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(Received for publication, October 13, 1995; and in revised form, January 12, 1996) From the
The involvement of pancreatic cholesterol esterase (bile
salt-stimulated lipase) in cholesterol absorption through the intestine
has been controversial. We have addressed this issue by using
homologous recombination in embryonic stem cells to produce mice
lacking a functional cholesterol esterase gene. Cholesterol esterase
knockout mice and their wild type counterparts were fed a bolus dose of
[
Cholesterol esterase, also called bile salt-stimulated lipase or
carboxyl ester lipase (abbreviated as CEL), ( While the high concentration of CEL in pancreatic
juice suggests that it may play a role in mediating nutrient
absorption, the precise physiologic function of the enzyme remains
controversial. Early studies with isolated intestinal cells suggested a
role for CEL in dietary cholesterol absorption(2) . However,
subsequent studies yielded contradictory results. For example, using
pancreatic diverted rats, Watt and Simmonds (3) showed normal
absorption and esterification of cholesterol. In contrast, using the
same experimental system, Gallo et al.(4) showed an
80% reduction in cholesterol absorption, which could be restored by
infusion of pancreatic juice containing CEL but not by juice depleted
of the enzyme. Cholesterol absorption has also been studied using a
variety of inhibitors. Bennett Clark and Tercyak (5) demonstrated a reduction in cholesterol transmucosal
transport in rats with inhibited acyl CoA:cholesterol acyltransferase
and normal pancreatic function, which suggested that acyl
CoA:cholesterol acyltransferase, and not CEL, was responsible for this
process. However, using similar inhibitors, Gallo et al. (6) showed no inhibition of cholesterol absorption, which again
suggested the involvement of CEL. In later studies, CEL inhibitors,
such as the phenoxyphenyl carbamates WAY-121,751 and WAY-121,898, were
shown to be effective inhibitors of cholesterol absorption in normal
and cholesterol-fed rats and dogs(7) . Thus, whole animal
studies have not consistently shown the importance of CEL in
cholesterol absorption. The possible role of CEL in mediating
intestinal absorption of cholesterol has also been investigated in
vitro without resolution. Bhat and Brockman (8) showed
that incubation of rat intestinal sacs with cholesterol-containing
micelles in the presence of CEL resulted in a 3-5-fold
enhancement of intracellular cholesterol and cholesteryl ester
accumulation compared with intestinal sacs incubated in the absence of
the enzyme. More recently, Lange and colleagues, using Caco-2 cells as
a model for intestinal epithelium, showed that CEL addition was
necessary for the transfer of exogenous cholesterol to a
``physiologically important pool'' that could be esterified
and assembled into lipoproteins(9) . In contrast to these
results, our laboratory could not demonstrate CEL-mediated uptake of
unesterified cholesterol by Caco-2 cells(10) . Our in vitro data were confirmed and extended in a recent publication by Fisher
and colleagues(11) . Both laboratories reported that the enzyme
was only effective in facilitating cellular uptake of esterified
cholesterol. In an attempt to resolve this controversy, we have used
the approach of gene targeting in embryonic stem (ES) cells to produce
mice lacking in CEL. The CEL(-/-) mice provide a unique in vivo model to assess the physiological function of the bile
salt-stimulated cholesterol esterase.
A 4.7-kb SacI DNA fragment, encoding
sequences from 540 bp upstream of exon 1 to intron 7 of the mouse
cholesterol esterase gene, was subcloned into a similarly-digested
PTZ18U plasmid. A 1.75-kb fragment containing a thymidine kinase
promoter-driven neomycin resistance gene (neo
Figure 1:
Diagram of the mouse cholesterol
esterase gene and targeting construct. Panel A, partial
restriction map and exon/intron arrangement of the mouse CEL gene.
Exons are indicated by boxes. Panel B, construct used
for targeting. neo
The presence of neo
For absorption studies, mice were
housed in metabolic cages where they had free access to food and water.
