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Human Molecular Genetics Pages 1347-1354  

Targeted disruption of the mouse lysosomal acid lipase gene: long-term survival with massive cholesteryl ester and triglyceride storage
Introduction
Results
   Generation of LAL-deficient mice
   LAL gene product analyses
   The phenotype of lal-/lal- mice
   Lipid analyses
   Histological analyses
Discussion
Materials And Methods
   Targeting vector (pKO-LAL) construction
   Gene targeting of embryonic stem cells
   Analysis of DNA
   Northern blot analysis
   Lysosomal acid lipase activity assays
   Western blot analysis
   Light and electron microscopic analyses of mouse tissues
   Free cholesterol, CE and TG analyses
Acknowledgements
References

Targeted disruption of the mouse lysosomal acid lipase gene: long-term survival with massive cholesteryl ester and triglyceride storage

Targeted disruption of the mouse lysosomal acid lipase gene: long-term survival with massive cholesteryl ester and triglyceride storage

Hong Du, Ming Duanmu, David Witte1 and Gregory A. Grabowski*

Divisions of Human Genetics and 1Pathology, Children's Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA

Received April 24, 1998; Revised and Accepted June 12, 1998

Lysosomal acid lipase (LAL) is essential for the hydrolysis of the triglycerides and cholesteryl esters in lysosomes. Its deficiency produces two phenotypes, a severe infantile-onset variant, Wolman disease (WD), and a later onset variant, cholesteryl ester storage disease (CESD). A mouse model with a LAL null mutation was produced by targeting disruption of the mouse gene. Homozygote knockout mice (lal-/lal-) produce no LAL mRNA, protein or enzyme activity. The lal-/lal- mice are born in Mendelian ratios, are normal appearing at birth, and follow normal development into adulthood. However, massive accumulation of triglycerides and cholesteryl esters occurs in several organs. By 21 days, the liver develops a yellow-orange color and is ~1.5-2.0× larger than normal. The accumulated cholesteryl esters and triglycerides are ~30-fold greater than normal. The lal+/lal- mice have ~50% of normal LAL activity and do not show lipid accumulation. Male and female lal-/lal- mice are fertile and can be bred to produce progeny. This mouse model is a phenotypic model of human CESD, and a biochemical and histopathologic mimic of human WD. The lal-/lal- mice provide a model to determine the role of LAL in lipid metabolism and the pathogenesis of its deficiency states.

INTRODUCTION

Lysosomal acid lipase (LAL) (EC 3.1.1.13) is the essential enzyme for the hydrolysis of cholesteryl esters (CEs) and triglycerides (TGs) that are delivered to lysosomes by low density lipoprotein (LDL) receptor-mediated endocytosis (1). By generation of free intracellular cholesterol, this enzyme contributes to the homoeostatic control of plasma lipoprotein levels and to the prevention of cellular lipid overload in liver, spleen and macrophages (2). Deficiency of LAL is associated with two phenotypes in humans: Wolman's disease (WD) and cholesteryl ester storage disease (CESD). WD is a severe infantile-onset phenotype leading to death usually before 1 year of age. Severe hepatosplenomegaly, malabsorption, steatorrhea, abdominal distention, adrenal calcification and failure to thrive are observed in the first few postnatal weeks. Massive, but variable, lysosomal storage of CEs and TGs is present in the liver, adrenal gland, small intestine and other organs (1,2). CESD is a milder, later-onset disease. CE deposition alone is present in many visceral tissues although hepatomegaly may be the only clinical manifestation. Survival beyond middle age can occur, and premature atherosclerosis develops in some patients (2). Almost all CESD patients have hypercholesterolemia (2), whereas most WD patients have normal plasma lipid levels. Severe deficiency of LAL activity is present in both diseases. Interestingly, CESD patients have only CE accumulation although LAL has activity toward both CEs and TGs.

The mouse and human enzymes (~75% identical and 95% similar amino acids) are encoded by genes (3-8) on chromosome 10 (q23.2-23.3, human) (9). The mouse and human genes have a similar exon-intron organization (8). The mLAL mRNA and protein expression patterns were similar to the tissue and cellular involvement in WD, i.e. high level expression in hepatocytes, splenic cells, small intestinal epithelial cells, and the zona fasciculata and zona reticularis of the adrenal cortex.

