Insulin-Like Growth Factor-I Affects Perinatal Lethality and Postnatal Development in a Gene Dosage-Dependent Manner: Manipulation Using the Cre/loxP System in Transgenic Mice
Jun-Li Liu,
Alexander Grinberg,
Heiner Westphal,
Brian Sauer,
Domenico Accili,
Michael Karas and
Derek LeRoith
Section on Cellular and Molecular Physiology (J.L.L., M.K.,
D.L.) Diabetes Branch and Laboratory of Biochemistry and
Metabolism (B.S.) The National Institute of Diabetes and
Digestive and Kidney Diseases
Laboratory of Mammalian
Genes and Development (A.G., H.W.) and Developmental Endocrinology
Branch (D.A.) The National Institute of Child Health and Human
Development National Institutes of Health Bethesda, Maryland
20892
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ABSTRACT
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Insulin-like growth factor-I (IGF-I) is essential
for cell growth, differentiation and postnatal development. A null
mutation in igf-1 causes intrauterine growth retardation
and perinatal lethality. The present study was designed to test the
lower limit of igf-1 gene dosage that ensures survival and
postnatal growth by using the Cre/loxP system. Mice with variable
reductions in IGF-I levels were generated by crossing
EIIa-cre transgenic mice and mice with loxP-flanked
igf-1 locus (igf-1/flox). EIIa-cre
mice express bacteriophage P1 Cre (causes recombination)
recombinase under the adenovirus promoter EIIa, during early embryonic
development before implantation, and cause genomic recombination of the
igf-1/flox locus. Mice with the most extensive
recombination die immediately after birth, while the survivors have
significant growth retardation in proportion to the reduction in their
igf-1 gene. Interestingly, this gene dosage effect on body
weight was not very significant before weaning. However, when the young
animals were weaned at 3 weeks, the igf-1 gene dosage was
the only independent predictor of the weight gain between 3 and 6 weeks
among the parameters tested. Although growth retarded, mice with
Cre-induced partial igf-1 deficiency were fertile and gave
birth to null mice. Thus Cre-induced genomic recombination using the
EIIa promoter occurs during development and creates distinct phenotypes
compared with the conventional null mutation. This variability allows
for postnatal survival and will enable one to begin to explore the role
of the endocrine vs. paracrine effects of IGF-I.
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INTRODUCTION
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Insulin-like growth factor I (IGF-I) is expressed by many
cells and tissues during embryonic and postnatal development and in
adult animals. IGF-I influences various biological processes through
binding to its membrane-anchored receptor, the IGF-I receptor (IGF-IR),
although at higher concentrations IGF-I can also activate the insulin
receptor. In addition, in vivo, IGF-I action is modulated by
a family of IGF-binding proteins present in the circulation. IGF-I,
secreted from the liver in response to GH, promotes postnatal growth in
bone, muscle, fat, and other tissues. Human and murine IGF-I deficiency
caused severe intrauterine growth retardation, perinatal lethality,
postnatal growth retardation, delayed development in brain, muscle,
bone, and lung, and infertility (1, 2, 3, 4), demonstrating the essential
role of IGF-I in normal growth and development. On the other
hand, overexpression of IGF-I in transgenic mice is coupled with
widespread tissue hypertrophy in the brain, heart, muscle, and
intestine (5, 6, 7, 8).
The conventional gene knockout approach potently demonstrated the
crucial role of IGF-I in intrauterine development and perinatal
survival but is unsuitable for postnatal studies on animal growth
because most of the mice die after birth (1, 2, 3). To study the lower
limit of IGF-I expression necessary for postnatal survival and the
effect of IGF-I deficiency on postnatal growth, the Cre/loxP system was
used to avoid perinatal lethality. We generated mice with the
igf-1 locus flanked by loxP (igf-1/flox)
repeats by homologous recombination and crossed them with
EIIa-cre transgenic mice, which express the bacteriophage P1
Cre recombinase under the adenovirus promoter EIIa during early
embryonic development before implantation. In this manner, we created
genetic mosaics whose IGF-I expression is partially abrogated, thus
allowing the correlation of the level of mosaicism with various
phenotypes and the level of IGF-I expression. As a result, Cre-induced
igf-1 recombination caused postnatal lethality in the most
severe cases of gene recombination and significant growth retardation
in the survivors who had lesser degrees of recombination of the
igf-1 locus. We demonstrate that igf-1 gene
dosage determines the level of IGF-I expression and the rate of pre-
and postnatal growth. Interestingly, we found a closer correlation of
the growth rate with serum IGF-I concentration than with the
level of liver IGF-I gene expression.
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RESULTS
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Mutagenesis of the igf-1 Locus
We designed a replacement vector that would enable deletion of the
entire exon 4 (encoding amino acid residues of 2670) upon Cre
expression and results in a truncated protein that has no functional
capabilities (Fig. 1
). A
positive-negative selection protocol was used to facilitate
identification of ES (embryonic stem cell) clones of homologous
recombination (9). Linearized vector DNA was introduced into recipient
ES cells by electroporation, and the cells were seeded and selected
with the appropriate combination of antibiotics on feeder layers of
mouse fibroblasts. A total of 125 clones were picked and expanded, and
their genomic DNA was analyzed by Southern blotting.

