(Received for publication, December 8, 1995; and in revised form, January 12, 1996)
From the
An 87-kilobase (kb) P1 bacteriophage clone (p649) spanning the mouse apolipoprotein (apo) B gene was used to generate transgenic mice that express high levels of mouse apoB. Plasma levels of apoB, low density lipoprotein cholesterol, and low density lipoprotein triglycerides were increased, and high density lipoprotein cholesterol levels were decreased in the transgenic mice, compared with nontransgenic littermate controls. Although p649 contained 33 kb of 5`-flanking sequences and 11 kb of 3`-flanking sequences, the tissue pattern of transgene expression was different from that of the endogenous apoB gene. RNA slot blots and RNase protection analysis indicated that the transgene was expressed in the liver but not in the intestine, whereas the endogenous apoB gene was expressed in both tissues. To confirm the absence of transgene expression in the intestine, the mouse apoB transgenic mice were mated with the apoB knockout mice, and transgenic mice that were homozygous for the apoB knockout mutation were obtained. Because of the absence of transgene expression in the intestine, those mice lacked all intestinal apoB synthesis, resulting in a marked accumulation of fats within the intestinal villus enterocytes. The current studies, along with prior studies of human apoB transgenic animals, strongly suggest that the DNA sequence element(s) controlling intestinal expression of the apoB gene is located many kilobases from the structural gene.
Within the past two years, transgenic mice expressing high
levels of human apolipoprotein B (apoB) ()have been
generated, both by our group (1) and by Callow and his
co-workers(2) . These mice were generated with a P1
bacteriophage clone that spanned the entire human apoB gene. The human apoB transgenic mice displayed several noteworthy features
that posed questions for subsequent studies. First, the tissue pattern
of transgene expression was decidedly abnormal. In multiple lines of
mice, the human apoB transgene was expressed at high levels in
the liver, but expression was undetectable in the
intestine(3, 4) . This finding was surprising in view
of the fact that the 80-kb transgene contained rather extensive
flanking sequences 19 kb 5` from the transcriptional start site and
17.5 kb 3` from the translation termination codon. One possible
explanation for absent transgene expression in the intestine was that
the intestinal tissue-specific regulatory element was located so far
from the structural gene that it simply was not contained within the
80-kb transgene. However, other potential explanations existed. One
possibility was that the human intestinal element was located
within the 80-kb fragment but that it was not recognized by the mouse
transcriptional machinery. An obvious means of testing the latter
possibility would be to analyze transgene expression patterns in mice
generated with a P1 clone spanning the mouse apoB gene.
A second issue that arose during the study of the human apoB transgenic mice was the finding of the high levels of triglyceride-rich low density lipoproteins (LDL) in the plasma of the mice(1) . Although we were initially tempted to ascribe the high levels of LDL to apoB overproduction by the liver, another potential explanation existed. Human apoB100 binds to the mouse LDL receptor with very low affinity, compared with mouse LDL(5) , so it is possible that the increased LDL levels could have been due, in large part, to defective clearance of the human LDL from the plasma. Indeed, this issue raised serious doubts as to whether transgenic expression of human apoB, with its defective binding to the mouse LDL receptor, could yield an accurate portrayal of the phenotype of apoB overproduction by the liver. An obvious way to obtain an accurate view of the phenotype of hepatic apoB overproduction was to develop and characterize transgenic mice that overexpress mouse apoB.
To address the issues posed by the human apoB transgenic mice, we obtained a P1 clone spanning the mouse apoB gene and used that clone to develop transgenic mice that expressed high levels of mouse apoB.
Figure 1: Map of p649, an 87-kb P1 clone spanning the mouse apoB gene. Probes A and B are 2593-bp NotI-MluI and 4758-bp MluI-SalI fragments, respectively, from the P1 vector. Probe C is a 1.1-kb EcoRI-HindIII fragment several kilobases upstream from exon 1(7) . Probe D is a 0.7-kb XbaI-HindIII fragment spanning from intron 24 to exon 25 of the apoB gene.
