Endocrinology Vol. 138, No. 10 4058-4063
Copyright © 1997 by The Endocrine Society
Targeted Disruption of Gnas in Embryonic Stem Cells1
William F. Schwindinger,
Kimberly J. Reese,
Ann M. Lawler,
John D. Gearhart and
Michael A. Levine
Division of Endocrinology and Metabolism (W.F.S., M.A.L.),
Department of Medicine and Division of Developmental Genetics (K.J.R.,
A.M.L., J.D.G.), Department of Gynecology and Obstetrics, The Johns
Hopkins University School of Medicine, Baltimore, Maryland 21205
Address all correspondence and requests for reprints to: William F. Schwindinger, Division of Endocrinology and Metabolism, Johns Hopkins University School of Medicine, Ross 863, 720 Rutland Avenue, Baltimore, Maryland 21205. E-mail: wschwind{at}welchlink.welch.jhu.edu
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Abstract
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Mutations in the gene encoding the stimulatory G protein of adenylyl
cyclase (G
s) are present in subjects with Albright
hereditary osteodystrophy, a syndrome of characteristic developmental
defects and, in some patients, resistance to multiple hormones that
stimulate cAMP accumulation (pseudohypoparathyroidism type Ia). As the
first step in generating a model of Albright hereditary osteodystrophy,
the gene encoding Gs (Gnas) was disrupted
in mouse embryonic stem (ES) cells by homologous recombination.
Northern blot analysis and immunoblot analysis demonstrated that
steady-state levels of Gs messenger RNA and
Gs protein in targeted ES cells were approximately 50%
of levels in untargeted ES cells. In response to 10 µM
forskolin and to various concentrations of isoproterenol (0.1–3.0
µM), cAMP accumulation was reduced in the
Gs knockout ES cell lines, relative to wild-type ES
cells and to five of six ES cell lines with randomly integrated
targeting vector. These results support the role of Gs
haploinsufficiency in reducing the ability of hormones to generate cAMP
in subjects with pseudohypoparathyroidism type Ia. The targeted
disruption of Gnas in mouse ES cells establishes an
in vitro system for further studies of the role of
Gs and cAMP coupled signal transduction in
differentiation and development.
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Introduction
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PATIENTS with Albright hereditary
osteodystrophy (AHO) have a variety of developmental defects, including
obesity, short stature, brachydactyly, and heterotopic ossifications.
In addition, many patients with AHO also manifest tissue resistance to
hormones that activate adenylyl cyclase (e.g. PTH), a
condition termed pseudohypoparathyroidism (PHP) type Ia. Cell membranes
from patients with AHO show deficient expression or function of
Gs, the -subunit of the heterotrimeric G protein that
stimulates adenylyl cyclase. Human Gs is encoded by the
GNAS1 gene located on chromosome 20q13.1–13.2 (1). It
contains at least 13 coding exons (2) and can be alternatively spliced
to yield at least 4 different protein isoforms with similar biological
activities (3). In addition to its role in stimulating adenylyl
cyclase, Gs may directly stimulate the opening of
calcium channels (4). Moreover, GNAS1 contains at least two
alternative first exons that encode proteins of unknown function (5, 6).
A variety of heterozygous defects in GNAS1 gene account for
generalized Gs deficiency in AHO, but leave unexplained
the considerable variability in the clinical and biochemical features
of affected subjects. Somatic features, such as brachydactyly or sc
ossifications, are subtle or absent in some patients. Moreover,
although deficiency of Gs leads to target organ
resistance to many hormones (e.g. PTH, TSH, glucagon, and
gonadotropins) that activate receptors coupled via Gs to
adenylyl cyclase, tissue responsiveness to other hormones
(e.g. vasopressin and ACTH) seems normal (7). Perhaps even
more confusing is the observation that patients with PHP type Ia may
have relatives with the same GNAS1 gene defect who lack any
features of hormone resistance, a condition termed pseudoPHP (8).
Attempts to address these biochemical conundrums by in
vitro studies have been hampered by the limited availability of
tissue samples from patients with Gs deficiency.
Surprisingly, cells and tissues from patients with PHP type Ia have not
consistently demonstrated decreased hormone responsiveness (see below).
As a first step to understanding the phenotypic variability of
Gs deficiency, we have used homologous recombination to
effect the targeted disruption of the gene encoding Gs
(Gnas) in mouse embryonic stem cells (ES cells). We report
here that loss of one Gnas allele leads to reduced
steady-state levels of Gs messenger RNA (mRNA) and
protein and results in decreased cAMP accumulation in response to
forskolin and isoproterenol.