LAL is a glycoprotein of 378 amino acids that is trafficked to the lysosome via the mannose-6-phosphate receptor system (10). The purified enzyme cleaves CEs and TGs in vitro using phospholipid/detergent systems (11). Ser153 has been defined tentatively as a part of the Asp-Ser-His catalytic triad common to many lipases (11,12). Other analyses of mutagenesis have indicated the potential for altering substrate preferences, i.e. CE versus TG, by substituting critical aspartate and/or cysteine residues (13,14). About 14 disease mutations have been described in LAL genes from WD and CESD patients (15-25). To date, heterologous expression of LALs of such mutations has not provided insight into the enzymatic basis of the differential storage in WD and CESD (11,15,22).

In this communication, we report the phenotype of mice bearing a disruption of the mLAL gene to include reproductively viable homozygotes with storage of TG and CE in a distribution like WD, but survival like CESD.

RESULTS

Generation of LAL-deficient mice

Following electroporation of the targeting vector (Fig. 1A) and blastocyst injection of targeted embryonic stem (ES) cells (Fig. 1B and C), 24 chimeric mice were identified by coat color (16 males and eight females). The males were bred to CF-1 females (non-agouti, white) and 10 males were germline transmitters. Among 150 chinchilla coat-colored mice, 71 were heterozygous for the lal- (disrupted), gene. The lal+/lal- offspring were intercrossed, and their offspring were analyzed by PCR analysis with primers E4f (m1) and HPRT (hp), and m1 and E5r(r5) to identify the genotypes, and confirmed by Southern blot analysis (Fig. 1D). A total of 244 F2 mice have been born and the offspring show Mendelian ratios for the respective genotypes: 63 lal+/lal+ (25%), 114 lal+/lal- (46.7%) and 67 lal-/lal- (27.5%). The homozygous LAL-deficient mice developed normally and are fertile. Crosses of lal-/lal- mice produced all lal-/lal- litters of 8-10 pups. These breedings indicate that there is no embryonic lethality associated with the lal-/lal- genotype.A

Figure 1. Targeting vector of mLAL. (A) Development of the LAL gene targeting vector and predicted results. The wild-type allele with exons 4-8 and associated restriction sites, the targeting construct and the targeted allele are shown. For PCR analysis, a 2.2 kb wild-type allele (with primers m1 and r5), a 1.6 kb targeted allele (with primers m1 and hp) and a 1.5 kb targeting construct (with primerS m2 and hp) are shown. For the genomic DNA analysis, probe A and BglII digestion were used. Probe B and BamHI digestion were used. The predicted wild-type, and targeted allele restriction fragment sizes for BamHI and BglII are shown. (B) Diagnostic PCR screening for DNAs from ES cells. PCR with hp and external primer m1 (lane E), and hp and internal primer m2 (lane I) were performed for each ES cell DNA sample. The PCR products were analyzed on a 1.2% agarose gel. (C) Southern analysis of DNAs from ES clones. (D) Southern analysis of DNAs from mouse tails.

LAL gene product analyses

Total RNA from the liver of lal-/lal- mice probed with the mLAL cDNA showed absence of the ~3 kb wild-type mRNA. No other transcripts were detected (Fig. 2A). In comparison, discrete specific bands at ~3 kb were present in extracts from lal+/lal+ and lal+/lal- genotypes. The LAL enzyme protein and activities in liver, spleen and small intestine from the mice with the various genotypes were determined from tissue homogenates of freshly sacrificed autopsy samples. Western analyses of tissue homogenates of lal-/lal- mice showed the absence of LAL (Fig. 2B). No LAL activity, above background, could be detected in the lal-/lal- mice, whereas the lal+/lal- mice have ~50-60% activity of the lal+/lal+ (Fig. 2C).

The phenotype of lal-/lal- mice

The lal-/lal- mice appear normal, and behave normally at birth and throughout life. Individual mice were weighed at ages 4-7 weeks, and the lal-/lal- mice had body weights that were the same in all genotypes, except that, in particular founder lines, lal-/lal- mice weighed ~15-20% less than their lal+/lal+ and lal+/lal- litter mates at age 4 weeks. No significant difference in body weight was observed between lal+/lal+ and lal-/lal- mice at age 8-8.5 weeks (Table 1). There are no other gross abnormalities in growth or development of these mice compared with their unaffected siblings. It should be noted that body weight and organ weights were significantly influenced by the founder line.

