Three independent lines of transgenic mice were established with 4 to 14 copies of a linearized P1 plasmid containing a 90-kb insert. The insert contained the whole human ABCG5
genomic sequences, about 30 kb of flanking sequence at the 3′ end of ABCG5
, and 6 kb of flanking sequence at the 3′ end of ABCG8
. Exons 6–13 of a gene of unknown function (CG1-60
; GenBank accession no. AF151818) were included in the insert at the 3′ end of ABCG5
. The mouse line (14
) with the highest copy number of the transgene (~14) was used for the studies described in this paper. Selected studies were also performed with another transgenic line (6
) containing approximately ten copies of the transgene to confirm observations made in the first line. No significant differences in litter size, body weight, liver weight, or liver enzymes were detected between transgenic and wild-type mice (Table ).
Comparison of body weight, liver weight, and plasma/serum chemistries in ABCG5/ABCG8 transgenic and wild-type mice
The human transgenes were expressed in the liver and in the small intestine (Figure b), which is similar to the expression patterns of the endogenous genes. Two major mRNAs for human ABCG5 (2.4 and 3.4 kb) and human ABCG8 (2.6 and 3.8 kb) that differ in their 3′-untranslated region were detected (22
). A third human transcript of higher molecular weight was detected in the liver and the jejunum of the transgenic animals (5.9 and 7.4 kb, respectively, for human ABCG5 and ABCG8); these larger mRNA species correlated directly with the amounts of the other two mRNA species. The expression of human ABCG5
in the liver and intestine of the transgenic animals was not associated with major changes in the expression levels of Abcg5
; however, the relative levels of expression of human ABCG5
and the endogenous murine genes differed in the liver and small intestine. The human transgenes were expressed at higher levels in the liver than in the small intestine, resembling the pattern seen in human tissues (4
). In contrast, the mouse genes were expressed at a higher level in the intestine than in the liver. These results suggest that cis
-acting elements present in the human transgene confer increased expression in the liver. An identical tissue-specific pattern and relative level of expression was seen in a different line of transgenic mice (6
) (data not shown). RT-PCR was performed to determine whether human ABCG5
were expressed in other tissues at levels that were too low to detect by mRNA blotting. Trace amounts of human ABCG5 and ABCG8 mRNA were detected in the ovaries of the transgenic mice. Neither the human transgenes nor the endogenous mouse genes were expressed in peritoneal macrophages of the transgenic animals (data not shown).
Figure 1 (a) The structure of the human ABCG5/ABCG8 transgene. A P1 clone (35B6; Incyte Genomics Inc.) containing a 90-kb insert was linearized with NotI, purified, and injected into fertilized embryos as described in Methods. The insert contains the entire coding (more ...)
The hepatic mRNA levels of mouse Abcg5 and Abcg8 were 1.5-fold higher in the female wild-type mice than in the male wild-type mice. No sex-specific differences in the expression levels of the endogenous genes were detected in the intestine (Figure ). Human ABCG5
was expressed in the livers at a 1.5-fold higher level in the female transgenic mice than in the male transgenic mice, whereas no sex-specific significant differences in the expression of human ABCG8
were seen. In mice (6
) with a lower copy number of the transgene (~10), expression of both ABCG5
was more than twofold higher in the liver of the female transgenic mice compared with the male transgenic mice (data not shown). In wild-type animals, the high-cholesterol diet increased expression levels of the endogenous mouse genes twofold in the liver and 1.5-fold in the intestine. In transgenic animals, no significant changes were seen in the levels of endogenous mRNA or the transgene mRNAs after 3 weeks on the high-cholesterol diet.
Figure 2 Northern blot analysis of ABCG5 and ABCG8 in male and female transgenic mice. Total RNA was isolated from the liver and jejunum of mice on a chow or 2% cholesterol diet. Equal amounts of total RNA from each mouse in each group were pooled and (more ...)