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Materials and Methods
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Cell culture
Mouse embryonic fibroblasts were maintained in DMEM supplemented
with 10% calf serum and nonessential amino acids. ES cells were
cultured in low-bicarbonate DMEM supplemented with 15% ES
cell-qualified FBS, to which was added sodium pyruvate, nonessential
amino acids, glutamine, penicillin-streptomycin, mouse leukemia
inhibiting factor (106 U/liter), and G418 (250 µg/ml)
(Life Technologies, Inc., Gaithersburg, MD). Cells were maintained at
37 C, 5% CO2, and 95% relative humidity. ES cells were
initially cultured, and ES cell clones were selected on a layer of
irradiated embryonic fibroblasts. Characterization of ES cell clones
was performed using cells that had been cultured on gelatin-coated
dishes.
Southern blot analysis
ES cells that had been cultured on irradiated fibroblasts in
24-well dishes or on gelatin-coated 100-mm dishes were suspended in 50
mM Tris-HCl (pH 8.0), 400 mM NaCl, 100
mM EDTA, 0.5% SDS and Proteinase K (0.6 mg/ml). After
overnight incubation at 55 C, high-molecular-weight DNA was extracted
with phenol-chloroform and ethanol precipitated. Aliquots of DNA (10
µg) were digested with PvuII (Life Technologies), resolved
on 0.8% agarose gels, and immobilized onto Nytran Plus (Schleicher &
Schuell, Keene, NH) by capillary transfer in 0.4 M NaOH,
0.6 M NaCl. Two oligonucleotides were synthesized that
corresponded to Gnas sequences upstream of the 5' end of the
targeting vector: CGGCTGTCTTCCTCTCGCTCTCGGCCGCTCGGGCTTTGAG-CTTCA and
GCGAACGGTTCTGCCAGCACCGCAGCCGCCACGG-CAGCTTCGGCA. Blots were incubated
for 2 h at 42 C in 6x SSPE (1x SSPE is 180 mM NaCl,
10 mM NaPO4, pH 7.7, 1 mM EDTA),
10x Denhardt’s, 1% SDS, 50 µg/ml sonicated and denatured salmon
sperm DNA, and hybridized overnight at 42 C in 50% formamide, 6x
SSPE, 1% SDS, 50 µg/ml salmon sperm DNA, with 2–5 x
106 cpm/ml of each [32P]5'-end labeled
oligonucleotide. Blots were washed to a final stringency of 0.1x SSPE,
1% SDS at 62 C. Hybridizing DNA fragments were detected after a 3-day
exposure to a storage phosphor screen (Molecular Dynamics, Sunnyvale,
CA).
Northern blot analysis
Trizol (Life Technologies) was used, according to the
manufacturer’s instructions, to prepare RNA from ES cells cultured on
gelatin-coated 100-mm dishes. Total RNA (20 µg) was resolved by
electrophoresis through 1% agarose-formaldehyde-MOPS gels and
transferred to Nytran Plus by standard techniques (9). Membranes were
prehybridized for 2 h in hybridization buffer (50% formamide, 5x
SSPE, 0.1% SDS, 5x Denhardt’s reagent, 100 µg/ml sonicated and
denatured salmon sperm DNA), and then hybridized to the
NcoI-SalI fragment of the human Gs
complementary DNA (cDNA) (pHGs8) (10) or an 18S ribosomal RNA (rRNA)
probe. The ubiquitously distributed 18S rRNA probe was used to correct
for variation in RNA loading. The cDNA probes were radiolabeled by
random primer extension (Multiprime, Amersham Corp., Arlington Heights,
IL). Blots were washed to a final stringency of 1x SSPE, 0.5% SDS at
42 C and exposed to a storage phosphor screen for 1 day.
Immunoblot analysis
ES cells were cultured on gelatin-coated 100-mm dishes and
harvested by hypotonic lysis in 5 mM HEPES (pH 8.0), 0.5
mM EDTA. Swollen cells were collected by repeated
pipetting, pelleted by centrifugation (27,000 x g for
10 min at 4 C), resuspended in 8 ml of 10 mM Tris HCl (pH
8.0), 1 mM EDTA, and broken by 20 strokes of a
loose-fitting dounce homogenizer (Wheaton, Millville, NJ) on ice.