Animals were allowed to adjust to the cages for at least 24 h before
beginning the test. On the day of the experiment, mice were
administered 50 µl of the test meal by gavage approximately
3-4 h before the beginning of their dark cycle. Feces were
collected for the following 24 h. The samples were homogenized in water
and then extracted with an equal volume of chloroform/methanol (2:1,
v/v). The aqueous phase was re-extracted once with chloroform. The
organic phases from each sample were combined, their volumes were
measured, and an aliquot was used for scintillation counting. Counting
efficiency was calculated using the external standard, channel ratio
method. Cholesterol absorption efficiency, determined as percentage of
administered dose absorbed, was calculated based on the formula
described by Grundy et al.(29) as follows: {1 -
((
The
electroporation of 5.5
Figure 2:
Southern blot analysis of wild type and
CEL gene-targeted ES colonies. A, representative colonies
digested with EcoRI or HindIII and hybridized with
the 5` probe. The wild type allele gives rise to a >30-kb fragment
with EcoRI, while the targeted allele yields a fragment of 6.7
kb due to EcoRI sites present in neo
Site-specific
integration of the targeting DNA at the CEL locus was confirmed by
additional Southern blot analysis with both the 5`- and 3`-flanking
probes. The addition of the 1.75-kb neo Two of the 11 cell lines
with proper CEL gene targeting were used to generate chimeric mice. One
cell line yielded only one chimeric mouse, which was female and had
only
Figure 3:
Southern blot analysis of wild type and
CEL gene-targeted mice. A, tail DNA digested with EcoRI. B, tail DNA digested with NcoI. Wild
type DNA yields a 9.2-kb fragment, while targeted DNA yields a 6.9-kb
fragment due to an NcoI site in the neo
To verify that the gene
targeting abolished expression of CEL protein, pancreatic homogenates
from control, heterozygous, and homozygous CEL gene-targeted mice were
examined for CEL expression using immunoblotting techniques (Fig. 4). The levels of CEL protein in pancreatic extracts of
the heterozygous animals were approximately half those of the wild type
mice. No CEL protein was detected in homogenates of the
CEL(-/-) mice. Furthermore, no CEL-immunoreactive
polypeptides of any size were detected, indicating that no fusion
protein or truncated protein was being produced as a result of the
modified gene. These extracts were also assayed for cholesteryl ester
hydrolytic activity. Table 1shows that cholesteryl oleate
hydrolysis is reduced 98% in the pancreatic extracts of
CEL(-/-) animals.
Figure 4:
Western blot of pancreatic extract from
wild type and CEL gene-targeted mice. Twenty µg of protein from the
100,000
In contrast to the
cholesteryl ester result, when unesterified
[
The presence of cholesteryl ester in the
core of an emulsion particle has been shown to increase the partition
of free cholesterol from the surface to the core(32) . Our
results show that CEL is necessary for digestion of this core
cholesteryl ester. In the absence of CEL, the free cholesterol may
remain sequestered and unabsorbed. To test this possibility, animals
were fed [ Published
literature indicates that dietary cholesterol and biliary cholesterol
may be absorbed from the intestine by different mechanisms (33) . Experiments were undertaken to determine the ability of
wild type and CEL gene-targeted mice to absorb unesterified cholesterol
presented in a vesicular complex with phospholipids, similar to that
present in the biliary tract.
[ The report that phenoxyphenyl carbamate inhibitors of CEL result in
delayed absorption of cholesterol (7) prompted additional
experiments to compare the rate at which the radiolabel from
cholesterol and cholesteryl esters appears in the serum of control and
CEL gene-targeted mice. In these experiments, the amount of
Figure 5:
Appearance of dietary cholesterol in the
circulation of wild type and CEL null mice.
[
To
examine the possibility that CEL plays a role in cholesterol absorption
when mice are fed a high fat, high cholesterol, atherogenic diet, we
studied the absorption of free and esterified cholesterol in wild type
and CEL-null mice fed this diet for 6 weeks. As shown in Table 4,
cholesteryl oleate absorption was reduced in the CEL-null mice, while
free cholesterol was absorbed similarly by the wild type and CEL
gene-targeted mice. The percentage of cholesterol absorbed was
decreased relative to normal diet in both the free cholesterol and
cholesteryl ester experiments due to the high level of cholesterol in
the atherogenic diet. In fact the total mass of absorbed cholesterol is
increased.
