From the Departments of Cell Biology and Medicine, Baylor College of Medicine, Houston, Texas 77030
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
Conventional knockout of the microsomal
triglyceride transfer protein large subunit ( Abetalipoproteinemia is an autosomal recessive disorder
characterized by the almost complete absence of circulating
apolipoprotein (apo)1 B-containing lipoproteins (1). In
addition to the lipoprotein abnormalities, patients with abetalipoproteinemia also suffer from
severe anemia with acanthocytosis, fat malabsorption, and progressive
neurodegenerative syndromes. Abetalipoproteinemia is caused by
mutations in the gene for the large subunit of the microsomal
triglyceride transfer protein (MTP) (2-5).
MTP is an integral protein in the endoplasmic reticulum. It was
originally described as a protein that accelerates the transport of
lipids (triglycerides, cholesteryl ester, and phosphatidylcholine) between synthetic membranes (6). MTP is a heterodimer composed of a
97-kDa subunit (designated Targeting Vector Construction--
A mouse 129 strain Generation of Germ Line Chimera--
An R1 ES cell line was
obtained from Dr. Andras Nagy at the University of Toronto. The cells
were expanded to passage 14 and used to generate knockout mice as
described previously (17). Three positive ES cell clones were injected
into blastocysts of C57BL/6J, and chimeric mice were obtained. They
were mated with C57BL/6J mice, and germ line transmission was confirmed
by Southern blot analysis. Most experiments were conducted on siblings
of F2 or F3 mice. The mice were weaned at 21 days and fed either a chow
diet (Teklad 7001) or a high fat cholesterol diet (ICN 960393)
containing 1.23% cholesterol and 17.84% fat.
RNase Protection Assay--
Total RNA from the liver and small
intestine were isolated using TriZOL (Life Technologies, Inc.). A
polymerase chain reaction product containing the first 350 base pairs
of the mouse Western Blot Analysis--
Liver and small intestine were
removed, and total proteins were extracted by a Wheaton No. 6 hand-held
homogenizer in buffer B (10 mM Hepes pH 7.4, 2.5 mM sodium phosphate monobasic, 250 mM sucrose,
5 mM EDTA, and 0.5 mM phenylmethylsulfonyl
fluoride). Microsomal fractions were isolated from extracts of liver
and small intestine by ultracentrifugation at 100,000 × g for 1 h. The pellet was resuspended in buffer B, and
the protein concentration was determined by a DC Protein Assay Kit from
Bio-Rad. Ten µg of microsomal protein was loaded onto a 4-15%
gradient polyacrylamide gel, and a rabbit anti-bovine Lipid Transfer Activity Assay--
One hundred µg of
microsomal protein was used to measure the MTP-mediated
[14C]triglyceride transfer activity as described in Jamil
et al. (18).
FPLC Analysis of Plasma Lipids--
Blood was collected after a
4-5-h fast, and total plasma cholesterol and triglyceride
concentrations were measured by enzymatic kits (Sigma Diagnostics). Two
hundred µl of pooled plasma from 3-4 animals was loaded on a FPLC
system with 2 Superose 6 columns connected in series (Pharmacia FPLC
System, Amersham Pharmacia Biotech). 0.5-ml fractions were collected
using an elution buffer (1 mM EDTA, 154 mM
NaCl, and 0.02% NaN3, pH 8.2) (19). Lipid contents in
individual fractions were determined with enzymatic assay kits (Sigma
Diagnostics). Very low density (VLDL), intermediate density (IDL), low
density (LDL), and high density (HDL) lipoproteins are well separated
by this technique (19).
Recombinant Cre-Adenovirus Treatment of Floxed Primary Hepatocyte Culture--
Hepatocytes were isolated from
AdCre1-treated mice by White's method (22) except that the perfusion
was done on an anesthetized animal through the portal vein instead of
on excised liver. A pulse-chase experiment using
[35S]methionine was done to determine apoB degradation
and secretion in these cells (23).
Triglyceride Secretion Rate--
Triglyceride secretion in
vivo was quantified by the intravenous administration of Triton
WR1339 (24). Plasma triglycerides were measured at 1, 2, 3, and 4 h after treatment; the triglyceride accumulation remained linear during
this time.
As reported by Raabe et al. (15), we produced MTP) gene is embryonic
lethal in the homozygous state in mice. We have produced a conditional
MTP knockout mouse by inserting loxP sequences flanking exons 5 and 6 by gene targeting. Homozygous floxed mice were born live with normal
plasma lipids. Intravenous injection of an adenovirus harboring Cre
recombinase (AdCre1) produced deletion of exons 5 and 6 and disappearance of
MTP mRNA and immunoreactive protein in a
liver-specific manner. There was also disappearance of plasma
apolipoprotein (apo) B-100 and marked reduction in apoB-48 levels.
