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Molecular and Cellular Biology, October 2000, p. 7292-7299, Vol. 20, No. 19
Laboratory of Molecular Pathology, Institute
of Molecular Biology, Academia Sinica, Taipei 115, Taiwan,1 and Eukaryotic
Transcriptional Regulation Section, Regulation of Cell Growth
Laboratory, National Cancer Institute-Frederick Cancer Research and
Development Center, Frederick, Maryland 217022
Received 10 April 2000/Returned for modification 6 June
2000/Accepted 21 June 2000
Knockout of C/EBP CCAAT/enhancer-binding proteins
(C/EBPs) are transcriptional regulators of the basic leucine zipper
family. Members of the C/EBP family (C/EBP Members of the C/EBP family also work in conjunction with each other or
in sequence to support development of a tissue and to maintain its
functions. For example, C/EBP In this study, we used a gene replacement strategy to generate a viable
and fertile C/EBP Generation of C/ebp
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
C/EBP
, When Expressed from the C/ebp
Gene Locus,
Can Functionally Replace C/EBP in Liver but Not in
Adipose Tissue
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
causes a severe loss of liver function and,
subsequently, neonatal lethality in mice. By using a gene replacement approach, we generated a new C/EBP-null mouse strain in which C/EBP
, in addition to its own expression,
substituted for C/EBP expression in tissues. The homozygous mutant
mice C/ebp
/ are viable and
fertile and show none of the overt liver abnormalities found in the
previous C/EBP-null mouse line. Levels of hepatic PEPCK mRNA are not
different between C/ebp/ and wild-type
mice. However, despite their normal growth rate, C/ebp/ mice have markedly reduced fat
storage in their white adipose tissue (WAT). Expression of two
adipocyte-specific factors, adipsin and leptin, is significantly
reduced in the WAT of C/ebp/
mice. In addition, expression of the non-adipocyte-specific genes for
transferrin and cysteine dioxygenase is reduced in WAT but not in
liver. Our study demonstrates that when expressed from the
C/ebp gene locus, C/EBP can act for C/EBP to
maintain liver functions during development. Moreover, our studies with
the C/ebp/ mice provide new insights
into the nonredundant functions of C/EBP and C/EBP on gene
regulation in WAT.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, C/EBP, Ig/EBP,
C/EBP
, and C/EBP
) recognize a common DNA-binding sequence
and display similar dimerization specificities (4, 9,
24). Although tissue expression patterns of these members often
overlap, recent studies using gene knockout analysis of C/EBPs in mice
revealed that C/EBPs differ significantly in their physiological
functions and in their downstream target genes. For example, mice
lacking C/EBP die shortly after birth due to severe
hypoglycemia and the absence of glycogen storage in liver
(23), whereas knockout of C/EBP causes defects in female
reproduction (18).
, -, and - are expressed at
defined times during adipogenesis, and each has a crucial role in
adipocyte development (4, 19, 25). The expression of
C/EBP and - precedes C/EBP and peroxisome proliferator activated receptor 
(PPAR) in adipogenesis, so it is believed that C/EBP and - are important in early adipocyte
differentiation as well as in activating expression of
C/EBP and PPAR. In contrast, C/EBP seems to play a critical
role in the later stage of adipogenesis by maintaining a differentiated
adipocyte phenotype, such as fat storage (5, 6).
-null mouse line in which C/EBP substitutes for
C/EBP in tissues during development. We show that C/EBP functions
for C/EBP in liver to maintain normal blood glucose levels but does
not act for C/EBP to regulate fat storage in white adipose tissue
(WAT). We also show that in WAT, C/EBP does not substitute for
C/EBP to stimulate expression of several factors, including adipsin,
leptin, and transferrin. These findings provide new insights into
nonredundant functions of C/EBP and - isoforms.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ mice.
The C/ebp genomic fragment used for preparing the
gene-targeting construct was described earlier (12). In this
targeting construct, the entire protein-coding region of
C/ebp (1,188 bp, from the start to stop codons) was
deleted and replaced with a 831-bp DNA fragment containing the mouse
C/EBP protein-coding region (Fig. 1).