Unbroken cells, nuclei, and mitochondria were removed by centrifugation
(500 x g, 10 min, 4 C). Crude membranes were collected
from the supernatant by centrifugation (27,000 x g for
30 min at 4 C) and stored at -70 C in 25 mM HEPES (pH
8.0), 1 mM dithiothreitol. Membrane proteins were resolved
by electrophoresis through 10% SDS-polyacrylamide gels, transferred to
Immobilon-P (Millipore, Bedford, MA), and probed with polyclonal
antisera to Gs (NEI-805, RM/1), Gq/11
(NEI-809, QL), and Gi1/i2 (NEI-801, AS/7) (Dupont NEN,
Boston, MA). Immunoreactive protein was detected with
125I-Protein A (Amersham Corp.) and quantified with a
PhosphorImager and ImageQuaNT Software (Molecular Dynamics).
cAMP accumulation
ES cells were seeded in 24-well dishes at approximately 2
x 105 cells per well and cultured for 24 h before
treatment. Growth medium was removed, and the wells were washed once
with 0.5 ml DMEM containing 10 mM HEPES (pH 7.5) before
addition of 0.2 ml DMEM containing 10 mM HEPES (pH 7.5),
0.5 mM isobutylmethylxanthine (IBMX), and various effectors
(as indicated below). After a 10-min incubation at 37 C (or at room
temperature), the reaction was terminated, and total cAMP was recovered
by addition of 0.1 ml of 0.3 N HCl. cAMP accumulation was
measured by RIA, as previously described (11). Total protein present in
the cell layer was determined by the BCA method (Pierce, Rockford, IL)
after solubilization in 0.1 N NaOH.
Statistics
Calculations were performed on an IBM compatible personal
computer using InStat v2.04a (Graphpad Software, San Diego, CA) or
STATA, release 5.0 (StataCorp, College Station, TX). Unless otherwise
noted, data are presented as mean ± SD. Most
comparisons were made by repeated-measures ANOVA, followed by
Bonferroni multiple comparison tests, when variation between the means
was significant (P < 0.05). For comparisons of the KO3
cell line with the six randomly integrated cell lines, cAMP
accumulation for all cell lines and concentrations of isoproterenol on
a given day was standardized by dividing by the mean cAMP accumulation
of the randomly integrated cell lines in response to 3 µM
isoproterenol for that day. The data were modeled using a
random-effects model as implemented in STATA. The random-effects model
was fit using generalized least squares. Analyses were done comparing
different cell lines measured multiple times at each concentration
value. The analyses were repeated using a generalized estimating
equations model of the data, which yielded the same results.
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Results
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Selection of targeted clones
We used a fragment of the mouse genomic DNA encompassing exon 1 of
Gnas (a gift of Dr. Robert Nissenson) to isolate a genomic
clone containing a portion of the mouse Gnas gene from a
129SvJ library (Stratagene Cloning Systems, La Jolla, CA). A 6-kb
BamHI fragment of this Gnas gene, extending from
2 kb upstream of the initiator ATG to the middle of exon 2, was
subcloned into pBluescript II KS+ (Stratagene) and used in the
construction of a targeting vector. A 500-bp fragment, extending from
the NcoI site at the initiator ATG to an Nru I site in
intron 1, was excised and replaced with the neomycin resistance gene
(neoR) from pMC1neo polyA (Stratagene). This
strategy will disrupt exon 1 of Gs. Disruption of exon 1
of Gnas is expected to produce the AHO phenotype, as human
subjects with mutations in the initiator ATG of GNAS1 have
decreased Gs protein expression and AHO (10).
Transfection of ES cells (J1, obtained from Dr. R. Jaenisch) was
accomplished by electroporation (240 V, 500 µF) in growth media,
using 5 µg of the BamHI fragment isolated from the
targeting vector. Transfected cells were grown on irradiated
neoR/+ embryonic fibroblasts and selected in 250
µg/ml G418 (Life Technologies). Colonies were picked 12–15 days
after transfection. After two subsequent passages, DNA was prepared
from a portion of the expanded colony, and the remainder was stored at
-80 C. DNA was digested with PvuII, which cuts upstream of
the 5' end of the targeting fragment, in the
neoR gene, and in exon 1 of Gnas.
Digestion with PvuII is predicted to yield a fragment of
either 2 kb from the wild-type Gnas gene or 2.4 kb from the
targeted Gnas gene (Fig. 1
).