Wild-type mice showed no response, and heterozygous mice, an
intermediate response, to AdCre1. Wild-type mice doubled their plasma
cholesterol level following a high cholesterol diet. This
hypercholesterolemia was abolished in AdCre1-treated
MTP
/
mice, the result of a complete absence of
very low/intermediate/low density lipoproteins and a slight reduction
in high density lipoprotein. Heterozygous mice showed an intermediate
lipoprotein phenotype. The rate of accumulation of plasma triglyceride
following Triton WR1339 treatment in
MTP
/
mice was
<10% that in wild-type animals, indicating a failure of
triglyceride-rich lipoprotein production. Pulse-chase experiments using
hepatocytes isolated from wild-type and
MTP
/
mice
revealed a failure of apoB secretion in
MTP
/
animals. Therefore, the liver-specific inactivation of the
MTP gene
completely abrogates apoB-100 and very low/intermediate/low density
lipoprotein production. These conditional knockout mice are a useful
in vivo model for studying the role of MTP in apoB biosynthesis and the biogenesis of apoB-containing lipoproteins.
INTRODUCTION
Top
Abstract
Introduction
References
MTP) and a 58-kDa subunit, which turned
out to be protein-disulfide isomerase (7). ApoB-100 and apoB-48
secretion from the cell requires the presence of functional MTP. In its
absence, all the newly synthesized intracellular apoB is degraded, and
little of it is secreted (8, 9). It is believed that MTP facilitates
the stabilization of newly synthesized apoB-100 (10) and apoB-48 (11)
in a narrow "window" shortly after completion of apoB translation
but before the addition of the major amount of lipids to the
lipoprotein particle (reviewed in Refs. 12-14). The pathophysiological
basis of abetalipoproteinemia is postulated to be an almost complete
failure of apoB secretion from the liver and small intestine because of
the absence of functional MTP in affected patients. An animal model
would be valuable in studying the molecular basis of the lipoprotein
abnormalities and elucidating the pathophysiological basis of the other
non-lipoprotein complications found in abetalipoproteinemia.
Unfortunately, the genetic inactivation of the
MTP locus in mice is
embryonic lethal in the homozygous state ((15) and see below). In this
communication, we report the production and characterization of a
viable mouse model of abetalipoproteinemia using a conditional knockout strategy.
MATERIALS AND METHODS
genomic library was purchased (Stratagene) and screened with mouse
MTP cDNA (16). Two overlapping clones encompassing exons 3 to 8 were used to construct two types of targeting vectors. For straight
replacement-type targeting construct, a neo cassette was inserted into
exon 7 of the
MTP gene between a SmaI and a
XhoI site (data available from L. C. upon request). The
conditional targeting construct was designed by inserting a neo-loxP
cassette in the XbaI site of intron 6 and a loxP fragment in
the BamHI site of intron 4 of the
MTP (Fig.
1A). A thymidine kinase cassette was ligated to the 5' end of the construct.
MTP cDNA were cloned into pBluescript KS vector and
used as a probe for RNase protection assay. The authenticity of the
clone was verified by sequencing, and the antisense strand RNA was
transcribed by using MAXIscript (Ambion). The assay was done using the
RPAII kit (Ambion) with 5 µg of total RNA following the instructions of the vendor's manual. A
-actin probe was used as an internal control in the assay.
MTP antiserum
(a gift from Dr. David Gordon of Bristol-Myers Squibb Pharmaceutical
Research Institute) was used for Western blot analysis.
MTP
Mice--
A replication-defective adenovirus containing recombinant
Cre recombinase, AdCre1, was a gift from Dr. Frank Graham (McMaster University, Hamilton, Ontario, Canada) (20). It was amplified in 293 cells and purified as described previously (21). Eight-week-old male
mice were injected with 3 × 109 plaque-forming units
of AdCre1 through a jugular vein. The AdCre1-treated mice were fed
either a normal chow or a high cholesterol diet before and after
injection. At day 10-21 after adenovirus administration, the mice were
sacrificed and studied.
RESULTS AND DISCUSSION
MTP
knockout mice by gene targeting in ES cells and found that inactivation of the
MTP locus in mice is embryonic lethal in the homozygous state.2 We have therefore
produced a conditional knockout construct shown in Fig.