The loxP-PGK.neo-loxP expression cassette was then inserted
into an SpeI site 159 bp downstream of the stop codon TAG.
The targeting vector contained 4.1 kb of homologous DNA upstream of the
C/EBP start codon and 3.8 kb of homologous DNA downstream of the
loxP-PGK.neo-loxP cassette.
View larger version (26K):
[in a new window]
FIG. 1.
Targeted modification of the C/ebp gene
locus. (A) C/ebp gene (top), targeted allele (middle),
and the expected Cre-loxP-mediated removal of the
PGK.neo transgene (bottom). (B and C) Southern blot analysis
of representative F2 (before crossing with the
EIIa-cre mice) mouse tail biopsies. Tail DNA was digested
with HindIII (B) and with HindIII and
BamHI (C) and probed with the probes shown in panel A. (D)
Southern blot analysis of representative F2 mouse tail
biopsies after crossing with the EIIa-cre mice. DNA was
digested with HindIII and probed with the probes shown
in panel A. +, wild-type allele; , targeted C/ebp
allele.
allele, termed C/ebp/+ (+, wild-type allele; , mutant
allele), were interbred to generate homozygous
C/ebp/ mice.
Diet experiments.
All mice used in this study were
F3 siblings derived from interbreeding the F2
C/ebp/+ mice. Mice were kept in a sterile
microisolator and were observed closely throughout the experiment. For
measuring the food intake, three mice of each sex and of each genotype
were placed separately in microisolators 2.5 weeks after birth. Fresh
food comprising a regular diet for mice (LabDiet, Richmond, Ind.) was
provided daily and the amount of food consumed was recorded daily. For the fasting experiment, 5-week-old mice were kept in a sterile microisolator and were provided with only sterile water for 24 h.
Southern blot analysis.
Isolated ES cell DNA or mouse tail
DNA was digested with the appropriate restriction enzyme,
electrophoresed in a 0.5% agarose gel, transferred to a nylon membrane
(GeneScreen Plus; Dupont), and hybridized with probes derived from the
C/ebp or C/ebp gene as indicated in Fig.
1A.
Histological analysis. Fat pads and livers were fixed in 4% phosphate-buffered saline-buffered paraformaldehyde solution for embedding in paraffin or were embedded immediately in tissue-freezing medium (OCT; Tissue-Trek, Miles, Inc.) and frozen in liquid nitrogen. Sections of 5 µm (for paraffin-embedded tissues) or 10 µm (for frozen tissues) were cut and mounted on silanized slides. The frozen tissue sections were stained with oil red O and counter-stained with hematoxylin as previously described (22). The paraffin tissue sections were stained with hematoxylin and eosin solutions (H&E staining).
Analyses of serum chemistries and levels of leptin and insulin. Mouse sera taken from various developmental stages were analyzed in an autodry chemical analyzer (SP4410; Spotchem) to monitor levels of serum glucose and other blood chemistries. Serum leptin and insulin levels were assayed with the respective kits (Crystal Chem, Inc., Chicago, Ill.) based on enzyme-linked immunosorbent assay according to the manufacturer's instructions.
Hemolytic assay. Serum factor D activity was monitored by hemolytic assay using rabbit erythrocytes and factor D-depleted human serum (1). In a total assay volume of 150 µl, 25 µl of mouse serum and 25 µl of factor D-depleted human serum were added to rabbit erythrocytes (107 cells in 100 µl of 5 mM Veronal-buffered saline-12 mM MgCl2, pH 7.4). After incubation at 37°C for 1 h, the hemolytic activity was determined, and the activity was expressed as a percentage of the activity in a lysed control (1).
LPS treatment.
Four-week-old
C/ebp/ mice were injected
intraperitoneally with 5 mg of lipopolysaccharide (LPS) per kg of body
weight to induce a generalized inflammatory response. Twelve hours
after the injection, livers were removed and snap-frozen in liquid
nitrogen for later RNA extraction.