Southern blot analysis, with oligonucleotide probes upstream of the 5'
BamHI site, was performed on DNA prepared from 167 colonies;
86 colonies showed no detectable hybridization (because of a low yield
of DNA or poor cutting by the restriction endonuclease), 74 colonies
showed only a single band (indicating random integration of the
neoR gene), and 7 showed an additional band of the expected
size, consistent with targeted disruption of the Gnas gene.
Three targeted ES cell clones (KO1–KO3) and 6 randomly integrated ES
cell clones (R1–R6) were used in these experiments (Southern blot is
shown in Fig. 2). Karyotype analysis of
clone KO3 was normal (40 chromosomes), clone KO1 had 41 chromosomes,
and clone KO2 had 39 chromosomes. Development of an abnormal karyotype
is a common occurrence with ES cells, as well as with other laboratory
cell lines. Many established cell lines routinely used for biochemical
analyses have abnormal karyotypes. The finding of a biochemical
abnormality shared by 3 ES cell lines with different karyotypes would
suggest that the biochemical abnormality is independent of the
karyotype.
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Figure 1. ES cells were transfected with a 6-kb
BamHI (B) fragment of the targeting vector in which an
0.5-kb fragment of Gnas extending from the
NcoI (Nc) site at the initiator ATG to an Nru I (Nr)
site in intron 1 was replaced with the neoR
gene (filled box). Homologous recombination of targeting
vector with genomic DNA will result in a 2.4-kb PvuII
(P) fragment, which can be distinguished from the untargeted 2.0-kb
PvuII fragment on Southern blots with oligonucleotide
probes (small boxes) upstream of the targeting vector.
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Figure 2. Southern blot analysis of ES cell clones. ES cell
DNA (10 µg) was digested with PvuII and resolved by
electrophoresis through 1.0% agarose gels. Oligonucleotide probes
upstream of the targeting fragment were used to detect a 2.0-kb
fragment in wild-type ES cells (J1), as well as in ES cell lines with a
randomly integrated targeting fragment (R1–R3). An additional 2.4-kb
fragment is seen in ES cells in which the targeting fragment underwent
homologous recombination with one allele of Gnas
(KO1–KO3).
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Northern blot analysis
Total RNA was prepared from three Gnas knockout ES cell
lines (KO1–KO3), 3 ES cell lines with randomly integrated targeting
vector (R1–R3), and untransfected ES cells. Northern blot analysis,
including correction for variation in RNA loading using the 18S rRNA
probe, indicated that the steady-state level of the 1.8-kb
Gs mRNA in the KO3 ES cell line was, on average, 43
± 13% of the levels in the R1–R3 ES cell lines, based on the average
of two independent experiments (Fig. 3).
Similarly, Gs mRNA levels in the targeted ES cells
(KO1–KO3) were, on average, 33 ± 8% of the level in the
nontransfected ES cells, based on a single experiment assayed in
duplicate (not shown).
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Figure 3. Northern blot of total RNA (20 µg) from three ES
cell lines with randomly integrated targeting vector (R1–R3) and one
targeted ES cell line (KO), hybridized with a fragment of the human
GNAS1 cDNA (upper panel) or of 18S rRNA
(lower panel). Note, approximately 50% reduction of
hybridization to Gnas in targeted clone, relative to
randomly integrated clones and equal loading of RNA. These blots are
representative of four similar experiments performed on total RNA from
ES cells, as described in the text.
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Western blot analysis
Plasma membranes were prepared from three Gnas knockout
ES cell lines (KO1–KO3), three other ES cell lines with randomly
integrated targeting vector (R1–R3), and untransfected ES cells. ES
cells express predominantly the 52-kDa form of Gs, but
the 45-kDa form also is present (Fig. 4).
Targeted ES cells did not express any additional or novel proteins that
were reactive with the Gs antisera (not shown).
Quantitative immunoblot analysis, with correction for variation in
protein loading calculated using immunoreactivity to antisera for
Gi1/i2, indicated that the steady-state level of
Gs protein in the KO3 ES cell line was 56 ± 16%
of the levels in the R1–R3 ES cell lines, based on the average of two
independent experiments, each assayed in duplicate (not shown).
Similarly, Gs protein levels in the targeted ES cells
(KO1–KO3), normalized to levels of Gq/11, were 52
± 11% of the level in the untransfected ES cells (Fig. 5), based on a single experiment assayed
in duplicate.