1A. It would insert two loxP
sequences encompassing exons 5 and 6. Deletion of these exons would be
predicted to inactivate the
MTP protein because it causes a shift in
the translation frame of the mRNA if the remaining exons are
correctly spliced into a mutant mRNA. Homologous recombination was
verified by digestion of genomic ES cell DNA with BamHI and
Southern blot analysis using a probe outside the targeting vector (Fig.
1A). The presence of a diagnostic 4-kb fragment instead of
the wild-type 6-kb fragment indicates insertion of the targeted vector
by homologous recombination. 14 of 124 clones (11%) that were G418-
and FIAU-resistant exhibited a pattern consistent with homologous
recombination. 3 of 14 ES cell clones were injected into C57BL/6J
blastocysts, yielding 8 chimeric mice with agouti coat color indicating
essentially 100% contribution of ES cells. Chimeric males were bred
with C57BL/6J females, and germ line transmission was observed in 5%
(2/40) of the progeny by Southern blot analysis of tail DNA (Fig.
1B). Heterozygous and homozygous floxed
MTP
/
mice were obtained by cross-breeding. These
animals were fertile and produced normal-sized litters. The birth
weights, growth, and development of wild-type,
MTP+/
,
and
MTP
/
mice were indistinguishable. The basal
plasma cholesterol and triglyceride levels are similar in the three
types of animals while they were on a regular chow diet (Table
I). Therefore, insertion of the loxP
sequences and the neo cassette did not appear to disrupt the function
of
MTP under basal conditions. Two weeks after these animals were
put on a high cholesterol diet, there was an approximate doubling of
the plasma cholesterol but little change in plasma triglyceride
concentrations. Again, the levels were not significantly different
among the three groups of animals (Table I).
View larger version (20K):
[in a new window]
Fig. 1.
Floxed conditional targeting of the mouse
MTP gene. A, exons 3-8 of the mouse
MTP gene are
indicated by black boxes. A PGKneobpA-lox
cassette (NEO) was inserted into the XbaI site of
intron 6, and another loxP sequence was inserted into a
BamHI site of intron 4. A pCM-TK-poly(A) cassette
(TK) was attached to the 5' end of the targeting construct.
loxP sequences are represented by triangles. Restriction
enzyme sites: B, BamHI; RV,
EcoRV; Xb, XbaI. An EcoRV
and BamHI genomic fragment 3' to the targeting construct
(represented by a horizontal bar under
Probe) was used as a probe for genotyping. The expected size
of the wild-type (6 kb) and the targeted (4 kb) alleles were indicated
as double arrowhead lines.
B, Southern blot analysis of tail DNA of animals of
different genotypes.
Plasma lipids in wild-type and floxed MTP knockout mice
and 4 from
/
; after AdCre1 treatment there were 4 for each genotype.
We examined the effect of the introduction of Cre recombinase on the
MTP gene and its expression in wild-type and knockout animals.
Induction of Cre recombinase expression was effected by the intravenous
administration of a purified recombinant adenovirus carrying the Cre1
recombinase (AdCre1) (20). We removed the liver and small intestine and
extracted DNA, RNA, and microsomes from these tissues 1.5-3 weeks
after AdCre1 treatment. By Southern blot analysis, we found that AdCre1
treatment produced a deletion of the floxed exons in the
MTP gene in
the liver. By EcoRV digestion, hybridization using the probe
shown produced a 6.5-kb band in the intact floxed
MTP gene (Fig.
2). Upon deletion of the floxed portion
of the gene, it produced a 3.5-kb band with the removal of the DNA
between the loxP sequences, which has an EcoRV site in exon
5. When we examined the blot from DNA extracted from the small
intestine, it was evident that the floxed
MTP locus remained intact.
The liver specificity is not unexpected because adenoviral vectors
administered intravenously are taken up preferentially by liver cells
(25). For heterozygous
MTP knockout mice, deletion of the floxed
MTP allele was also detected in the liver but not the small
intestine (data not shown).
|
We next examined liver and small intestine MTP mRNA levels
following AdCre1 administration. We used RNase protection assay with an
MTP cDNA fragment and a
-actin cDNA fragment as internal control. As shown in Fig. 3A,
the
MTP mRNA band is essentially undetectable in the liver of
Cre-treated
MTP
/
mice, whereas the
MTP mRNA
level in treated
MTP+/
mouse liver was only slightly
reduced. In comparison, the concentration of
MTP mRNA in the
small intestine of mice of all three genotypes and in the liver of
wild-type mice was unaffected by AdCre1 administration. Therefore,
there was good correlation between
MTP mRNA expression and the
presence of an undisrupted
MTP gene, and Cre-induced deletion of
exons 5 and 6 led to the absence of detectable
MTP mRNA. Since
the antisense probe used corresponds to a region of the
MTP gene 5'
to the missing exons, these results suggest that in the AdCre1-treated
mouse liver, if the disrupted
MTP gene were transcribed at all, the
RNA transcript was so unstable and its steady-state concentration so
low that it was undetectable by RNase protection.