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Figure 1. Targeting of the Mouse igf-1 Locus
A, Restriction map of the wild-type igf-1 gene in the
region containing exon 4. The restriction sites are: B,
BamHI; E, EcoRI; H,
HindIII; and K, KpnI. B, Schematic
structure of the targeting vector. L: loxP sites. C, Expected
mutagenesis to the igf-1 locus upon homologous
recombination. At the bottom of each diagram, positions of the
hybridization probes and PCR primers are indicated. The 1-kb ruler is
approximate.
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In the initial screening digested with BamHI and hybridized
to the 3'-external probe, two clones (JL-43 and JL-115) exhibited a
hybridization band of 11 kb, in contrast to 10 kb wild type allele, a
sign of correct recombination (Fig. 2A
).
When digested with KpnI, a 9.5-kb segment of
igf-1 locus was hybridized to both 5'- and 3'-external
probes. Due to insertion of an extra KpnI site on the
vector, these two correctly targeted clones exhibited additional
shorter bands of expected sizes when hybridized to both probes (Fig. 2B
). Both clones, JL-43 and JL-115, were injected into C57BL/6
blastocysts and transferred into pseudopregnant mothers. Male chimeric
mice were bred against female C57BL/6 mice. The mice with germline
transmission of loxP-flanked igf-1 locus
(igf-1/flox) (F1) were intercrossed to generate
homozygous igf-1/flox mice (F2), identified by
PCR assay and Southern blot analysis of their tail DNA (Fig. 3
).

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Figure 2. Characterization of ES Clones by Southern Blot
Analysis
BamHI- and KpnI-digested genomic DNA from
control ES cells (nos. 44, 45, 69, 70, 114, 116, and 125) and two
correctly targeted clones (nos. 43 and 115) were hybridized to 5'
(pB2-BH) and 3' (pH3-HE) external probes as indicated. The nature and
size of the hybridization fragments (in kilobases) are shown.
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Figure 3. Germline Transmission of loxP Recombination
F1, Crossing of chimeric founder mice with C57BJ/6
generated F1 offspring. To detect the presence of loxP
integration, PCR was performed on tail DNA specimen with primers IA-8,
IA-6, and ID-3 (as illustrated in Fig. 1 ). Both wild-type (350 bp,
IA-8/ID-3) and igf-1/flox bands (200 bp, IA-6/ID-3) from
the PCR reaction are depicted from mice of a representative litter.
F2, Heterozygous igf-1/flox mice were
allowed to interbreed to generate F2 offspring. For some
representative individual mice, their genomic DNA, digested with
HindIII, was probed with pSP-3 in a Southern blot.
Wild-type band is 6.5 kb, while igf-1/flox locus is 4.9
kb, due to the presence of an internal HindIII site on
the targeting vector.
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Offspring from interbreeding of heterozygous igf-1/flox mice
(F1) showed Mendelian inheritance of the mutant
igf-1 alleles. Of eight litters (64 mice) analyzed, 19% of
the mice were homozygous igf-1/flox, 56% were heterozygous,
and the remaining 25% were wild type. This indicates that the
introduction of loxP sequences and neo did not cause any
major abnormalities. Mice with both homo- and heterozygous
igf-1/flox loci are fertile and appear normal and healthy. A
study of five litters revealed a slight growth retardation (<10%) in
homozygous igf-1/flox mice (data not shown).
Cre Causes igf-1 Recombination in Heterozygous
igf-1/flox Mice
EIIa-cre transgenic mice, which express Cre recombinase
in early embryonic development, were used to develop mosaic mice of
igf-1 recombination. All the offspring (Cre F1) from
crossing male EIIa-cre and female heterozygous
igf-1/flox mice carry one allele of cre, and
half of them have their igf-1 exon 4 flanked by loxP
sites. PCR analysis of tail DNA in mice bearing igf-1/flox
revealed various degrees of exon 4 deletion. When primers ES-1/ID-3
were used (Fig. 1B
), the wild-type PCR product is approximately 1 kb
and the igf-1/flox locus is approximately 2 kb (which cannot
be amplified under the assay condition), whereas Cre-induced exon 4
recombination is detected as a 0.2-kb band (Fig. 4
). Southern blot analysis confirmed
the conversion from the igf-1/flox fragment of 4.9 kb to the
recombinant of 2.8 kb, when DNA was digested with HindIII
and hybridized to pSP-3 internal probe (Fig. 4
). This indicates that
one copy of the cre transgene is sufficient to induce
targeted gene recombination. Incomplete recombination may be attributed
to delayed and/or mosaic expression of the cre.