To
prepare p649 DNA for microinjection, plasmid DNA was isolated from Escherichia coli strain NS3529 (6) and cleaved with MluI. A 95-kb MluI fragment containing the entire
87-kb insert and 8 kb of P1 vector sequences (
2200 bp 5` and
5800 bp 3`) was purified from a pulsed-field agarose gel and
dialyzed against a microinjection buffer(6) . After adjusting
the DNA concentration to 3 ng/µl, the DNA was microinjected into F2
murine zygotes (C57BL/6J
SJL), both at the Gladstone Institute
of Cardiovascular Disease and at DNX Transgenics.
Founder 6691 was mated to mice heterozygous
for an apoB knockout mutation (7) to generate mouse apoB transgenic mice that were heterozygous for the knockout
mutation. Those mice were intercrossed to generate mouse apoB transgenic mice that were homozygous for the knockout mutation
(genotypes
MBTg,apoB
or
MBTg
,apoB
). Mice
that were homozygous for the knockout mutation were detected by
demonstrating a ``double dose'' of the neo gene
(inserted into the apoB locus in the knockout mice), as
compared with the ``single dose'' of neo in mice
heterozygous for the knockout mutation. For these studies, tail DNA was
digested with HindIII, and Southern blots were probed
simultaneously with a neo probe and a cDNA probe for the
-tocopherol transport protein(8) . The intensity of the neo signal relative to the
-tocopherol transport protein
signal was assessed using a Phosphorimager (Fuji Bio-Imaging Analyzer,
BAS 1000 with MacBAS, Fuji Medical Systems, USA, Inc., Stamford, CT).
For the RNase protection assays, total RNA was isolated from liver and intestine (duodenum) of F1 mice from lines 6690 and 6691 by homogenization in guanidine isothiocyanate (10) using the RNAzol(TM) method (Tel-test, Inc., Friendswood, TX). A 245-bp antisense RNA transcript was prepared with XbaI-linearized plasmid pBSmB245 and T7 RNA polymerase, using the MAXIscript(TM) In Vitro transcription Kit (Ambion). Plasmid pBSmB245 contains a XbaI/MscI fragment from the 5` portion of exon 26 of the mouse apoB gene. The RNase protection assay was performed using the RPA II Ribonuclease protection assay kit (Ambion). The samples were electrophoresed on denaturing 6% polyacrylamide gels. Dried gels were analyzed by autoradiography and with the phosphorimager.
To analyze the distribution of cholesterol, triglycerides, and apoB within the plasma lipoproteins, plasma (250 µl pooled from six animals of the same genotype) was fractionated on a Superose 6 10/30 column (Pharmacia Biotech Inc.) using the automated Bio-Rad Biologic System (Bio-Rad). The column was eluted at a flow rate of 0.5 ml/min, and 96 fractions (0.25 ml each) were collected in a 96-well plate. Consecutive fractions were pooled to obtain 48 fractions (0.5 ml each), and cholesterol and triglyceride concentrations in each fraction were measured using colorimetric methods(4) . The distribution of mouse apoB in each fraction was analyzed on Western blots of 4% polyacrylamide-SDS gels, using the rabbit antibody to mouse apoB and the Enhanced Chemiluminescense Western blotting detection reagents (Amersham Corp.).
To generate transgenic mice expressing mouse apoB,
the 95-kb MluI fragment of p649 was microinjected into murine
zygotes (Fig. 1). Six founder mice were identified by Southern
blot analysis and the two with the highest levels of plasma apoB, 6690
and 6691, were selected for breeding and further analysis (Fig. 2, A and B). Using Southern blots of
pulsed-field agarose gels, we demonstrated that both of the transgenic
lines contained intact copies of the transgene (Fig. 2C). In line 6690, the transgene copy number was
assessed by phosphorimager analysis of a Southern blot as 20. High
copy numbers were also found in the human apoB transgenic mice
generated with a P1 bacteriophage clone(1) .