The results of the current study show that disruption of the
CEL gene has no significant effect on the ability of mice to absorb
unesterified cholesterol from the gastrointestinal tract. Similar
results were observed regardless of the physical characteristics of the
substrate or the dietary conditions of the animals. Furthermore,
similar results were observed when cholesterol absorption was
determined based on the amount of nonabsorbed cholesterol present in
the feces or on the appearance of the radiolabeled cholesterol in the
serum. These observations demonstrate that CEL is not necessary for
cholesterol flux across the intestinal epithelium. In contrast to its
effect on unesterified cholesterol absorption this study shows that CEL is necessary for intestinal absorption of esterified
cholesterol. However, since cholesteryl esters constitute only a small
fraction of total cholesterol in the diet and are absent from the bile (34) , these results strongly suggest that CEL does not play a
determining role in the absorption of either dietary or biliary
cholesterol. Although CEL does not appear to be essential for
intestinal absorption of unesterified cholesterol, results of this
study demonstrate unequivocally that CEL plays a primary role in
absorption of cholesteryl esters. These results are consistent with in vitro studies from this laboratory and others that showed a
role of CEL in facilitating the uptake of esterified cholesterol but
not unesterified cholesterol by intestinal
cells(10, 11) . Curiously, our fecal sterol
experiments indicate a base-line level of cholesteryl ester absorption
that is independent of CEL (Table 2). However, pancreatic
extracts from CEL(-/-) mice lack significant esterolytic
activity (Table 1). Also, the amount of radiolabel appearing in
the serum of cholesteryl ester-fed, CEL-null mice was not consistent
with this base line (Fig. 5). One explanation for this
discrepancy is that a minor pathway for absorption of these nutrients
exists and that our serum assay was insufficiently sensitive.
Alternatively, some cholesteryl ester may remain associated with cell
membranes or lipid vesicles of the intestinal epithelium due to
hydrophobic interactions. The current study, showing normal
absorption of unesterified cholesterol in CEL-null mice, resolves the
discrepancy in previously published data. Our results are consistent
with those of Watt and Simmonds(3) , which showed that
unesterified cholesterol absorption was independent of pancreatic
proteins. Gallo et al. (4) showed that CEL-depleted
pancreatic juice could not restore cholesterol absorption in
pancreatic-diverted rats; however, intestinal lymph flow in the
CEL-depleted group was severely compromised in those experiments (11) . Our current results are also in agreement with in
vitro data that showed no CEL requirement for unesterified
cholesterol uptake by Caco-2 cells(10, 11) . The
CEL-stimulated uptake in Caco-2 cells reported by Lopez-Candales et
al.(9) , while intriguing, did not reflect physiologically
relevant levels of cholesterol in the gastrointestinal tract. Until
the availability of an animal model lacking in CEL, such as the
gene-targeted mice described here, the most physiologically relevant
experiments regarding the role of this enzyme in cholesterol absorption
were performed by feeding animals CEL inhibitors. Two classes of such
inhibitors, phenoxyphenyl carbamates (7) and the
lipstatins(35) , were reported to reduce cholesterol absorption
in normal and cholesterol-fed animals. However, the lipstatins were
shown to also inhibit pancreatic lipase(35) . Thus, their
inhibitory effect on cholesterol absorption may be related to the
inhibition of lipid emulsion hydrolysis and the release of unesterified
cholesterol for diffusion through the mucosa (36) . In support
of this possibility was the observation that tetrahydrolipstatin had no
effect on intestinal uptake of unesterified cholesterol from
phospholipid-bile salt mixed micelles(35) . Although the
carbamate inhibitors were reported to be more specific for CEL and did
not inhibit the activity of pancreatic lipase or acyl CoA:cholesterol
acyltransferase in vitro, other possible side effects that can
modulate cholesterol absorption in vivo may exist in the
carbamate-treated animals. Although our data show that CEL does not
participate in free cholesterol absorption, the wide range of
absorption values (37-87%) as well as additional studies with
inbred strains of mice support the hypothesis that cholesterol
absorption is regulated by at least one gene. ( Although
CEL does not play a primary role in free cholesterol absorption, its
abundance in the intestinal lumen suggests that it may play a different
role in the absorption of lipid-based nutrients. This enzyme may
complement other lipolytic enzymes to increase the efficiency of
dietary fat absorption by the small intestine. For example, CEL has
been shown to be more efficient than pancreatic lipase in the
hydrolysis of long chain polyenoic fatty
acids(43, 44) . CEL is also capable of hydrolyzing
phospholipids and lysophospholipids (45) and thus may play a
role in the assimilation of dietary phospholipids. More importantly, a
pancreas-derived, vitamin-ester hydrolytic activity has been ascribed
to CEL(46, 47) , suggesting its importance in the
absorption of fat-soluble vitamins, which are primarily esterified in
dietary sources. The presence of CEL in the milk of many mammalian
species has led to the proposal that the milk CEL is critically
important for digestion of milk triglycerides, the major source of
energy in infants, before the maturation of the pancreas(48) .