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Figure 4. Genomic Analysis of Cre F1 Offspring,
Derived from Crossing EIIa-Cre and Heterozygote
igf-1/floxed Mice
A, PCR products of cleaved igf-1/flox (0.2 kb) and
wild-type IGF-I gene (1 kb). B, Southern blot of the conversion of
igf-1/flox (4.9 kb) into the cleavage product (2.8 kb).
The conversion is not complete, indicating a mosaic expression of
cre. C, All the offspring have cre gene
in their genome as demonstrated by Southern blot analysis.
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Neonatal Lethality and Growth Retardation Induced by
igf-1 Recombination
To study the effect of cre expression in homozygous
igf-1/flox mice, five pairs of mice heterozygous for both
EIIa-cre and igf-1/flox were crossed to generate
Cre F2. Each of three dams gave birth to 2 dead pups each
(total of 6) in addition to 711 pups per litter, which survived for
further study. Genomic DNA was prepared from 5 of the dead pups. By PCR
and Southern blot analysis, all of them have almost complete (
95%)
recombination of the igf-1 (Fig. 5
) and were positive for the
cre (data not shown). We were unable to prepare intact RNA
from these pups to determine the level of igf-1
expression.

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Figure 5. Near-Complete Recombination of igf-1
Locus Caused Postnatal Death of Cre F2 Pups
Mice heterozygous for both EIIa-cre and
igf-1/flox were crossed to generate Cre F2.
Some dams gave birth to pups that died immediately after birth. Genomic
DNA, digested with HindIII, was analyzed by Southern
blot with 32P-labeled pSP-3. A, B, C, and D represent the
dead pups while A48, A27, and A32 represent their parents.
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To examine tissue igf-1 expression, fetuses at days 1920
of gestation from two mothers were removed by cesarean section, their
weights were determined, and their DNA and RNA were prepared. Out of 19
fetuses, 4 were homozygous igf-1/flox, and all of them were
cre positive. Overall, there was 90100% recombination on
igf-1 exon 4 (Fig. 6
), their
body weights were only 69% of the wild type control (Table 1
), and IGF-I mRNA was only 17% (Fig. 7
and Table 2
), indicating that Cre-induced
igf-1 recombination caused intrauterine growth retardation.
Therefore, in the most severe cases of igf-1 recombination,
a phenotype similar to the previous reports of generalized
igf-1 gene knockout is observed (2, 3).

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Figure 6. Genotyping of Cre F2 Fetus or the
Surviving Mice at 3 Weeks
As in Fig. 3 (F2), wild-type (WT) mice only have a 6.5-kb
band, igf-1/flox (L/L) mice only have a 4.9-kb band,
while heterozygous (W/L) have both bands. Upon Cre action, there are
varying degrees of recombination of igf-1/flox allele
(4.9 kb) to 2.8 kb.
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Figure 7. IGF-I mRNA Expression Is Dramatically Reduced by
Cre Expression in igf-1/flox Mice
A, A representative RNase protection assay is shown. The level of IGF-I
mRNA is diminished in W/L mice and almost abolished in L/L mice. To
demonstrate equal loading of total RNA, a protected band for 18S rRNA
is illustrated at bottom. B, A representative Northern blot of IGF-I
mRNA from 6-week-old mice liver, illustrating the 0.7-kb major form and
7-kb minor form. Their abundance is incomparable due to differential
exposure. Compared with their wild-type littermates, IGF-I mRNA is
decreased in W/L and dramatically reduced in L/L mice upon Cre
activation, while actin-mRNA is not affected (see Table 2 ).
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Table 2. IGF-I Gene Dosage-Dependent Reduction in Liver
IGF-I mRNA and Serum IGF-I Concentration Induced by EIIa-cre
Expression
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Cre-Induced, Gene Dosage-Dependent Growth Retardation and Survival
of igf-1/flox Mice
Most of the Cre F2 offspring survived, including some
cre-positive homozygous igf-1/flox mice. Their
body weights were measured at 3 and 6 weeks after birth, and their
genotypes were analyzed by PCR and Southern blots. The level of
igf-1 expression was determined by Northern blot analysis of
total RNA prepared from the liver of 6-week-old mice. The animals were
grouped by their igf-1 dosage determined by Southern blots.
Compared with their wild-type littermates, mice with Cre-induced
homozygous igf-1/flox recombination were significantly
smaller at birth (69%) and grew at a slower rate [i.e.
body weight 72% and 68% of wild-type controls at 3 and 6 weeks,
respectively (Table 1
)]. In the survivors, the igf-1/flox
locus was incompletely cleaved, and they maintained 28% of
igf-1 expression compared with their wild-type controls
(Table 2
). There was no significant change in the appearance and the
relative weights of the organs examined, including brain, heart, liver,
kidney, lung, and spleen (data not shown). Mice with a single-copy
recombination of igf-1 demonstrated marginal slower growth
(8185% of control, P < 0.01) and had significantly
impaired IGF-I expression (67% in fetus head and 62% in adult liver
compared with their wild-type littermates) (Tables 1
and 2
).