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Figure 5. cAMP accumulation in one targeted ES cell line
(heavy line), R1 cell line (dotted line),
and five other ES cell lines with randomly integrated targeting vector
(light lines) during a 10-min incubation at 37 C, in the
presence of 500 µM IBMX, with the indicated doses of
isoproterenol. Data are presented in terms of percent of maximal
response (which is defined as the average response of the untargeted
cells to 3 µM isoproterenol) and represent the mean
± SE of six independent experiments for the KO line or
three independent experiments for each random line.
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cAMP accumulation
At each concentration of isoproterenol tested, cAMP accumulation
was reduced in the KO3 cell line relative to five of the six ES cell
lines with a randomly integrated targeting vector (R2–R6) (Fig. 5
). In
a random-effects model ANOVA, cAMP accumulation in both the KO3 and R1
ES cell lines was significantly less than it was in the R2–R6 cell
lines at 1 µM and 3 µM isoproterenol
(P < 0.001); cAMP accumulation in the KO3 cell line
also was significantly less than it was in the R2–R6 cell lines at 0.3
µM isoproterenol (P < 0.05). cAMP
accumulation was not significantly different between any of the ES cell
lines at 0.1 µM isoproterenol or in the absence of
isoproterenol. It is unclear why cAMP accumulation in the R1 ES cell
line did not differ from that in the KO3 cell line, considering that
the R1 line did not have reduced levels of Gs mRNA or
protein. The results obtained with the R1 line introduce the important
caveat that factors other than Gs deficiency may be
responsible for the reduction in cyclase activation.
cAMP accumulation also was measured in three targeted ES cell lines
(KO1–KO3) and compared with that in untransfected ES cells (Fig. 6). In the absence of effector, basal
levels of cAMP accumulation were not significantly different between
the untransfected ES cells and any of the three targeted ES cell
clones. cAMP accumulation was less in each of the targeted ES cell
lines than it was in the untransfected ES cells, in response to
forskolin and to all concentrations of isoproterenol. However, these
differences were statistically significant (P < 0.01)
only for two of the three targeted ES cell lines (KO2 and KO3).
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Figure 6. cAMP accumulation in three targeted ES cell lines
(KO1–KO3) and untransfected ES cells (wt) during a 10-min incubation
at 37 C, in the presence of 500 µM IBMX, with the
indicated doses of isoproterenol or with 10 µM Forskolin.
Data are presented as pmol cAMP/mg protein·min and represent the mean
and SD of three independent experiments.
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To obtain a time course, cAMP accumulation was measured in KO3 cell
line and three cell lines with randomly integrated targeting vector
(R4–R6), during incubation with 1 µM isoproterenol, for
varying periods of time between 3 and 15 min (Fig. 7). Incubations were done at room
temperature in an effort to accentuate differences in cAMP accumulation
at short times of incubation. At all time points, cAMP accumulation in
the KO3 cell line was less than in the R4–R6 cell lines (on average,
54 ± 2% of the levels in the R4–R6 lines); however, these
differences reached statistical significance (P <
0.05) only for longer times at incubation (>3 min).
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Figure 7. cAMP accumulation in the Gs
knockout ES cell line (KO3) (squares), compared with the
average of three cell lines (R4–R6) with randomly integrated targeting
vector (diamonds), during incubation with 1
µM isoproterenol at room temperature for the indicated
periods of time. Data are presented as percent maximal response and
represent the mean and SD of three independent
experiments.
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Discussion
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Targeted disruption of one Gnas allele in ES cells
leads to an approximately 50% reduction in steady-state levels of
Gs mRNA and Gs protein. Thus, this model
reproduces the findings of reduced levels of Gs protein
in skin fibroblasts and erythrocyte membranes of patients with AHO who
have nonsense mutations or small deletions in the GNAS1 gene
(reviewed in 12 . These findings indicate that the expression of
Gs is not up-regulated, either transcriptionally or
posttranscriptionally, in the setting of Gs deficiency.
By contrast, a previous study had demonstrated that the level of
Gs protein can be down-regulated posttranscriptionally
(13). Transgenic mice with increased expression of Gs in
cardiac myocytes (under the control of the -myosin heavy-chain
promoter), showed a 38-fold or an 8-fold increase in Gs
mRNA but only a 2.8-fold or 1.4-fold increase in Gs
protein, respectively (13).