Finally, in addition to its presence in the gastrointestinal tract, CEL
is also found to be synthesized by the liver and is present in
serum(12, 26, 49, 50, 51) ,
where its level is correlated to that of serum cholesterol and
LDL(52) . Thus, CEL may play a role in the modulation of
lipoprotein structure and metabolism. The knockout mice described in
this report will provide a useful tool to address the role of CEL in
various aspects of lipid absorption and metabolism.
Volume 271,
Number 12,
Issue of March 22, 1996 pp. 7196-7202
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H]cholesterol and a trace amount of
[
-
C]sitosterol by gavage. The ratio of the
two radiolabels excreted in the feces over a 24-h period was found to
be similar in the control and cholesterol esterase-null mice. Similar
results were observed when the radiolabeled sterols were supplied in an
emulsion with phospholipid and triolein or in lipid vesicles with
phosphatidylcholine. Cholesterol absorption results were similar
between the control and cholesterol esterase-null mice regardless of
whether the animals were fed a low fat diet or a high fat/high
cholesterol diet. The rate of [H]cholesterol
appearance in the serum of the gene-targeted mice paralleled that
observed in control animals. In contrast to these results, when
experiments were performed with [H]cholesteryl
oleate instead of [H]cholesterol, a higher amount
of the H radiolabel was found excreted in feces and
dramatically less of the radiolabel was detected in the serum of the
cholesterol esterase-null mice in comparison with that detected in
control animals. Serum cholesterol levels were not significantly
different between control and cholesterol esterase-null mice fed either
control or an atherogenic diet. These results indicate that cholesterol
esterase is responsible for mediating intestinal absorption of
cholesteryl esters but does not play a primary role in free cholesterol
absorption.
)is a lipolytic
enzyme capable of hydrolyzing triacylglycerol, phospholipid,
lysophospholipid, and cholesteryl esters. The enzyme is synthesized in
the acinar cells of the pancreas and is stored in zymogen granules.
Cholesterol esterase is released into the intestinal lumen upon food
ingestion and constitutes 1-2% of total protein in pancreatic
juice(1) .
Cloning of the Mouse Cholesterol Esterase Gene and
Production of the Targeting Vector
A strain 129 mouse genomic
library made in -DASH phage vector was obtained from Dr. Thomas
Doetschman at our institution and used to isolate the mouse CEL gene.
The 2-kb full-length rat cholesterol esterase cDNA (12) was
used as the probe for screening the library. A positive clone that also
hybridized with probes corresponding to both the 5`-flanking region and
the 3`-flanking region of the rat CEL gene was selected for further
characterization. Restriction mapping, Southern hybridization with
various cholesterol esterase cDNA fragments, and partial nucleotide
sequencing were performed to determine the intron and exon locations of
the mouse CEL gene.
) was
isolated from SspI/HincII-digested pMC1Neo
(Stratagene) and subcloned into the unique BalI site in exon 4
of the 4.7-kb SacI clone (Fig. 1). A plasmid containing neo inserted in the same orientation as the CEL
gene was selected for the gene-targeting experiment. After CsCl
purification, the targeting vector was digested with SacI, and
the 6.5-kb DNA fragment containing the disrupted CEL gene sequence was
purified by agarose gel electrophoresis.
(shaded box) was
inserted into the BalI site in exon 4 of the 4.7-kb SacI fragment. Panel C, PstI/SacI
5` probe used for screening ES colonies and mice for homologous
recombination. Panel D, BglII/SalI 3` probe
used for screening ES colonies and mice for homologous recombination.
Restriction enzymes indicated are as follows: BalI (B), EcoRI (E), HindIII (H), NcoI (N), SphI (P), SacI (S), and XbaI (X). Arrows indicate the approximate positions of the primers used for
polymerase chain reaction analysis of mice.
Targeted Disruption of the Cholesterol Esterase Gene in
ES Cells
Gene targeting experiments were performed using the R1
ES cell line derived from the 129 mouse strain by Nagy et al. (13) . Cells were grown and passaged as described(14) .
When ES cells were grown at high densities, the medium was supplemented
with leukemia inhibitory factor (LIF) at 500
units/ml(15, 16, 17) . On the day of the
experiment, 5.5
10
ES cells in 0.5 ml of culture
medium were electroporated in the presence of 3 pmol of the targeting
DNA, using an IBI GeneZapper 450 set to deliver 200 microfarads at 800
V/cm. Surviving cells (50%) were cultured on G418-resistant
feeders (a kind gift from Dr. Tom Doetschman of this institute) in
selection medium containing G418 at 300 µg/ml (added 24 h after
electroporation) for 3 days and 250 µg/ml thereafter. After 7 days,
colonies resistant to G418 selection were picked and expanded
individually in 24-well dishes. After 3 or 4 days of growth,
approximately half of the cells in each colony were used to isolate DNA
while the remaining cells were maintained for colony expansion and
freezing.