In contrast to the dramatic decrease in the level of liver IGF-I mRNA,
the change in serum IGF-I concentration is more moderate and correlates
more closely with postnatal growth rate. Thus, mice with two alleles of
Cre-induced igf-1 recombination (gene dosage 0.10.4)
demonstrate liver IGF-I mRNA at 28% of the level of their wild-type
littermates, whereas the level of circulating IGF-I is 71% and their
growth rates were 6872% of that of the control mice (Tables 1
and 2
).
Tissue Variations in Cre-Induced igf-1 Recombination
and IGF-I Gene Expression
One explanation for the relative high level of serum IGF-I (71%
of control) in the face of markedly reduced liver IGF-I mRNA levels
(28% of control) is that circulating IGF-I may be derived from
extrahepatic tissues. We therefore analyzed brain, kidney, muscle,
liver, and lung from two litters of wild-type, heterozygous, and
homozygous igf-1/flox mice (two to three each). Genomic DNA
was prepared, and Cre-induced igf-1 recombination was
studied by Southern blots. There was no significant difference in the
pattern and degree of gene recombination induced by Cre expression,
among all tissues examined (including the tails), indicating that the
promoter EIIa was activated well ahead of tissue differentiation to
have ensured homogenous recombination (data not shown).
To determine the level of IGF-I gene expression, total RNA from these
tissues was used for ribonuclease (RNase) protection assay. While there
was a >65% decrease in the liver IGF-I mRNA, the extrahepatic tissues
had only
a 4050% decrease in IGF-I mRNA in igf-1/flox
mice (data not shown). In a study involving 51 mice, Northern blots of
liver RNA revealed a 72% reduction in the major form (
0.7 kb) of
IGF-I mRNA and a more moderate (
48%) decrease in its minor form
(
7 kb) (Fig. 7
and Table 2
). Interestingly, in extrahepatic tissues,
the major IGF-I mRNA species are
7 kb, which showed a similar modest
reduction (
4050%) upon Cre expression (Northern blots, data not
shown).
Correlation of Growth Rate, Liver IGF-I mRNA Level, and Serum IGF-I
Concentration with igf-1 Gene Dosage
The results of linear correlation analysis demonstrate that the
Cre-induced decrease in igf-1 dosage directly affects the
level of igf-1 expression, circulating IGF-I levels, and the
rate of animal growth (Table 3
). Very
strong correlation (r = 0.80) was found between
igf-1 gene dosage and liver IGF-I mRNA level. Significant
(P < 0.001), but less strong correlation
(r = 0.460.68) was observed between body weights at 3
and 6 weeks and the igf-1 gene dosage, liver IGF-I mRNA, and
serum IGF-I levels. The correlation was stronger at 6 weeks of age when
compared with 3 weeks of age (Table 3
).
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Table 3. Linear Correlation of Growth Rate, Liver IGF-I
mRNA Level, and Serum IGF-I Concentration with igf-1 Gene
Dosage
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To further explore the statistical relationship between body weight and
the level of mRNA, serum IGF-I, and igf-1 gene dosage,
forward stepwise multiple linear regression analysis was performed. As
a large sample size is needed for this extensive analysis and as there
are obvious differences in body weights between males and females (at 6
weeks), we performed this analysis on the males (females were not
distributed evenly between subgroups to allow informative analysis). As
shown in Table 4
, body weight at 3 weeks
could be independently predicted by the serum IGF-I level. The impact
of liver mRNA level was insignificant, and igf-1 gene dosage
was excluded from the model. The situation is somewhat different when
body weight at 6 weeks was predicted. Here again, the principal
component of the model was the serum IGF-I level, but liver mRNA level
accounts for a significant proportion of body weight prediction. When
the gain of body weight (36 weeks) was analyzed as a dependent
variable, the only independent parameter predicting this gain was
igf-1 gene dosage.
Mice with Cre-Induced Partial igf-1 Deficiency Are
Fertile and Give Birth to igf-1 Null Mutants
As growth retardation is often associated with infertility, we
tested 1) whether our igf-1/flox mice are fertile after
Cre-induced recombination, 2) whether the knockout allele can be
transmitted into germline, and 3) whether we can generate viable
igf-1 null mice. Results of these breeding experiments
demonstrated that homozygous igf-1/flox mice (with
igf-1 gene dosage
0.5), although growth retarded, are able
to reproduce. Two pairs gave birth repeatedly to an average of seven
pups per litter. Interestingly, about half of the igf-1 null
mice (accounting for
one quarter of the offspring) survived up to
46 weeks, although with severe growth retardation. At 3 weeks, null
mice weighed only 4.5 ± 0.2 g (n = 3) in comparison to
12.0 ± 0.2 g (n = 4) for their heterozygous littermates
(igf-1/flox/igf-1/null). IGF-I deficiency was confirmed by
PCR and Southern blots on genomic DNA and by RNase protection assay for
liver and brain total RNA (Fig. 8
).

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Figure 8. IGF-I Null Mutation Generated by Interbreeding Mice
with Cre-Induced Partial IGF-I Deficiency
Homozygous igf-1/flox mice (with igf-1
gene dosage 0.5) were allowed to reach maturity and to intercross.
