. Non-additiveinheritanceof glucose
phosphate isomerase activity in mice heterozygous at theGpi-1sstructural
locus. Genet. Res. 54:27–35.
30. Wilmut, I. 2002. Are there any normal cloned mammals? Nat.Med.8:215–
216.
31. Young, L. E., K. Fernandes, T. G. McEvoy, S. C. Butterwith,C.G. Gutierrez,
C. Carolan, P. J. Broadbent, J. J. Robinson, I. Wilmut, and K.D.Sinclair.
2001. Epigenetic change in IGF2R is associated with fetalovergrowthafter
sheep embryo culture. Nat. Genet. 27:153–154.
VOL. 23, 2003 CHARACTERISTICS OF ES CELL TETRAPLOID MICE 3989