The mean fasting plasma cholesterol levels were not significantly different in the male transgenic and control mice (Figure , left top) but were significantly lower in the female transgenic mice (60.2 vs. 75.7 mg/dl). Pooled fasting plasma samples from chow-fed transgenic and wild-type mice were subjected to fast protein liquid chromatography to determine the distribution of lipoprotein cholesterol (16
). No significant difference in the fractionation profile was seen in the two groups of male mice (Figure , bottom). Although female transgenic mice had reduced plasma levels of HDL-cholesterol, which accounts for the reduction in plasma cholesterol in these animals, transgene expression did not influence the size distribution of lipoprotein particles.
Figure 3 Levels of plasma sterols and lipoprotein profiles of ABCG5 and ABCG8 transgenic mice. Male and female 12-week-old transgenic mice and their littermate controls (n = 4–7 in each group) were maintained on a chow diet. After a 4-hour fast, (more ...)
Plasma levels of other neutral sterols were measured by gas chromatography (Figure , right top). The mean plasma levels of sitosterol in fasted mice were reduced by 64% and 79% in the male and female transgenic animals, respectively. Marked reductions in another major plant sterol, campesterol, were also detected. In contrast to the plant sterols, the levels of lathosterol, a biosynthetic precursor to cholesterol, were significantly increased in the transgenic animals, suggesting that cholesterol synthesis was elevated (8
To determine the effect of ABCG5 and ABCG8 transgene expression on whole-body cholesterol turnover, the daily output of neutral sterols in the feces was measured in transgenic mice and their littermate controls. The amount of neutral sterol in the feces (μmol/d/100 g body weight) was threefold higher in transgenic male mice, and sixfold higher in the transgenic female mice. No significant differences in fecal bile acid excretion were found. The daily stool output (g/100 g body weight) did not differ between transgenic and control mice. Total fecal fat was significantly higher in female transgenic mice but not in male transgenics (Figure ).
Figure 4 The fecal lipid content and composition of ABCG5 and ABCG8 transgenic mice. Feces were collected from 9- to 10-week-old transgenic mice and their littermate controls (n = 10 in each group) for 3 days while they consumed a chow diet. The daily (more ...)
The increased neutral sterol excretion in the transgenic mice may be caused by an increased biliary cholesterol secretion and/or by decreased cholesterol absorption. To determine which of these processes was affected, the concentrations of cholesterol, phospholipids, and bile acids were first measured in bile from transgenic and wild-type animals (four to five in each group). The genotypes of the animals could be accurately assigned by visual inspection of the bile; the bile from transgenic animals was opaque, whereas that of the control animals was clear (Figure a). The bile was subjected to centrifugation (12,000 g
for 1 minute), which resulted in a phase separation with the opaque material floating to the top of the tube over a clear yellow infranatant (Figure a, right). Pooled bile samples (n
= 5) were delipidated (23
) and assayed for protein. The concentration of protein in the two bile samples differed slightly (6.1 vs. 7.3 mg/ml). Microscopic examination of the bile revealed large amounts of amorphous material in the transgenic animals that was not present in the bile from littermate wild-type controls. The bile from the transgenic animals was inspected using a microscope with a polarized filter, and no crystals were observed (data not shown). The most striking difference between the bile of transgenic and that of wild-type mice was the concentration of cholesterol, which was more than fivefold and more than sevenfold higher in the male and female transgenic mice, respectively. There was a modest but significant (P
< 0.05) increase in phospholipid concentration of the bile from the male transgenic animals, but no significant differences in bile acid concentration between transgenic and wild-type animals (Figure b).
Figure 5 A comparison of the appearance of the bile (a) and the biliary lipid concentrations and composition (b) in the ABCG5/ABCG8 transgenic mice and littermate controls. The gallbladder bile was collected from 12-week-old wild-type (n = 4 in each group) (more ...)