Figure 2: Southern blot identification of transgenic mice generated with the 95-kb MluI fragment of p649. Mouse tail DNA was digested with EcoRI and fractionated on 0.8% agarose gels. A Southern blot using probe A (see Fig. 1) is shown in A; a blot using probe B is shown in B. C, Southern blot of a pulsed-field agarose gel using probe D (see Fig. 1), demonstrating the expected 88-kb NruI fragment in the genomic DNA of F1 transgenic mice from lines 6690 and 6691. The NruI fragment containing the endogenous apoB gene was >200 kb and was not resolved on this pulsed-field gel.
Transgenic mice
from line 6691 had increased amounts of -migrating lipoproteins on
an agarose gel (Fig. 3A), and Western blots
demonstrated increased levels of mouse apoB (Fig. 3B). Using chemical techniques, the
concentration of mouse apoB in the plasma of the nontransgenic mice was
10 mg/dl. Quantitative analysis of the Western blots of agarose gels
with a phosphorimager revealed a 3-fold increase in the amount of
plasma apoB in the transgenic animals (line 6691) compared with
nontransgenic controls. Therefore, based on the analysis of the Western
blots, we estimate that the concentration of apoB in the plasma of the
transgenic mice (line 6691) was
30 mg/dl. The plasma apoB levels
in transgenic line 6690 were approximately 2-fold higher than those in
line 6691,
60 mg/dl. The increased amounts of apoB in the plasma
of the transgenic mice were associated with significantly increased
amounts of plasma triglycerides and significantly decreased amounts of
HDL cholesterol, compared with nontransgenic littermates (Fig. 4). Fractionation of the plasma by fast phase liquid
chromatography (FPLC) revealed that the transgenic mice had increased
amounts of LDL cholesterol (Fig. 5A). The FPLC studies
revealed that the LDL of the transgenic animals was strikingly enriched
in triglycerides (Fig. 5B), accounting for the
increased plasma triglyceride levels in these animals. Western blots of
the FPLC fractions revealed differences in the quantity of apoB48 and
apoB100 in transgenic and nontransgenic animals, but there were no
significant differences in the particle size distribution of apoB100
and apoB48 (Fig. 5C). Most of the apoB48 and apoB100
were located in LDL-sized lipoproteins.
Figure 3:
Agarose
gel electrophoresis of the plasma of mouse apoB transgenic
mice (line 6691). A, an agarose gel of the plasma of two
transgenic mice (MBTg) and two nontransgenic controls. The gel was
stained with Fat Red 7B. B, a Western blot of the agarose gel,
using a I-labeled rabbit antiserum to mouse
apoB.
Figure 4:
Lipids
and lipoproteins in transgenic and nontransgenic mice. A,
comparison of the lipids and lipoproteins in six 10-week-old female
transgenic mice and six female nontransgenic littermate control mice. B, comparison of the lipids and lipoproteins in
MBTg,apoB
mice
and age- and sex-matched nontransgenic control mice. Bars are
means, and lines represent standard
deviations.
Figure 5: Analysis of the distributions of cholesterol (A), triglycerides (B), and apoB48 and apoB100 (C) in the plasma of 8-week-old mouse apoB transgenic mice. Pooled plasma samples from six female transgenic mice and six female nontransgenic littermate controls were size-fractionated on an FPLC column, as described under ``Materials and Methods.'' Cholesterol and triglycerides were determined by an enzymatic assay (panels A and B), and the distributions of apoB48 and apoB100 were determined by Western blots of SDS-polyacrylamide gels using a rabbit antibody to mouse apoB and the Enhanced Chemiluminescense Western blotting detection reagents (Amersham Corp.) (panel C). The angled appearance of the apoB48 and apoB100 bands in the lower blot in panel C was due to the angled placement of the gel on the blotting paper before electrophoretic transfer to nitrocellulose.