Targeted disruption of one Gnas allele in mouse ES cells is
associated with an approximately 50% reduction in cAMP accumulation in
response to stimulation with the ß-adrenergic receptor agonist,
isoproterenol. By contrast, studies of tissues from patients with PHP
type Ia have produced somewhat conflicting results (reviewed in 14 . The initial biochemical characterization of adenylyl cyclase
activity of renal tissue from a patient with PHP type Ia demonstrated
normal responsiveness to PTH (15). A subsequent study of renal
membranes from a second PHP type Ia patient showed similarly normal
basal and PTH-stimulated adenylyl cyclase activity, but activity was
markedly reduced, compared with normal, when measured in the presence
of low ATP concentrations or when guanine nucleotides were not added to
the assay (16). Cultured bone cells from a patient with PHP type Ia
have shown normal responses to PTH (17), and transformed lymphoblasts
from patients with PHP type Ia have shown normal adenylyl cyclase
activity (18). By contrast, thyroid membranes from a single patient
with PHP type Ia have shown reduced responsiveness to TSH (19), and
adipocyte plasma membranes have shown a blunted adenylyl cyclase
response to isoproterenol (20). In cultured skin fibroblasts, we found
decreased adenylyl cyclase responses to PGE1 (11), whereas
others found normal responsiveness (21). More recent studies also have
demonstrated reduced metabolic responses to isoproterenol by intact
cultured fibroblasts from patients with PHP type Ia (22). These
experimental differences may reflect receptor-specific differences in
the amount of Gs protein necessary for normal coupling
to adenylyl cyclase. Alternatively, cell specific differences in the
expression of other components of the cAMP signal transduction cascade
may account for cell specific modification of the generalized 50%
reduction in Gs expression. Finally, cell specific
differences in the expression of GNAS1, which arise as a
result of imprinting (23) or other mechanisms, may account for variable
hormone resistance in patients with only one functional
GNAS1 allele.
In the presence of the phosphodiesterase (PDE) inhibitor IBMX, cells
with targeted disruption of one allele of Gnas accumulated
less cAMP than ES cells with randomly integrated targeting vectors at
all time points between 1 and 15 min. However, at later time points,
targeted ES cells accumulated as much cAMP as the untargeted ES cells
had accumulated at earlier time points. This observation suggests that
PDE activity can influence tissue responsiveness to protracted hormone
stimulation and may provide one possible explanation for tissue
specific differences in hormonal responsiveness. Tissues with low
levels of PDE activity might be able to accumulate sufficient cAMP to
manifest a normal response to a protracted or chronic hormonal signal,
despite a 50% reduction in the level of Gs protein,
whereas tissues with high levels of PDE activity might not be able to
overcome a 50% deficiency of Gs.
We have established several ES cell lines with a targeted disruption of
Gnas in which the effect of Gs deficiency can
be analyzed more fully . This represents the first step to generating a
transgenic mouse model of AHO. To date, we have been unsuccessful in
our efforts to establish a knockout mouse. The KO3 ES cell line was
introduced into C57BL/6 mouse embryos on 2 occasions and gave rise to a
total of 17 chimeric mice (7 males and 10 females). In matings to
C57BL/6 mice, we obtained 40 viable progeny from the male chimeras that
were derived from the injected ES cells, as indicated by their agouti
coat color. However, none of these mice carried the targeted
Gnas allele. None of the progeny of the female chimeras were
derived from the injected ES cells. Chimeric mice generated from 2
additional ES cell clones with a targeted disruption of Gnas
are currently under study.
A mouse with Gs deficiency will permit further
characterization of the cause of tissue specific differences in hormone
resistance in PHP type Ia and investigation of the molecular basis for
the distinct phenotypes of PHP type Ia and pseudoPHP. Moreover, ES
cells with a targeted disruptions of both alleles of Gnas
can be selected for by culture in the presence of high concentrations
of G418 (24) or generated by direct targeting of the second allele
(25). Because ES cells will differentiate in vitro into a
variety of cell lineages and will develop into all tissues of chimeric
mice after injection in mouse embryos, these ES cells will permit
investigation of the role of Gs and cAMP coupled signal
transduction in differentiation and development.
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Acknowledgments
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We are grateful to Randall Spencer for technical assistance in
isolating the Gnas gene from the 129SvJ library and to Josef
Coresh, M.D., Ph.D., for assistance with the statistical analysis of
the data.
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Footnotes
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1 This work was supported by General Clinical Research Center Clinical
Associate Physician Award RR-00722–22S1 (to W.F.S.); NIH, NCRR,
OPD-GCRC Grant RR-00052; and by NIH Grant DK-34281 (to M.A.L.). This
work was presented in part at the 10th International Congress of
Endocrinology in San Francisco, California. 
Received March 24, 1997.
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Abstract
Introduction