RNA extraction and Northern blot analysis. Frozen mouse tissues were homogenized in TRIzol RNA reagent (GIBCO-BRL), and total RNAs were isolated according to the manufacturer's protocol. Total RNA (10 µg) was denatured, electrophoresed, transferred to a nylon membrane, and probed with cDNA probes using standard protocols.
Western blot analysis.
Tissues were homogenized in a 10×
volume of 10 mM Tris-HCl (pH 7.0)-1% Triton X-100 containing the
protease inhibitors phenylmethylsulfonyl fluoride and pepain. Fifty
micrograms of total protein was electrophoresed in a 10% denaturing
polyacrylamide-bis gel and transferred to a nitrocellulose membrane.
The membrane was blocked in phosphate-buffered saline containing 5%
(wt/vol) nonfat milk and 0.2% Tween 20, incubated with the primary
rabbit immunoglobulin G against mouse C/EBP (Santa Cruz, Santa Cruz,
Calif.) and developed by using peroxidase-labeled goat anti-rabbit
immunoglobulin G (Santa Cruz).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Generation of C/EBP knockin homozygous
C/ebp/ mice.
The C/EBP
knockin targeting vector was constructed to replace the entire
protein-coding region of C/ebp with that of
C/ebp in the C/ebp gene locus (Fig. 1A). A
loxP-PGK.neo-loxP cassette was inserted downstream of the
stop codon to serve as the targeting selection marker. After
transfection of the linearized targeting vector DNA into ES cells, 1%
of the G418-resistant ES clones were found to carry a mutant
C/ebp allele in which the protein coding region of
C/ebp was deleted and replaced with that of
C/ebp (Fig. 1A). Mice having germ line transmission of
the mutant allele C/ebp/+ were produced.
The C/ebp/+ mice were interbred to
generate homozygous C/ebp/ mice.
Deletion of the C/EBP protein-coding region in the manipulated C/ebp gene locus was confirmed by Southern analysis with
a 1.2-kb DNA probe (probe I) containing the entire protein-coding
region and a 2-kb probe (probe II) covering the full-length C/EBP
transcript (Fig. 1B). In addition, the presence of the C/EBP
protein-coding region in the C/ebp gene locus was
confirmed by probing the C/ebp/ mouse
genome with a C/EBP cDNA probe (probe III) (Fig. 1C). Furthermore, to avoid the potential interference of the
PGK.neo marker gene on the expression of C/EBP
mRNA (the transcript encoded by the mutant allele),
C/ebp/ mice were then bred with a
cre transgenic mouse line to remove the PGK.neo
gene from the targeted C/ebp locus (11). The
resulting F1 mice heterozygous for the
C/ebp allele and lacking the marker gene
were selected for interbreeding to yield F2
C/ebp/ mice (Fig. 1D). The absence of
the C/EBP mRNA and the presence of C/EBP
mRNA (1.8 kb) in C/ebp/ mice
were further confirmed by probing total RNAs from various tissues with
the respective cDNA probes (Fig.
2 and
3).
|
|
Normal growth and hepatic functions in
C/ebp/ mice.
C/ebp/ mice were viable and grossly
normal. C/ebp/ mice did not differ from
their wild-type littermates in their growth rate as measured by body
weight gain (Fig. 1A). Both sexes of
C/ebp+/+ mice are fertile. Liver
dysfunction and other reported abnormalities associated with the
previous knockout of C/EBP (3, 12, 23), such as severe
hypoglycemia at birth, were not found in
C/ebp/ mice.
C/ebp/ mice had normal blood glucose
levels and other blood chemistry, such as albumin (Table
1), indicating that liver functions in C/ebp/ mice are not defective. In
addition, histological analysis of livers from
C/ebp/ mice did not suggest abnormality
(Fig. 2B).
|
/
mice lack C/EBP but have a concomitant gain of C/EBP expressed
from the C/ebp gene locus (shown as C/EBP) (Fig.