Southern Blot Analysis
Colonies were screened for
homologous recombination by Southern blot analysis. Genomic DNA was
prepared from each colony(18) , digested with various
restriction enzymes according to the manufacturer's conditions,
fractionated on 0.7-0.8% agarose gels, and transferred (19) to Nytran Plus membranes (Schleicher & Schuell, Inc.).
Blots were prehybridized, hybridized, and washed according to the
method of Church and Gilbert(20) . Hybridization probes were
purified from low melting agarose gels and labeled to a specific
activity of 1
10
dpm/µg using a random
primer kit from Ambion, Inc.Generation of Chimeric Mice
Mice with targeted
disruption of the CEL gene were produced according to standard
procedures using C57BL/6 blastocysts and (C57BL/6 C3H/HeN) F1
pseudopregnant females as foster mothers(21) . Chimeric mice
were identified by the degree of agouti coat color on the black
background. Male chimeric mice were mated with female Black Swiss mice
(Taconic Farms, Germantown, NY) to test for germ line transmission of
the ES cell genome. Agouti pups carrying the modified allele were
identified by polymerase chain reaction analysis of ear punch DNA.
Heterozygotes from different parents were mated to obtain mice with
homozygous disruption of the cholesterol esterase gene. Mice were
maintained in a temperature and humidity-controlled room with a 12-h
light/dark cycle and were allowed food (Teklad LM485 rodent diet) and
water ad libitum. All animals of the F
generation
were fed this same diet supplemented with retinol and tocopherol
(Sigma) at a dose designed to deliver 10 units/day/mouse of retinol and
0.2 units/day/mouse of tocopherol. These unesterified forms of vitamins
A and E were provided to compensate for any deficiencies in vitamin
ester absorption resulting from the loss of CEL activity. Some animals
were fed an atherogenic diet (TD 88051, Harlan Teklad), which was based
on Purina Mouse Chow 5015 and also contained 7.5% cocoa butter (15.75%
final fat content), 1.25% cholesterol, and 0.5% sodium cholate. This
diet was also supplemented with unesterified vitamins. Animals were fed
the atherogenic diet for at least 6 weeks before analysis.Mouse Genotyping by Polymerase Chain Reaction
Ear
punch tissue was incubated at 55 °C in 25 µl of digestion
buffer (18) and 400 µg/ml proteinase K for at least 4 h.
After digestion, samples were briefly vortexed, the hair was allowed to
settle, and 2 µl of the suspension was diluted into 50 µl of
HO, boiled for 5 min, and immediately placed on ice. One
µl of the boiled sample was used within 4 h for polymerase chain
reaction analysis using the procedure described by Kim and
Smithies(22) . Primer concentrations were each 2.5
µM, nucleotide triphosphates were each 200
µM, and one unit of Taq polymerase (Life
Technologies) was used per reaction. Reactions (30 µl) were
prepared on ice, preheated to 90 °C for 1 min, and subjected to 33
cycles of amplification consisting of 30 s at 94 °C and 2 min at 65
°C. Samples were analyzed on 1.5% agarose gels. was determined using the primers described by
Kim and Smithies(22) , which amplify a 555-bp portion of this
gene. The CEL gene was analyzed with these primers and an additional
set of primers that amplifies exon 4. The upstream CEL primer,
5`-CCCTTTCAGTGTCCCACAACCT-3`, and the downstream CEL primer,
5`-TCACTATTCCCGCTCTTACAGTC-3`, amplify a 244-bp fragment from the wild
type exon 4 but do not amplify the targeted allele because of the
insertion of neo between their cognate sequences.
Identical conditions were used for both primer sets but in separate
reactions. A positive result with the exon 4 primers only was scored as
wild type. Positive results with both sets indicated a heterozygote,
and a positive result with the neo primers only
was scored as a homozygous knockout.Cholesterol Ester Lipase Determination
Levels of
CEL protein in the mouse pancreas were determined by immunoblot assay.
The mice were euthanized, and the abdominal cavities were opened. The
pancreas was removed and homogenized on ice in a solution containing 10
mM sodium phosphate, pH 6.2, 0.1 M NaCl, 1 mM EDTA, 0.02% sodium azide, 1.5% glycerol, and 0.02% soybean trypsin
inhibitor (Sigma). The homogenate was centrifuged at 4 °C for 1 h
at 100,000 g to precipitate particulate fractions.