To assess levels of apoB mRNA in the liver and intestines of transgenic animals (line 6691), we used RNA slot blots (Fig. 6). Quantitative analysis of the RNA slot blots using a phosphorimager indicated that the livers of the transgenic mice had a 3-fold increase in the amount of apoB mRNA (Fig. 6A). However, no increase in the amount of intestinal apoB mRNA was observed in the transgenic mice (Fig. 6B).
Figure 6: RNA slot blot demonstrating the relative amounts of apoB mRNA in the livers (A) and intestines (B) of transgenic and nontransgenic mice. With one of the nontransgenic mice, the 0.25-µg liver RNA sample was not loaded onto the membrane.
To further evaluate the issue of apoB mRNA levels, we performed RNase protection studies of liver and intestine RNA samples, using a riboprobe from exon 26 of the mouse apoB gene (both the 6690 and 6691 lines). In the nontransgenic animals, as well as the transgenic animals, we observed the expected 245-bp protected fragment. This fragment was of equal intensity in transgenic and nontransgenic animals (Fig. 7). In addition, the liver RNA from transgenic animals yielded protected fragments of approximately 151 and 91 bp. DNA sequencing revealed that the transgene contained two single-nucleotide substitutions, separated by one nucleotide, in exon 26 of the apoB gene (the exact DNA sequence changes were determined and are given in the legend to Fig. 7). Thus, the smaller protected fragments in the RNase protection experiment represented apoB mRNA derived from the transgene, which had been cleaved at the mismatched bases. In a separate RNase protection assay, the apoB mRNA levels were normalized to the amount of mRNA for GAPDH (data not shown). Quantitative analysis of apoB mRNA levels (relative to GAPDH mRNA levels) using a phosphorimager indicated a 3-fold increase in hepatic apoB mRNA in the transgenic mice (total radioactivity in the 245-, 151-, and 91-bp bands) compared with the nontransgenic mice (the 245-bp band). With the intestinal RNA, the endogenous 245-bp protected fragment was observed in both transgenic and nontransgenic mice; however, the 151- and 91-bp bands (indicative of transgene expression) were not observed.
Figure 7: RNase protection assay of liver and intestinal RNA from transgenic and nontransgenic mice. RNase protection assay of liver and intestinal RNA from transgenic (line 6691) and nontransgenic mice. The protected fragment arising from the endogenous apoB gene was 245 bp. Because the transgene contained two single-nucleotide substitutions in exon 26, the apoB mRNA arising from the transgene was cleaved into two smaller fragments (151 and 91 bp). The hepatic apoB mRNA levels in transgenic line 6690 were approximately twice as high as those in line 6691 (data not shown). The 151- and 91-bp protected fragments were caused by two single-nucleotide substitutions in exon 26 of the transgene, a G to A transition at nucleotide 225 of exon 26, and a C to A transversion at nucleotide 227. These residues are homologous to human apoB cDNA residues 4569 and 4571 (17) . The polymorphisms did not change a restriction endonuclease site but did change a GTC-Val codon to an ATA-Ile codon (homologous to human apoB100 amino acid residue 1454).
To evaluate intestinal transgene
expression further, the transgenic mice (line 6691) were mated with apoB knockout mice to obtain mice that carried the transgene
and were homozygous for the knockout of the endogenous apoB gene (genotype
MBTg,apoB
). In
the presence of the mouse apoB transgene, we demonstrated
homozygosity for the apoB knockout mutation by showing that
these animals had a double dose of the neo gene, relative to a
signal from an unrelated single-copy gene (Fig. 8, A and B). The
MBTg
,apoB
mice
had a striking phenotype. Within 24 h of birth, these animals developed
a completely white abdominal cavity and manifested growth retardation (Fig. 9). The white abdominal cavity was due to the failure to
synthesize apoB in the intestines, which caused a massive
accumulation of lipids in the villus enterocytes (Fig. 10). Of
note, the MBTg
,apoB
mice had increased levels of triglycerides and decreased levels
of HDL cholesterol, compared with nontransgenic mice (Fig. 4B). FPLC fractionation of the plasma of the
MBTg
,apoB
mice
revealed that these animals had an increased amount of LDL cholesterol,
compared with nontransgenic animals, and a marked reduction in HDL
cholesterol (Fig. 11A). The LDL of the
MBTg
,apoB
mice
was enriched in triglycerides (Fig. 11B). The size
distribution of apoB48- and apoB100-containing
lipoproteins was similar in the nontransgenic mice and the
MBTg
,apoB
mice (Fig. 11C).