2C). The expression pattern of C/EBP in
C/ebp/ mice did not differ from that of
C/EBP in wild-type littermates, indicating that the expression
activity of the C/ebp gene locus was not affected by our
gene replacement approach. Similarly, the mRNA levels of
C/EBP expressed from its own gene locus were not altered by the
presence of C/EBP (Fig. 2C). Furthermore, the levels of hepatic
C/EBP protein were correlated with the mRNA levels
(C/EBP or C/EBP) in each genotype at the embryo stage (day
18), but at the age of 7 weeks, the levels of C/EBP protein in liver
or WAT were not different between genotypes (Fig. 2D).
The previously described C/EBP-null mice died shortly after
birth, possibly due to severe hypoglycemia and lack of glycogen storage in liver (12, 23). The molecular mechanism that
caused neonatal death might have involved a defect in gene
expression of phosphoenolpyruvate carboxykinase (PEPCK)
(23), a key enzyme in gluconeogenesis. We therefore examined
the PEPCK mRNA levels in livers of
C/ebp/ mice during development. As
expected, PEPCK mRNA was present in the livers of
C/ebp/ mice, and the timing and levels
of its expression were similar to those of wild-type mice (Fig. 2C).
This indicates that the PEPCK gene expression is not affected in the
livers of C/ebp/ mice. To further
support this observation, C/ebp/ mice
were subjected to fasting. After fasting for 24 h,
C/ebp/ mice did not appear different
from their wild-type littermates in viability and mobility, and both
kinds of mice developed a similar degree of hypoglycemia (Table 1).
We next examined the acute-phase response in livers of
C/ebp/ mice. C/EBP was previously
found to be absolutely required for the acute-phase response in
neonatal mice (3). However, in response to LPS treatment,
C/ebp/ mice did not differ from
wild-type mice in the up-regulated expressions of hepatic acute-phase
responsive genes, such as haptoglobin and serum amyloid A (Fig.
2E), suggesting that the acute-phase response is not affected in
livers of C/ebp/ mice.
Reduced fat storage in WAT of
C/ebp/ mice.
Although
C/ebp/ and wild-type mice had similar
body weights, the size and appearance of their WAT were strikingly
different (Fig. 3A). WAT in C/ebp/ mice
appeared small and yellowish, whereas WAT in
C/ebp/+ and wild-type littermates was
significantly enlarged and appeared whitish (Fig. 3A, panels a and b).
Nuclear DNA contents of the epididymal fat pads were not different
between C/ebp/ mice and their wild-type
littermates (data not shown). This suggests that the reduced fat mass
in C/ebp/ mice is not due to a
reduction in fat cell numbers. On the other hand, histological analysis
showed that the size of adipocytes and the degree of lipid accumulation
were markedly reduced in WAT of C/ebp/
mice (Fig. 3A, panels c through f), indicating that the lipid accumulation in WAT of C/ebp/ mice is
inhibited. By contrast, C/ebp/ mice had
60% more brown adipose tissue (BAT) than their wild-type littermates
(Fig. 3B, panels a and b), and the adipocytes in their BAT appeared
more vacuolated, presumably containing more lipid droplets (Fig. 3B,
panels c and d).
/ mice is not due to a decrease of
lipid supply in circulation. As shown in Table 1, the levels of serum
triacylglyceride were not different between C/ebp/ and wild-type mice. In addition,
despite their lipodystrophic status,
C/ebp/ mice did not develop fatty
liver as analyzed by oil red staining of frozen liver sections (data
not shown).
Reduced mRNA levels of leptin and adipsin in WAT of
C/ebp/ mice.