The fractional absorption of cholesterol was then measured by the fecal dual-isotope ratio method in the transgenic and wild-type mice. The intestinal absorption of cholesterol was reduced about 50% in both male and female transgenic animals compared with their wild-type counterparts (Figure a). The decrease in fractional absorption of cholesterol could not be attributed to a change in the amount or composition of bile acids, as bile acid pool size and the ratios of cholic acid and muricholic acid were not significantly different in transgenic and wild-type mice (Figure , b and c).
Figure 6 (a) The fractional absorption of dietary cholesterol in the ABCG5/ABCG8 transgenic mice. A total of six male and six female 9- to 10-week-old transgenic mice and their littermate controls were gavaged with [14C] cholesterol and [ (more ...)
Susceptibility to diet-induced hepatic cholesterol accumulation was determined next in transgenic and wild-type mice fed a high-cholesterol (2%) diet for 3 weeks. The mean levels of hepatic cholesterol are shown in Figure a. On a chow diet, the cholesterol content was similar in the transgenic and wild-type animals. As expected, ingestion of a high-cholesterol diet was associated with an increase in hepatic cholesterol in both the male and the female wild-type animals. In contrast, the levels of cholesterol in the liver did not change significantly in the transgenic mice fed a high-cholesterol diet.
Figure 7 (a) The hepatic cholesterol levels in the ABCG5/ABCG8 transgenic and wild-type mice. Individually housed male and female 12-week-old mice of the indicated genotypes were fed a powdered chow diet (0.02% cholesterol) or the same diet containing (more ...)
Quantitative real-time PCR was used to measure the mRNA levels of key genes of cholesterol metabolism in the livers of chow- and cholesterol-fed mice (Figure b). The data are expressed relative to the level of the transcript in the wild-type male animals on a chow diet. High-level expression of human ABCG5 and ABCG8 appeared to selectively increase mRNA levels of genes participating in cholesterol synthesis (HMG-CoA reductase; HMGCR) without major changes in the levels of mRNAs encoding proteins involved in transcriptional regulation of cholesterol (sterol regulatory element–binding proteins-1c and -2; SREBP-1c and SREBP-2), bile acid synthesis (cholesterol 7α-hydroxylase; CYP7A1), hepatic cholesterol uptake (LDL receptor; LDLR), and cholesterol trafficking (ABC transporter A1; ABCA1). We also checked the hepatic mRNA levels of scavenger receptor class B type I (SR-BI), fatty acid synthase, sterol 12α-hydroxylase, short heterodimer partner, farnesoid X receptor, and Na+-taurocholate–cotransporting polypeptide, and no significant changes in these mRNAs were seen between transgenic and wild-type chow-fed animals (data not shown).
As expected, ingestion of a high-cholesterol diet was associated with reduced expression of HMGCR
and increased mRNA levels of CYP7A1. The levels of SREBP-1c mRNA, which is an LXR target (24
), were also increased. In general, expression of the transgene attenuated the effect of cholesterol feeding on suppression of cholesterol-regulated genes.
To examine the effect of robust expression of ABCG5
on cholesterol synthesis in the liver and intestine, we measured in vivo cholesterol synthesis using tritiated water (22
). Cholesterol synthesis in the liver was increased in both the male (twofold) and the female (about fourfold) transgenic mice (Figure ), which is consistent with the larger increase in HMGCR mRNA levels in the female transgenic animals. Cholesterol synthesis was increased threefold in the duodenum of female transgenic mice, decreased in the jejunum and ileum of male transgenic mice, and decreased in the ileum of female transgenic mice. No significant differences in fatty acid synthesis were found between the transgenic and wild-type mice (data not shown).
Figure 8 In vivo cholesterol synthesis rates in the ABCG5/ABCG8 transgenic mice and wild-type controls. Four-month-old transgenic and control mice (n = 6 in each group) maintained on a chow diet were injected intraperitoneally with 40 mCi of [ (more ...)