Twenty-five µg of the 100,000 g supernatant
fraction from each sample was analyzed by electrophoresis on a 10%
SDS-polyacrylamide gel(23) . The electrophoresed samples were
either stained with Coomassie Blue or transferred to nitrocellulose (24) for immunoblotting with affinity-purified rabbit anti-rat
cholesterol esterase and I-labeled anti-rabbit IgG
(Amersham Corp.) as described(25) . CEL activity was determined
as described (26) using 0.1 µg of the above supernatant, 15
mM bile salt, and 10 µM cholesteryl
[
C]oleate (35 µCi/µmol).Cholesterol Absorption Studies
The single-dose,
dual-isotope feeding method, originally described by Zilversmit and
Hughes (27) and validated for measuring cholesterol absorption
in mouse by Dueland et al.(28) , was used to determine
cholesterol absorption efficiency in control and CEL gene-targeted
mice. Test meal was prepared either as a lipid emulsion or as sonicated
vesicles. Emulsified substrate was prepared by mixing 35 µCi of
[H]cholesterol (50 Ci/mmol, DuPont NEN) or
[H]cholesteryl oleate (50 Ci/mmol, Amersham) and
7 µCi of [-C]sitosterol (56 mCi/mmol,
Amersham) with 2 mg of cholesterol, 8 mg of phosphatidylcholine (PC),
and 50 mg of triolein in organic solvent. After evaporating the solvent
under a nitrogen stream, 1 ml of deionized water was added, and the
sample was emulsified by 5 min of sonication in a Bransonic-32
bath sonicator. When cholesteryl ester absorption was being studied,
the mix also included 0.2 mg of unlabeled cholesteryl oleate. For some
of the cholesteryl ester studies, the emulsion consisted of 50 µCi
of [
H]cholesteryl oleate, 1 µCi of
[-C]sitosterol, 120 µg of cholesterol,
12 µg of cholesteryl oleate, 960 µg of PC, and 5 µg of
triolein emulsified in 600 µl of water. Vesicles were prepared by
sonicating 10 µCi of [H]cholesterol, 1
µCi of -[C]sitosterol, 5 µg of
cholesterol, and either 5 or 35 µg of phosphatidylcholine in 1 ml
of water for 1 min with a Heat Systems Ultrasonics W-380 probe
sonicator (fine tipped probe).
H-dpm/C-dpm) in
feces/(H-dpm/C-dpm) administered)}
100. Total recovery of the
[-C]sitosterol over the 24-h period ranged
from 66 to 97%. Differences in cholesterol absorption between groups
were evaluated for statistical significance by Mann-Whitney rank sum
and Student's t tests using SigmaStat software from
Jandel Corporation.Serum Cholesterol Analysis
Blood was collected
from euthanized animals by severing the inferior vena cava. Samples
were allowed to clot on ice, and serum was collected after
centrifugation. Total cholesterol levels were determined using
commercially available enzymatic kits from Sigma.
Targeting of the Cholesterol Esterase Gene in Mouse
Embryonic Stem Cells
Restriction mapping and Southern blot
analysis revealed that the mouse cholesterol esterase gene is highly
homologous to the rat CEL gene and contains 11 exons interrupted by 10
introns(30) . A 4.7-kb SacI fragment that contains
exon 1 to intron 7 was used to construct the targeting DNA as described
under ``Experimental Procedures'' and schematically outlined
in Fig. 1B. The insertion of neo into exon
4 of the CEL gene disrupts the CEL coding sequence immediately
preceding the active site domains of cholesterol
esterase(25, 30) . Additionally, any truncated
polypeptide derived from the disrupted CEL gene could not be secreted
by the pancreas due to the absence of the exon 11 domain, which is
important for this function(31) . Thus, homologous
recombination of the endogenous CEL gene with this targeting construct
will provide an animal with a CEL-null phenotype. 10 mouse ES cells with the
targeting DNA resulted in approximately 4,800 G418-resistant colonies.