To understand
the molecular mechanism underlying the resistance of fat accumulation
in WAT of C/ebp/ mice, we first
examined the expression of numerous genes whose functions are related
to adipocyte differentiation and maturation. The mRNA levels
of C/EBP expressed from its endogenous gene locus (as a 1.5-kb
transcript) appeared to be up-regulated in WAT of C/ebp+/+ mice (Fig. 3C). The levels of
C/EBP expressed from the C/ebp gene locus (as a 1.8-kb
transcript) were similar to those from the C/ebp gene
locus. mRNA levels of other transcription regulators involved
in adipocyte development, such as PPAR and adipocyte determination
and differentiation-dependent factor (ADD1, also called SREBP-1), were
not different in WAT of C/ebp/ and
wild-type mice. Similarly, aP2 and glucose transporter IV that are
expressed in mature adipocytes were not affected in WAT of
C/ebp/ mice. Expression of enzymes
involved in fatty acid synthesis, such as fatty acid synthetase and
stearyl-coenzyme A-desaturase, were not significantly different between
wild-type and C/ebp/ mice. These
results indicate that the expression of the above factors important in
adipogenesis is not inhibited in WAT of
C/ebp/ mice. However, mRNA
levels encoding two factors expressed specifically in mature
adipocytes, adipsin (factor D) and leptin (product of the ob
gene), were significantly reduced in WAT of
C/ebp/ mice (Fig. 3C). Interestingly,
unlike expression of adipsin, whose mRNA level was markedly
reduced in both WAT and BAT, leptin expression was not significantly
affected in BAT of C/ebp/ mice (Fig.
3D). The uncoupling protein 1 mRNA levels were not affected
in BAT of C/ebp/ mice (Fig. 3D).
/ mice were
reduced to reflect their mRNA levels in WAT, a
hemolysis assay was carried out to determine the adipsin activity
in sera of C/ebp/ mice. Indeed,
hemolytic activity in sera of C/ebp/
mice was reduced to 50% of that in sera of wild-type mice (Fig. 4A). Similarly, based on an enzyme-linked
immunosorbent assay, serum leptin levels in
C/ebp/ mice were reduced to 60% of the
normal level (Fig. 4A). On the other hand, serum insulin levels in
C/ebp/ mice were not different from
those in wild-type littermates (Fig. 4A). Despite their normal body
weight, C/ebp/ mice consumed more food
(on average, 9% more) than wild-type mice, as revealed by measuring
their food intake for 6 consecutive weeks (Fig. 4B). Since leptin has
an inhibitory effect on food intake, this moderately higher food
consumption by C/ebp/ mice might be
associated with the decreased leptin levels in circulation.
|
/ mice is
associated with the lack of C/EBP or with the gain of C/EBP,
C/ebp/ mice were bred with the C/EBP
knockout heterozygotes generated by the Cre-loxP system
(12). Mice carrying one allele of
C/ebp and one allele of the
Cre-loxP-mediated C/EBP deletion (designated C/ebp/
mice) were selected and
compared to the C/ebp/+ mice, in which a
wild-type C/ebp allele remained. The morphology and
histology of WAT in C/ebp/
mice were similar to those in C/ebp/
mice, while the WAT of C/ebp/+ mice was
not different from that of wild-type mice (data not shown). Similarly,
the levels of adipsin and leptin mRNA were reduced
significantly in WAT of C/ebp/ mice but
not in WAT of C/ebp/+ mice (Fig. 3E).
These results indicate that C/EBP is required to maintain
normal expression of adipsin and leptin genes and that C/EBP,
regardless of its level of expression, cannot substitute for C/EBP
for this function in WAT.
WAT-specific reduction of CDO and transferrin gene expression.
Adipsin and leptin are adipocyte-specific factors (5). To
further elucidate the nonredundant function of C/EBP in
adipose tissues, we examined the expression of other genes whose
expression is not restricted to adipocytes and whose expression is
regulated by C/EBP proteins. Transferrin and cysteine dioxygenase
(CDO) are expressed at a high level in liver, and both are expressed at
a low level in other tissues, including adipose tissues (10, 20). In addition, C/EBP proteins play an important role in
regulating their expression (16, 20, 26). As shown in Fig.