Figure 8:
Generation of transgenic mice that carried
the transgene and were homozygous for the knockout mutation in the
endogenous apoB gene. A, Southern blot, using a P1
vector probe (probe B in Fig. 1), demonstrating the
presence of the mouse apoB transgene. B, Southern
blot probed simultaneously with a neo probe and a probe for
-tocopherol transport protein (
-TTP).
Figure 9:
Photograph of two 6-day-old mice. The
mouse on the left (genotype
MBTg,apoB
) lacks apoB expression in the intestine. Approximately one-third of
the MBTg
,apoB
mice survived until they were weaned. After weaning onto a chow
diet, they resumed normal growth and appeared
healthy.
Figure 10:
A
hematoxylin- and eosin-stained section of the duodenum of a 10-day-old
MBTg,apoB
mouse
and a wild-type mouse. The intestinal pathology in
MBTg
,apoB
mouse (A) is virtually identical to that observed in the human
disease, abetalipoproteinemia(18) . In addition to fat
accumulation in the duodenum, fat accumulation was also observed in the
jejunum, ileum, and colon. B, a hematoxylin- and eosin-stained
section of the duodenum of a 10-day-old wild-type mouse. Histological
examination of the livers of wild-type mice showed mild to moderate
amounts of fat, whereas no fat was found in the livers of
MBTg
,apoB
mice.
Figure 11:
Analysis of the distribution of
cholesterol (A), triglycerides (B), and apoB48 and
apoB100 (C) in the pooled plasma of two transgenic mice that
were homozygous for the knockout mutation (MBTg, apoB
) and six nontransgenic control
mice. Pooled plasma samples were size-fractionated on an FPLC column as
described under ``Materials and Methods.'' Cholesterol and
triglycerides were determined by an enzymatic assay (panels A and B), and the distributions of apoB48 and apoB100 were
determined by Western blots of SDS-polyacrylamide gels using a rabbit
antibody to mouse apoB and the ECL detection system (panel C).
For panel C, the control plasma sample that was used was from
a apoB
mouse, which is a mouse that is
homozygous for a targeted apoB mutation that prevents the
formation of apoB48 (R. V. Farese Jr. and S. G. Young, unpublished
results). The slightly angled appearance of the apoB48 and apoB100
bands in panel C was due to the angled placement of the gel on
the blotting paper before electrophoretic transfer to
nitrocellulose.
In this study, we used a 95-kb MluI fragment from a mouse apoB P1 clone, p649, to produce transgenic mice that overexpress mouse apoB. As judged by Southern blots of pulsed-field gels of high molecular weight liver DNA, intact copies of the transgene were incorporated into the genomes of the transgenic lines that we investigated. Documentation of transgene expression was straightforward, even though the transgenic animals also expressed apoB from their endogenous apoB alleles. The apoB transgene used in this study was isolated from a strain RIII genomic library, and it contained two single nucleotide substitutions within the 5` portion of exon 26. The existence of this pair of DNA sequence polymorphisms made it possible to document transgene expression unequivocally using RNase protection assays. The RNase protection studies, as well as the RNA slot blot studies, indicated that the hepatic levels of apoB mRNA in transgenic line 6691 were increased 3-fold, compared with nontransgenic mice.