One-fourth of the colonies were picked and expanded individually in
24-well dishes. A total of 268 colonies were selected for Southern blot
analysis to screen for homologous recombination between the targeting
DNA and the resident CEL gene. For the initial screening, ES colony DNA
was digested with EcoRI and hybridized with an 1100-bp PstI/SacI DNA fragment corresponding to genomic
sequence 5` from the targeting DNA (Fig. 1C). As shown
in Fig. 2A, the wild-type allele gives rise to a fragment
>30 kb in length, while a correctly targeted gene yields a fragment
6.5 kb in length due to the insertion of two EcoRI sites present
in the thymidine kinase promoter of neo
(Fig. 1B). To confirm that the putative targeting
events had taken place as planned, additional aliquots of the ES colony
DNA were digested with XbaI and hybridized with an
1100-bp BglII/SalI DNA fragment corresponding to
sequences 3` from the targeting DNA (Fig. 1D). Fig. 2B shows that the wild type allele yields a 7.2-kb
fragment, while the correctly targeted allele yields a 9.0-kb fragment
due to the insertion of 1.75 kb of DNA corresponding to the selectable
marker cassette. Of the 268 colonies screened, 11 were positive in both
tests. The overall targeting efficiency was 4.4%.
.
When digested with HindIII, the wild type allele is 6.7 kb,
while the targeted allele is 8.5 kb due to the presence of neo. B, representative colonies digested
with SphI and XbaI and hybridized with the 3` probe.
Wild type DNA yields an 18-kb fragment with SphI, while
targeted DNA yields a 7.5-kb fragment due to an SphI site
present in neo. When digested with XbaI,
the wild type DNA yields a 7.3-kb fragment, and the targeted DNA yields
a 9.1-kb fragment due to the insertion of neo. W, wild type; T,
targeted.
cassette
to the endogenous CEL gene resulted in an 8.3-kb HindIII
fragment that hybridized with the 5` probe in addition to the 6.5-kb
band observed for the controls (Fig. 2A). Using the 3`
probe, a 7.5-kb SphI band resulted from the insertion of an SphI site in neo in targeted clones in
addition to the 18-kb SphI band observed for the wild type
allele (Fig. 2B). These hybridization patterns were
consistent with those predicted for the site-specific insertion of the neo cassette into exon 4 of the endogenous CEL
gene (Fig. 1). A total of eight enzymes, informative with either
the 5` or 3` probe, were used to confirm that the gene targeting had
occurred as planned. In addition, a neo-specific
probe was used to confirm that the targeting DNA had inserted in only
one site in the genome (data not shown).5% agouti coat color. However, the second cell line produced
22 chimeric mice (from 119 injected and reimplanted blastocysts), all
with extensive agouti coat color. Nineteen of these were male, and 15
of the 19 were able to transmit the modified gene to their offspring.
Progeny from these test matings (chimerics
Black Swiss), which
carried the modified allele, were bred to generate homozygous knockout
animals. Southern blot analysis of the genomic DNA from representative
wild type, heterozygous, and homozygous CEL-targeted mice is shown in Fig. 3. Because of an apparent restriction fragment length
polymorphism between the ES cells and the outbred mice used in the
initial breeding, XbaI was not informative, and NcoI
was used as a diagnostic enzyme for the 3` end of the recombination.
This enzyme yields a 9.5-kb fragment from the wild type allele and a
6.5-kb fragment from the targeted allele due to the insertion of the NcoI site in the neo gene (see Fig. 1for details).
gene. Wild type, +/+; heterozygotes, +/-;
homozygous knockout, -/-. The 4-kb band seen in panel B is due to spurious hybridization and has not been seen in other
experiments.
General Characteristics of CEL Gene-targeted
Mice
The targeted allele was transmitted by the chimeric males
at approximately the expected frequency (40%, 39 CEL+/- mice
of 97 agouti pups), indicating no obvious disadvantage to embryos or
neonates heterozygous at the CEL locus. Furthermore, the crosses
between these CEL(+/-) animals yielded progeny with the
expected 1:2:1 ratio of wild-type to heterozygous to homozygous
knockout genotypes (37:89:40). Both homozygous and heterozygous CEL
gene-targeted mice grew normally and appeared to be healthy by
inspection. The serum cholesterol levels for CEL(-/-) mice
fed control diet (104.1 ± 3.33 mg/dl) or 24 weeks of atherogenic
diet (356 mg/dl ± 21.6) were not significantly different (p = 0.2420) from those of their wild type littermates (114.9
± 6.76 and 399 ± 17.6 mg/dl).
g supernatant fraction of wild type and
CEL-targeted mouse pancreas was run in duplicate on a 10%
SDS-polyacrylamide gel electrophoresis gel and either stained with
Coomassie Blue (A) or transferred to nitrocellulose and
reacted first with rabbit anti-rat CEL and then with I-labeled goat anti-rabbit IgG (B).