The offspring have two genotypes: L/- (heterozygous
igf-1/flox/igf-1/null), which has one intact allele of
igf-1 exon 4 (Southern blot, panel A), and normal expression of IGF-I
gene in the liver and brain (RNase protection assay, RPA, panels B and
C); and -/- (igf-1 null), which has the entire exon 4
excised (panel A) and complete absence of IGF-I mRNA in both the liver
and brain (panels B and C). The blots were representatives of three
experiments; 18S rRNA was also probed to demonstrate the equal loading
of total RNA.
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DISCUSSION
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Cre ("causes recombination") is a 38-kDa recombinase that
recognizes loxP repeats, deletes the region in between, and rejoins
genomic DNA in bacteriophage P1 (10). Gu et al. (11)
demonstrated that the Cre/loxP system can mediate partial T
cell-specific deletion of DNA polymerase ß in mice. More recently,
Tsien et al. (12, 13) have generated Cre-induced
NMDAR1 gene deletion restricted to CA1 region of the
hippocampus. The mutant mice grow into adulthood without gross
developmental abnormalities. More precise testing showed that they lack
NMDA receptor-mediated synaptic currents and exhibited impaired
spatial memory learning. Thus, in such cases, undesirable developmental
and behavioral consequences caused by conventional gene knockout were
circumvented.
Using homologous recombination, we have established a mouse strain in
which the exon 4 of igf-1 is flanked by loxP sites. We have
targeted the entire exon 4, which encodes for amino acid residues
2670 of the peptide, including part of the domain B and entire
domains C, A, and D. Previously Liu et al. (1, 2) replaced
only part of the same exon encoding the residues 5170, whereas
Powell-Braxton et al. (3) targeted exon 3, which encodes
only part of domain B of the molecule.
Our results from the mice with the most severe igf-1
recombination are similar to previously reported null mutations of the
mouse igf-1 (1, 2, 3). Using the newly developed Cre/loxP
system in transgenic mice, we created near- complete recombination of
the target locus, which caused postnatal lethality, growth retardation,
and dramatic decreases in igf-1 expression. Linear
correlation analysis demonstrated that igf-1 gene dosage
strongly defines the IGF-I mRNA level in liver as expected.
Surprisingly, body weight at 3 weeks was predicted by only the serum
IGF-I level but not by igf-1 gene dosage or the level of
IGF-I mRNA in liver. At 6 weeks the mRNA level became a significant
parameter for the forward stepwise model. This discrepancy could be
explained by the consumption of mothers milk by the pups up to 3
weeks of age. It is known that the content of IGF-I in the milk of most
mammals is high (14). However, the role of milk-derived IGF-I in the
growth of young animals is unclear. Although milk-borne IGF-I may not
be essential for neonatal development, it has been reported that milk
proteins protect IGF-I from degradation in neonatal rat intestine and
oral consumption of IGF-I via milk is associated with the absorption of
the growth factor into the bloodstream. Therefore therapeutic use of
oral IGF-I has been proposed as an effective intervention for preterm
neonates and those with compromised intestinal function (15, 16, 17). Our
data are in agreement with this finding since once the animals were
weaned, the body weight gain was predicted exclusively by gene dosage,
which suggests that this effect could have been masked by oral IGF-I
delivery. In addition, persistent presence or even compensatory
increase of IGF-II in the perinatal period may also compensate for the
growth deficiency in the first 3 weeks of life.
Another novel observation from our study was that the decrease in liver
IGF-I mRNA levels was much more dramatic than the reduction in serum
IGF-I concentration. This result contradicts the generally accepted
hypothesis that liver is the main source of postnatal IGF-I production
and is the major source of circulating, endocrine IGF-I that controls
body growth. A number of possible explanations may explain the
discrepancy between liver IGF-I mRNA levels and circulating IGF-I
levels in our genetically engineered animals: 1) Enhanced translational
activity may compensate for lower mRNA levels. 2) Circulating IGF-I may
be metabolized more slowly, perhaps as a result of changes in
IGF-binding proteins. 3) Extrahepatic tissues may, in fact, play a
major role in contributing to the circulating IGF-I levels. Indeed, we
favor the last possibility, despite the fact that it differs from
conventional notions. Interestingly, recombination of the
igf-1 by Cre, as detected on Southern blots, was
similar in extrahepatic and hepatic tissues. Thus, we must conclude
that igf-1 expression in these extrahepatic tissues was
maintained despite the severe reduction in intact igf-1
gene. One possible explanation for this discrepancy is that in all the
extrahepatic tissues we examined, IGF-I was apparently translated
mainly from the higher (
7 kb) mRNA species, which may be less
affected under the circumstances of low gene dosage. On the other hand,
liver IGF-I production may be more sensitive to changes in
igf-1 gene dosage with an effect on the lower mol wt mRNA
species (
0.7 kb). These differences between liver and extrahepatic
regulation of IGF-I expression have been previously described
(18, 19, 20, 21).