One of the
most intriguing aspects of these studies was that the mouse transgene,
which contained 33 kb of 5`-flanking sequences and 11 kb of 3`-flanking
sequences, was not expressed in the intestines, an organ where the
endogenous gene is normally expressed at high levels. This finding,
which initially was shown by RNA slot blot and RNase protection
studies, was further analyzed by breeding the transgenic mice with the apoB knockout mice. Transgenic mice that were homozygous for
the knockout mutation
(MBTg,apoB
) could
not synthesize apoB in the intestines and consequently developed a
massive accumulation of fats within the villus enterocytes of the small
and large intestines.
In a prior study(1) , we found that a human apoB gene construct containing 19 kb of 5`-flanking sequences and 17.5 kb of 3`-flanking sequences did not confer intestinal expression of the apoB gene in transgenic mice. Although we suspected that the human transgene simply lacked the DNA sequences controlling intestinal expression of the gene, those studies did not allow us to exclude the possibility that the mouse was simply incapable of recognizing and utilizing the human sequences that control intestinal expression of the apoB gene. The current studies using a mouse transgene argue strongly against the latter possibility and, together with the human transgene studies, suggest that the intestinal element is probably located more than 33 kb 5` of the structural gene or more than 11 kb 3` from the gene. One hypothesis to explain the distant location of the cis-acting DNA sequence element controlling intestinal apoB expression is that the element might be shared with a neighboring gene. Sharing a tissue-specific element with an adjacent gene would not be unprecedented. For example, apoE and apoCI are known to share a DNA sequence element that governs liver expression of both genes(14) .
The phenotype of the
MBTg,apoB
mice
was similar to that of the
HuBTg
,apoB
mice
that we described earlier(15) , both in terms of the intestinal
pathology and the lipoprotein phenotype. However, two findings with the
MBTg
,apoB
mice
deserve to be underscored. First, those animals had higher levels of
triglycerides in the plasma, compared with nontransgenic animals,
despite the fact that they had severe intestinal pathology. Because fat
absorption is markedly reduced in these mice(15) , the high
plasma triglyceride levels are probably maintained by prodigious rates
of de novo lipogenesis in the liver. Second, the
MBTg
,apoB
mice
had markedly decreased levels of HDL cholesterol. This finding is
consistent with the low levels of HDL cholesterol that are observed in
human patients with abetalipoproteinemia (16) and suggests a
critical role for normal chylomicron formation in the maintenance of
normal plasma concentrations of HDL.
The studies of the mouse apoB transgenic mice helped to clarify the lipoprotein phenotype associated with increased apoB production by the liver. High levels of human apoB expression in transgenic mice resulted in a large increase in the amount of LDL cholesterol in the plasma, as well as decreased levels of HDL cholesterol(1, 4) . However, since human apoB binds very poorly to the mouse LDL receptor(5) , one could make a strong argument that the LDL accumulation in these animals might be caused, in large part, by defective clearance of the human LDL from the plasma. The current study, involving overexpression of mouse apoB in mice, provides a portrayal of hepatic apoB overexpression that is free of caveats regarding the intrinsic metabolic properties of a heterologous apoB. The transgenic mice that overproduce mouse apoB in the liver had increased plasma levels of LDL cholesterol and decreased levels of HDL cholesterol, strongly suggesting that these findings represent hallmark metabolic features of apoB overexpression by the liver.
Finally, our
prior studies of the human apoB transgenic mice revealed that
those animals had a marked triglyceride enrichment of the
LDL(1) . This unexpected finding suggested the possibility that
lipolysis of triglycerides within the human apoB-containing
lipoproteins might be defective in the mouse. The fact that we observed
triglyceride enrichment of the LDL in the mouse apoB transgenic mice casts serious doubt on that potential explanation.
It seems more likely that the triglyceride enrichment of the LDL might
be due to relatively low levels of hepatic triglyceride lipase in the
mouse ()or to the production of small, triglyceride-rich
nascent lipoproteins by the liver(1) . This topic deserves
further investigation.