|
To examine whether these changes at the DNA and mRNA levels are
reflected at the protein level, we isolated microsomes from the liver
and small intestine and performed immunoblot analysis using an MTP
antibody. As shown in Fig. 3B, a
MTP immunoreactive band
was easily detectable in wild-type mouse liver and small intestine,
being slightly more intense in the latter. Following AdCre1 treatment
there was a reduction in intensity of the immunoreactive band in the
liver, but not small intestine, of the heterozygous floxed
MTP+/
mice. In the homozygous floxed
MTP
/
mice, the Cre gene transfer to the liver
completely eliminated the
MTP band. Interestingly, there seemed to
be a concomitant increase in the intensity of the
MTP band in the
small intestine following AdCre1 treatment.
MTP has a relatively
long half-life (4.4 days in HepG2 cells (26)). These results indicate
that in mice, within 10 days of acute interruption of
MTP gene
transcription, there is essentially no immunoreactive
MTP left in
the liver cells.
We determined the MTP activity in the microsomes isolated from
AdCre1-treated wild-type, floxed heterozygous MTP+/
,
and floxed homozygous
MTP
/
animals. The results
shown in Fig. 3C reveal that the MTP triglyceride transfer
activity of microsomes isolated from the liver of heterozygous floxed
MTP+/
(3.7%) mice is reduced to about half that of
the wild-type (7.3%), and the activity of the homozygous floxed
MTP
/
mice is almost down to background level
(1.3%). In contrast, the intestinal MTP triglyceride transfer activity
in all three groups of animals is very similar (7.8%, 8.6%, and
7.7%, respectively, for wild-type, heterozygous, and homozygous
knockout animals). Therefore, in the liver and small intestine there is
good correlation between
MTP protein expression and MTP functional
activity, which suggests that
MTP, and not protein-disulfide
isomerase, is limiting under these conditions.
The plasma lipid levels of the different types of mice before and after
AdCre1 treatment are shown in Table I. Before adenovirus administration, the mice were put on a high cholesterol diet, and their
plasma cholesterol and triglyceride levels were similar in the three
groups of animals. Following AdCre1 injection, there was a significant
reduction in the plasma cholesterol concentration in the homozygous
MTP
/
mice, which was significantly lower than that
in wild-type animals. The cholesterol level in the heterozygous animals
was intermediate between those of wild-type and homozygous knockout
animals. The plasma triglyceride level was not statistically different
between the three groups of animals, although it tended to be lower in the homozygous mice.
Because functional MTP is required for apoB biogenesis (12, 14), we
analyzed the plasma for apoB-100 and apoB-48 expression by immunoblot
analysis. We took plasma from these mice 2 weeks following AdCre1
treatment. In animals that were on regular chow (Fig. 3D,
left panel), there was no difference in wild-type
and heterozygous knockout animals; both had clearly detectable apoB-48, but barely detectable apoB-100. In homozygous knockout animals, apoB-100 was undetectable and apoB-48 was markedly reduced and barely
detectable. In wild-type animals that were fed a high cholesterol diet
(Fig. 3D, right panel), plasma
apoB-100 and apoB-48 were clearly detected on the blot, with apoB-48
being a much more intense band than the apoB-100 band. In comparison,
the AdCre1-treated heterozygous floxed
MTP+/
mice had
mildly reduced apoB-48 and markedly reduced apoB-100 bands. When we
analyzed the plasma from AdCre1-treated homozygous floxed
MTP
/
mice, we found a marked reduction in the
intensity of the apoB-48 band and an almost complete disappearance of
the apoB-100 band. Therefore, specific inactivation of the
MTP locus
in the liver leads to an essentially complete absence of circulating
apoB-100 and a marked decrease in plasma apoB-48. These changes would
be consistent with the annulment of apoB-100 and apoB-48 production by
the liver with the preservation of apoB-48 production in the small
intestine in the AdCre1-treated homozygous floxed
MTP
/
mice.