In the present study, Cre-induced igf-1 knockout was
achieved in mice of two generations (Cre F1 and
F2). We demonstrated significant target gene recombination
in Cre F1 mice that carry only one allele of
igf-1/flox and cre transgene. Interbreeding of
Cre F1 generated at least five genotypes of Cre
F2 mice: homozygous igf-1/flox with or without
cre transgene; heterozygous igf-1/flox with or
without cre; and wild-type igf-1 locus in which
case the presence of cre gene is irrelevant. The pattern of
Cre activity judged by the recombination of the igf-1 locus
is quite variable. There was near complete (
95%) recombination,
which caused perinatal lethality, whereas incomplete deletion to the
igf-1/flox locus was associated with postnatal survival and
growth retardation. As listed in Tables 1
and 2
, we generated 11 mice
with homozygous igf-1/flox locus, 7090% gene deletion
(gene dosage 0.10.4 group), which survived up to 6 weeks.
Surprisingly, in this group five mice actually did not carry
cre transgene. How then did igf-1 recombination
occur? If the gene deletion occurred before the formation of the
gametes, the recombination has to be all or none. If, on the other
hand, gene recombination occurred after fertilization of the Cre
F2, the cre transgene should not be lost during
development. Therefore, we propose that these mice are derived from
haploid gametes (oocytes) that inherited Cre protein from their diploid
precursor cells via the cytoplasm during meiosis (without itself
carrying a cre transgene). Limited Cre activity in those
gametes and fertilized eggs will create incomplete (mosaic)
recombination of the igf-1 locus. While this has to be
tested, it remains a reasonable explanation since the EIIa promoter has
been shown to be activated in undifferentiated stages of murine
oogenesis and preimplantation development (22).
Growth retardation caused by genetic defects is often associated with
infertility, such as mice lacking the IGF-I, vitamin D receptor, and
ATM genes (1, 2, 3, 23, 24). In the case of igf-1 targeting,
homozygous mice are sterile and growth retarded while heterozygous
(gene dosage 0.5) are basically normal in both growth and fertility. We
were interested in testing the effect of growth retardation caused by
Cre-induced igf-1 recombination on fertility. In our
studies, mice with igf-1 gene dosage
0.5 are still
fertile. Therefore, infertility is not always the consequence of growth
retardation. The same experiment also demonstrated that the
Cre-recombined allele can be transmitted through the germline and
produce null mice.
IGF-I is widely expressed throughout development and in adult life.
Since the total knockout of the igf-1 gene has been known to
cause profound defects in early development and postnatal viability,
these animals cannot be used to address specific questions such as 1)
what is the role of the circulating (endocrine) IGF-I in postnatal
growth compared with local tissue IGF-I production? 2) how does IGF-I
deficiency causes infertility? and 3) what is the role of
tissue-specific expressed IGF-I? The Cre/loxP system provides a more
powerful tool in answering these questions. Flanking the
igf-1 locus by loxP sites opens a new chapter so that the
gene can be manipulated just by expressing Cre in a chosen target
tissues and at defined stages using tissue-specific promoters. Toward
that direction, a tissue-specific promoter can activate the
cre transgene at a defined stage in the development of the
target tissue, such as T cell-specific lck promoter and neuron-specific
-calmodulin kinase II promoter (11, 12). A silent cre
transgene (Mx1-Cre) can be induced upon application of interferon
(25). Furthermore, cre transgene has been successfully
expressed in the liver after intravenous injection of virus-Cre
constructs (26). The current study therefore demonstrates that the
Cre/loxP system is suitable to generate genomic recombination of the
mouse igf-1 gene in a controlled manner.
In summary, we developed a conditional igf-1 knockout system
using the Cre/loxP model. The igf-1/flox mice appear healthy
and demonstrated virtually normal growth rates. Expression of
cre transgene created partial to near complete recombination
of the igf-1 locus, which caused postnatal lethality, growth
retardation, and dramatic decreases in igf-1 expression and
decrease in serum IGF-I concentration. After cre expression,
we demonstrated igf-1 gene dosage-dependent survival,
growth, and decreases in mRNA and protein levels. Compared with those
with null mutations, our mice with partial igf-1 deficiency
are viable, have milder growth retardation, and are fertile, therefore
allowing gene dosage-dependent studies. To dissect complex roles
(endocrine, paracrine, autocrine) of a growth factor such as IGF-I, the
Cre/loxP system can be extended to generate tissue- and developmental
stage-specific knockout mice.
 |
MATERIALS AND METHODS
|
---|
Targeting Vector and Generation of Mice with loxP-Flanked
igf-1 Locus
Mouse genomic DNA fragments encompassing exon 4 and flanking
sequences of igf-1 were prepared from
-phage clones 19
and 7, kindly provided by Drs. P Rotwein and R. D. Palmiter,
respectively (20, 27). A 6.5-kb HindIII fragment (from
19) was used as the targeting region; immediately downstream, a
0.8-kb HindIII/EcoRI fragment (pH3-HE from
19)
was used as 3'-external probe; and immediately upstream, a 1-kb
BamHI/HindIII fragment (pB2-BH from
7) was
used as 5'-external probe in screening homologous recombination (Fig. 1
). The relative position of the fragments was confirmed by restriction
mapping, Southern blot analysis, and partial sequencing and compared
with the previous partial characterization of this region (20, 27).