5, mRNA levels for both CDO and
transferrin were not reduced in liver but were significantly
decreased in WAT. Again, this result indicates that C/EBP can
substitute for C/EBP in regulating CDO and transferrin gene
expression in liver but not in WAT.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
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Our approach of knocking C/ebp into the
C/ebp gene locus allows C/EBP to be expressed from the
endogenous C/ebp gene locus to replace C/EBP in
tissues during development. The resulting homozygous mutant mice, which
are C/ebp/, lack C/EBP but have a
concomitant gain of C/EBP in tissues. C/ebp/ mice are viable and fertile. The
neonatal lethality and other adverse physiological effects that were
associated with the knockout of C/EBP are overcome in
C/ebp/ mice. However,
C/ebp/ mice have a significant
reduction in their fat accumulation and gene expression in WAT, a
defect that is not overcome by our gene replacement approach, thus
providing a unique system for studying in detail the regulatory role of
C/EBP in maintaining fat cell functions.
In C/ebp/ mice, during embryonic and
early postnatal developments the levels of hepatic C/EBP
mRNA expressed from the C/ebp allele appear to
be significantly higher than those of C/EBP mRNA expressed
from its endogenous allele. In the earlier C/EBP knockout studies
(12, 23), the normal expression of C/EBP was not able to
compensate for C/EBP deficiency and thus overcome those adverse
physiological effects in liver. It is therefore possible that C/EBP,
during the early developmental stage, is not expressed at a level
sufficient to compensate for C/EBP deficiency in the livers of the
knockout mice. In contrast, in C/ebp/
mice, the C/EBP insufficiency is overcome due to the higher expression from the C/ebp allele. Nevertheless, the
mechanism of how C/EBP functions for C/EBP in the livers of
C/ebp/ mice awaits further study.
C/EBP is required for adipogenesis of both WAT and BAT (4,
25). However, its regulation in maintaining mature fat cells and
fat cell function is not yet fully understood, especially in adipose
tissues in animals. Adipsin is a serine protease involved in generating
the active complement C3a, also known as acylation-stimulating protein
(15). Acylation-stimulating protein stimulates triglyceride synthesis in adipocytes (2). Therefore, the decrease of
adipsin production might be responsible in part for the defect of
lipid accumulation in WAT of C/ebp/
mice. However, the molecular mechanism underlying the prevention of fat
accumulation in WAT C/ebp/ mice awaits
further elucidation.
Previously, leptin expression was found to be modulated by
C/EBP in humans (14), whereas the regulatory role of
C/EBP on adipsin gene expression is not yet clear. The reduced
expression of both adipsin and leptin genes in the WAT of
C/ebp/ mice but not in the WAT of
C/ebp/+ mice indicates that C/EBP is
the major trans-activator for both genes in mice. Although
C/EBP appears to function for C/EBP in maintaining the expression
of hepatic PEPCK, it does not substitute for C/EBP to activate
adipsin and leptin gene expressions in WAT. On the other hand, the
expression of other C/EBP-regulated genes, such as those for SREBP
and aP2 (5, 13), whose functions are also related to the
adipocyte maturation, were not affected in the WAT of
C/ebp/ mice. This indicates a
gene-dependent mechanism that determines whether C/EBP can
substitute for C/EBP in regulating transcription of a gene.
Similarly, the finding that the expression of the transferrin and CDO
genes was affected in WAT but not in liver suggests a tissue-specific
mechanism that determines whether C/EBP can substitute for
C/EBP in regulating expression of certain genes in a tissue. Taken together, our studies with the
C/ebp/ mice indicate that the gene
regulation by C/EBP proteins likely involves a complex mechanism that
is tissue and target gene dependent. Further studies with
C/ebp/ mice should provide new insights
into the mechanism of the functional specificity of C/EBP on
regulating gene expression.
Adipose tissues play an important role in regulating energy balance in
mammals (17). Obesity is a disorder of energy balance and is
a significant risk factor for many serious illnesses, such as heart
disease, arthritis, and diabetes. C/EBP plays a critical role in
lipid accumulation in adipose tissues. Therefore, understanding the
C/EBP regulatory mechanism of fat storage in adipose tissues might
ultimately lead to development of therapeutic approaches to preventing
the onset of obesity and its associated pathologies. The lean
C/ebp/ mice provide an excellent system
to further uncover the mechanism and to explore therapeutic strategies.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan. Phone: 886-2-26517983. Fax: 886-2-26517983. E-mail: mbying{at}ccvax.sinica.edu.tw.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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