Cholesterol Absorption Studies
The role of CEL in
mediating absorption of dietary cholesterol and cholesteryl esters was
assessed by comparing the amount of radiolabeled sterol excreted in the
feces after its infusion into the stomach of normal and homozygous CEL
gene-targeted mice. Since a role for CEL in the absorption of
cholesteryl esters is not in dispute, the initial study used
[H]cholesteryl oleate in a lipid emulsion with
cholesterol, triolein, and phospholipid as the substrate. A trace
amount of -[C]sitosterol was added as a
marker to normalize the amount of nonabsorbed sterol recovered in the
feces. The results showed that absorption of the cholesteryl ester was
reduced in the CEL(-/-) mice to 35% of the wild type
value (Table 2). The experiment was performed using emulsions
with either normal (2:0.2:8:50, FC:CE:PC:TG, w/w/w/w) or low
(10:1:80:5) triglyceride content. Similar results were obtained in both
tests, indicating that the triglyceride content of the test meal had
only a limited effect. The absorption of cholesteryl ester in
heterozygous animals was found to be similar to wild type despite the
reduced CEL protein level in their pancreas.
H]cholesterol was included in the emulsion
instead of the cholesteryl oleate, no significant difference was found
between wild type and CEL-null mice in absorption of the radiolabeled
sterol (Table 3). Interestingly, males were found to absorb
significantly less (59.6 ± 3.05%) cholesterol than females (72.0
± 1.61%), but this difference was independent of their
CEL genotype. For clarity, results from male and female mice are
combined in Table 3.
H]cholesterol in an emulsion that
contained unlabeled cholesteryl ester along with phospholipid and
triglyceride. Table 3also shows that the presence of the
cholesteryl ester in the core had no effect on the ability of
CEL(-/-) mice to absorb the free cholesterol.-C]Sitosterol was used as a marker of
recovery as described above. The results showed that, regardless of the
ratio of cholesterol to phospholipid used to prepare the lipid
vesicles, there was no significant difference between the ratios of
[H]cholesterol to
[-C]sitosterol recovered in the feces of
wild type versus CEL gene-targeted mice (Table 3). H in 15 µl of serum was determined at various times
after gastric infusion of emulsified radiolabeled sterol. The infusion
of the unesterified [H]cholesterol resulted in
the progressive appearance of the radiolabel in the serum of both wild
type and CEL gene-targeted mice with a maximum at 10 h (Fig. 5) and a slow decline thereafter. No significant
difference in the rate of radiolabeled cholesterol appearance in the
serum was observed between the two groups of animals in this case. In
contrast, when the radiolabel was supplied as emulsified
[
H]cholesteryl oleate, the serum level of the
radiolabel after 12 h was 8-fold higher in the CEL(+/+)
mice than in the CEL-null mice. In fact, very little radiolabel was
detected in the serum of the gene-targeted mice (Fig. 5).
H]cholesterol (FC) or
[H]cholesteryl oleate (CE) was administered in an
emulsion with PC, TG, and a trace amount of
[-C]sitosterol. Thirty µl of whole
blood (FC, first experiment, solid lines) or 15 µl of
plasma (all other points) was counted at various times after
the label was given. For FC, each point represents the average of two
animals. For the cholesteryl oleate experiment, each point represents
the average of six animals. 
and 
, wild type and FC;
and
, CEL(-/-) and FC; , wild type and CE;
, CEL(-/-) and CE. Open and closed
symbols represent different experiments with
FC.
)The recent
report by Kirk et al. (37) on the responsiveness of
different strains of mice to dietary fat and cholesterol with respect
to cholesterol absorption and serum lipid parameters also supports this
hypothesis. Candidate genes potentially involved in this process
include the cholesterol transfer protein(38) , which may be the
same as, or closely related to, sterol carrier
protein-2(39, 40) , acyl CoA:cholesterol
acyltransferase(5, 41) , pancreatic
lipase(36) , and liver fatty acid binding protein(42) .
Additional experiments are necessary to investigate the physiological
role of these and other proteins in cholesterol absorption.
), neomycin resistance gene; FC, free
cholesterol; CE, cholesteryl ester; PC, phosphatidylcholine; TG,
triglyceride.)
We gratefully acknowledge the expert contribution of
Dr. John Duffy, Moying Yin, Sharon Pawlowski, Ilona Ormsby, Bing-Tao
Fan, and Maureen Luehrmann, who operate the core facility that
generated chimeric animals from our CEL gene-targeted ES cells.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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