The replacement-type targeting vector (Fig. 1B
) was made by inserting
the first loxP site (from pMC-lox-Neo) into intron 3 at the
BamHI site of the 6.5-kb HindIII fragment, on a
pBluescript KS+ backbone (Stratagene, La Jolla, CA). The bacterial
neomycin-resistance gene (neo) along with the second loxP
site [1.2 kb from pGH-1, K. Rajewsky (11)] was inserted into intron 4
at the EcoRI site. As a result, the two direct repeats of
loxP sites flanked a 0.9-kb genomic region including the entire exon 4
(182 bp), which encodes residues 2670 of mouse IGF-I (1, 20). The
targeting vector contained 2.5 kb of homologous DNA upstream (left arm)
of the first loxP site and 2.8 kb of homologous DNA downstream (right
arm) of the second loxP site. A herpes simplex virus thymidine kinase
gene [HSV-tk, 1.2 kb from pGH-1, K. Rajewsky (11)] was
inserted upstream to the left arm. A unique SacII site at
the 3'-end of the right arm was used to linearize the construct.
The targeting vector was electroporated into ES cells derived from 129
sv mice maintained on subconfluent embryonic fibroblasts. ES clones,
resistant to both geneticin (G418) and gancyclovir, were picked and
expanded. The ES cells harboring the homologous recombined construct
were determined by Southern blotting using both 5'(pB2-BH) and
3'(pH3-HE) probes external to the homologous region (Fig. 1C
). The
correctly targeted ES cell clones were expanded and injected into
C57BL/6 blastocysts and transferred into pseudopregnant mothers. Male
chimeric mice were bred against female C57BL/6 mice. The F1
mice with germline transmission of the igf-1/flox locus were
allowed to interbreed to generate homozygous igf-1/flox
(F2) mice, identified by PCR assay and Southern blot
analysis of their tail DNA.
Breeding Mice of EIIa-cre with
igf-1/flox
EIIa-cre mice, on a FVB/N genetic background, carry
the Cre recombinase gene driven by the adenovirus EIIa promoter (28).
They express Cre in oocytes and in early mouse embryos before
implantation (22, 28). Initially, female mice heterozygous for
igf-1/flox locus were bred with male mice homozygous for the
EIIa-cre transgene. Recombination of igf-1 exon 4
in offspring was assayed by PCR and Southern blotting of tail DNA. Mice
heterozygous for both EIIa-cre and igf-1/flox
locus (Cre F1) were interbred to obtain mice with
homozygous igf-1/flox alleles (Cre F2). The
expression of Cre in these mice induces deletion of
igf-1 exon 4 and impaired expression of IGF-I, as shown in
Results. Some Cre F2 mice were further allowed
to breed to test their fertility and generate mice with
igf-1 null mutation. Offsprings were ear tagged at P21 for
identification and weaned, and tail biopsies were taken for DNA
analysis.
Southern Blot Analysis
Genomic DNA from ES cells was prepared by an overnight digestion
at 37 C in a lysis buffer (100 mM Tris HCl, pH 8.5, 5
mM EDTA, 0.2% SDS, 200 mM NaCl, 0.1 mg/ml
proteinase K) and precipitation in isopropanol (29). The DNA was
digested overnight with either BamHI or KpnI,
fractionated by electrophoresis through 0.8% agarose gels, transferred
to a maximum strength Nytran membrane (Schleicher & Schuell, Keene,
NH), and hybridized with 32P-labeled 5'- or 3'-RNA probes
synthesized with the Riboprobe Systems (Promega, Madison, WI). To
calculate the extent of igf-1 recombination induced by Cre,
Southern blots were analyzed by PhosphorImager 400E (Molecular
Dynamics, Sunnyvale, CA).
Mouse tail DNA was prepared similarly in a lysis buffer (10
mM Tris, pH 8.0, 100 mM EDTA, 0.5% SDS, 0.2
mg/ml proteinase K) but at 55 C, and purified DNA was digested
overnight with HindIII and processed for Southern blot using
riboprobes synthesized with template DNA of pBS-1 or pSP-3 (Fig. 1
).
For detection of the cre transgene, tail DNA was digested
with BamHI and probed with a
XhoI/BamHI fragment of Cre coding region (from
pMC-Cre) (11). Southern blots were performed from DNA prepared from
other tissues including brain, kidney, liver, lung, and muscle.