We next analyzed the plasma lipoproteins in the different groups of
mice by FPLC analysis (Fig. 4). Plasma
was obtained from wild-type, heterozygous floxed
MTP+/
, and homozygous floxed
MTP
/
mice 10 to 21 days following AdCre1 treatment. In chow-fed animals, in
comparison with wild-type, the
MTP
/
mice displayed a
complete absence of the VLDL and IDL/LDL peaks (Fig. 4A,
top panel) and a markedly reduced HDL peak. In
heterozygous animals, only minor reductions were observed in the VLDL
and IDL/LDL regions, with no change in the HDL region compared with
wild-type animals. Among animals that were fed a high cholesterol diet, wild-type mice developed a prominent VLDL peak, a substantial IDL/LDL,
and a prominent HDL peak. In the Cre-treated homozygous floxed
MTP
/
mice, there was essentially a complete absence
of detectable lipoproteins in the VLDL and IDL/LDL fractions. There was
a slight reduction in the HDL peak. Therefore, the markedly reduced
apoB-100 production resulting from the almost complete inhibition of
MTP activity in the
MTP
/
mice was sufficient to
abrogate a VLDL/IDL/LDL response to high cholesterol diet feeding. The
heterozygous
MTP+/
mice, which had intermediate
MTP
mass and MTP activity (Fig. 3), displayed an intermediate lipoprotein
phenotype (Fig. 4A, middle panel). The
plasma lipoprotein triglyceride was entirely in the VLDL region (Fig.
4A, bottom panel) in wild-type
animals. It was essentially abolished in
MTP
/
mice.
|
To explore the mechanism of the marked VLDL deficiency in the
MTP
/
mice, we measured the rate of triglyceride
production in these animals. The intravenous administration of Triton
WR1339 inhibits the catabolism of triglyceride-rich lipoproteins. The
accumulation of plasma triglyceride following Triton treatment reflects
the triglyceride secretion rate from the liver and intestine. Because the animals were on a fat-free diet during this experiment, the secretion came exclusively from the liver. As shown in Fig.
4B, the triglyceride secretion rate in
MTP
/
mice (0.09 ± 0.04 mg/min/100 g) was
reduced to less than one-tenth that in the wild-type controls
(1.02 ± 0.19 mg/min/100 g). The rate in heterozygous knockout
mice was intermediate (0.52 ± 0.07 mg/min/100 g). Thus, the
absence of VLDL/IDL/LDL in the knockout animals was a result of failure
of production, not increased catabolism. Pulse-chase experiments on
cultured hepatocytes isolated from wild-type and homozygous knockout
animals revealed that there was complete failure of secretion of apoB
in
MTP
/
animals, which would account for the absence
of VLDL production in these animals. Albumin production was normal
(Fig. 4C).
In conclusion, we have produced a viable abetalipoproteinemia gene
knockout mouse model using a Cre/loxP strategy. We found that the
liver-specific disruption of the MTP gene was sufficient to
completely abrogate the plasma VLDL/LDL response to a high cholesterol
diet. Because conventional knockout of the
MTP gene is embryonic
lethal in the homozygous state, the conditional knockout mice will be a
valuable model for studying the metabolic defect and pathophysiology of
abetalipoproteinemia as well as the role of MTP in apoB biosynthesis
and the biogenesis of apoB-containing lipoproteins in
vivo.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Frank Graham (McMaster University, Hamilton, Canada) for providing AdCre1, Drs. David Gordon and Haris Jamil (Bristol-Myers Squibb Pharmaceutical Research Institute) for providing antibody to MTP and helpful discussions, Dr. Kazuhiro Oka and Maria Merched-Sauvage for assistance with AdCre1 production, Dr. Hye-Jeong Lee for primary hepatocyte culture, and Sylvia Ledesma for expert secretarial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant HL-16512 (to L. C.) and Fellowship F32-L09738 (to B. H.-J. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by the Karolinska Institute/Baylor College Exchange
Program and by the Henning and Johan Throne-Holsts Foundation.
§ To whom correspondence should be addressed: Baylor College of Medicine, MS112A, One Baylor Plaza, Houston, TX 77030.
2 B. H.-J. Chang, M. Nakamuta, and L. Chan, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
apo, apolipoprotein;
MTP, microsomal triglyceride transfer protein;
MTP, MTP large
subunit;
VLDL, very low density lipoproteins;
IDL, intermediate density
lipoproteins;
LDL, low density lipoproteins;
HDL, high density
lipoproteins;
kb, kilobase(s);
PAGE, polyacrylamide gel
electrophoresis;
FPLC, fast protein liquid chromatography;
FIAU, 1-(2'-deoxy-2'-fluoro-
-D-arabinofuranosyl)-5-iodouracil.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|