PCR Analysis
PCR was performed on tail DNA preparations to identify which
mice have loxP sites integrated into their genome. Using primers IA-8,
IA-6, and ID-3 (see Fig. 1B
for their positions), the PCR reactions
were conducted after Perkin Elmers recommendation, with extra
MgCl2 (1.5 mM) added. The reaction cycles are:
94 C, 1 min; 56 C, 1 min; 72 C, 1 min; 25 cycles.
To detect Cre-induced recombinants, primers ES-1 and ID-3 were used
(Fig. 1B
). The cycles are: 94 C, 1 min; 56 C, 1 min; 72 C, 1.5 min; 25
cycles.
To detect the presence of the Cre-coding sequence in mouse genome, PCR
was performed with primers Cre-5 and Cre-3, which amplify a 0.6-kb
fragment of the cre gene. The cycles are: 94 C, 1 min; 67 C,
1 min; 72 C, 1 min; 25 cycles. The primer sequences are (5' to 3'):
IA-8: AGTGATAGGTCACAAAGTTCC
IA-6: AAACCACACTGCTCGACATTG
ID-3: CACTAAGGAGTCTGTATTTGGACC
ES-1: AGCCTCTCAACTAAGACAATA
Cre-5: AATGCTTCTGTCCGTTTGCCGGT
Cre-3: CCAGGCTAAGTGCCTTCTCTACA
RNA Preparation, Northern Blot, and RNase Protection Assay
IGF-I gene expression in adult liver was studied by Northern
blot analysis (30). Briefly, 1015 µg total RNA, prepared using
RNAzol B (Tel-Test Inc, Friendswood, TX), were electrophoresed on a 1%
agarose gel containing 5% formaldehyde, transferred to a
maximum-strength Nytran membrane (Schleicher & Schuell), and hybridized
to a 32P-labeled-antisense riboprobe synthesized by T7 RNA
polymerase using BamHI-linearized template DNA pMI-4 (exon
4) (Fig. 1A
) (27). The blot was washed and exposed to X-Omat AR film at
-70 C, and the specific bands of expected sizes were illustrated. To
demonstrate equal loading of total RNA on the gel, the blots were
stripped and reprobed with a 32P-labeled ß-actin
riboprobe (Ambion, Austin, TX). The abundance of the hybridization
signals was analyzed by PhosphorImager 400E and corrected by the amount
of ß- actin mRNA.
In other tissues that express low levels of IGF-I, RNase protection
assay was performed (31). Briefly, 50 µg total RNA were hybridized to
the 32P-riboprobes for exon 4 and 18S rRNA (Ambion)
overnight at 45 C, treated with RNase A, RNase T1, proteinase K, and
phenol/chloroform, and precipitated. Protected probes were denatured,
electrophoresed on an 8% polyacrylamide gel, and exposed to X-Omat AR
film for 12 days. The protected bands corresponding to IGF-I mRNA and
18S rRNA were scanned in an Agfa Arcus II scanner, and densitometric
analysis was performed using the MacBAS v2.31 program (Fuji Photo Film
Co., Tokyo, Japan). The level of IGF-I mRNA was corrected by the
amount of 18S rRNA and expressed as relative abundance to wild-type
control samples.
Growth Rate, Serum IGF-I Concentration, and Statistical
Analysis
The growth rate of mice with mutant igf-1 was
determined by measuring the body weight at 3 weeks (when animals were
weaned and ear tags were applied) and 6 weeks after birth. Animals are
grouped according to their igf-1 dosage derived from their
genotype (wild-type or igf-1/flox) and the extent of
Cre-induced recombination, determined by PCR, Southern blots, and
densitometry.
For serum IGF-I determination, 6-week-old mice were fasted overnight
and killed by decapitation and blood was collected. The serum was
extracted, to remove IGF-I binding proteins, and assayed by RIA using a
kit purchased from Diagnostic Systems Laboratories, Inc. (Webster, TX)
(32). The antibody used is specific for rat IGF-I and does not
cross-react with human IGF-I or IGF-II. All manipulations were approved
by the Animal Care and Use Committee of the National Institute of
Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD.
Statistical analysis was performed using Statistica Software Package
(StatSoft, Inc., Tulsa, OK). Levels of IGF-I mRNA, serum IGF-I, and
igf-1 gene dosage were entered into a forward stepwise
multiple regression analysis to determine their independent
relationship to the body weight.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Drs. H. Gu and K. Rajewsky for
providing the plasmids pGH-1 and pMC-Cre, Dr. Peter Rotwein for
providing mouse IGF-I genomic constructs
19 and pMI-4, Dr. R.
D. Palmiter for the mouse IGF-I genomic library
7 and Drs. A. Koval,
A. Butler, and C. Hernandez-Sanchez for DNA constructs, Cre probe, and
RNase protection assay, respectively, and V. Blakesley and Marc Reitman
for helpful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Derek LeRoith, M.D., Ph.D., Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Room 8S235A, Building 10, Bethesda Maryland 20892-1770. E-mail: derek{at}helix.nih.gov
J.-L. Liu is supported by a fellowship award from Medical Research
Council of Canada.
Received for publication February 25, 1998.
Revision received April 10, 1998.
Accepted for publication May 12, 1998.
 |
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