Figure 2. Analyses of LAL gene products by northern and western blot, and enzyme assays. (A) Northern analysis of LAL mRNA from mouse liver. Total RNA (30 µg) from mouse livers (+/+, wild-type; +/-, heterozygote; -/-, targeted) were loaded. The membranes were hybridized with a full-length mLAL cDNA probe. A human [beta]-actin cDNA was used as control. (B) Western blot analysis of mLAL protein extracted from mouse liver. The same amount of total protein (14 µg) was loaded for each lane. The Escherichia coli-expressed hLAL was used as positive control (lane 2). (C) LAL enzyme activity assay of mouse liver extract using [14C]cholesteryl oleate as substrate.

At 4 and 8 weeks of age, various tissues from lal-/lal- mice were obtained following sacrifice. The liver ranged from yellow to orange in color (Fig. 4A). The hepatic color progresses from yellow to a more orange color with age. The liver weights in the lal-/lal- mice were greater than those from age-matched lal+/lal+ mice (Table 1, Fig. 4A). The livers of the lal+/lal- mice are similar to those from lal+/lal+ mice in appearance color, size and weight (Table 1, Fig. 4A). The spleen of the lal-/lal- mice also showed a pale yellow color and appeared slightly larger in size, but this was not significant (Table 1). There were no gross abnormalities in other tissues from lal-/lal- mice including small intestine, kidney, brain and adrenal glands.

Table 1. Mouse body and tissue weight
Genotype Age (days) Body weight (g) Liver (g) Spleen (g) Kidney (g)
lal+/lal+ (n = 7)a 53-56 28.3 ± 2.3 1.8 ± 0.3 0.11 ± 0.03 0.3 ± 0.04
lal+/lal- (n = 8)a 53-56 29.5 ± 3.4 1.9 ± 0.1 0.15 ± 0.04 0.3 ± 0.03
lal-/lal- (n = 9)a 53-56 28.5 ± 4.1 3.4 ± 0.2 0.3 ± 0.3 0.2 ± 0.06
aNumber of animals analyzed.

Lipid analyses

Total lipids from liver, spleen and small intestine were analyzed by thin-layer chromatography (TLC). The results are presented for 6-week-old mice (Table 2). Total CEs in lal/lal- livers were estimated at ~32-fold greater than those in lal+/lal+ livers. The levels of CEs in lal+/lal- mouse liver were similar to those in lal+/lal+ controls. TG concentrations were estimated at ~35-fold increased in lal-/lal- compared with those in lal+/lal+ mice. Accumulations of di-acylglycerol and mono-acyl glycerides and free cholesterol were also noted on TLC. The CEs also accumulated in spleen and small intestine (Fig. 3B and C). The CE levels in spleen and small intestine were ~10- and ~2-fold higher in the affected mice. Interestingly, splenic TG levels were significantly decreased in lal-/lal- mice compared with lal+/lal+ mice. In lal+/lal- mice, the TG levels were ~3-fold increased. Repeated analyses of lal-/lal- mice from different chimera founders showed similar results.


Figure 3. Histology analyses of mouse tissues. (A) Gross appearance of liver and spleen from lal-/lal- (left), lal+/lal- (middle) and lal+/lal+ (right). Only the homozygote mutant liver shows yellow discoloration from lipid accumulation. The spleen below also shows subtle discoloration. (B) Photomicrograph illustrating lal+/lal+ liver (left) and lal-/lal- liver (right). The mutant liver shows enlarged vacuolated hepatocytes which are compressing the sinusoidal spaces. (C) Oil red-O staining for lipid in the lal+/lal+ (left) and lal-/lal- liver (right). Only the mutant liver shows extensive red droplets indicating lipid accumulation. (D) Unstained frozen section of the lal-/lal- liver illuminated with polarized light. The hepatocytes show bright white birefringent crystal-like cytoplasmic inclusions that were not present in either the lal+/lal- or lal+/lal+ liver (not shown). (E) Adrenal section from lal+/lal+ (left) and lal-/lal- (right). In the affected adrenal, there is an expanded pale zone (arrows) involving the innermost layer of the adrenal cortex adjacent to the medulla. This pale zone is a result of lipid accumulation in the cells. (F) Oil red-O staining of the lal+/lal+ (left) and mutant adrenal (right). Although the cortex shows lipid staining droplets in both the wild-type and mutant adrenals, the pale zone in the mutant (E) shows large cells with abundant lipid accumulation (arrows) at the interface between cortex and medulla (me). (G) Birefringent inclusions under a polarized light source at this same interface in the affected adrenal. (H) Oil red-O stain of small bowel mucosa from lal+/lal+ (left) and the lal-/lal- (right) mice. The villi from the mutant mouse are slightly enlarged and show lipid accumulation (red droplets, arrow) in the lamina propria. Magnification: (B) ×450; (C-H) ×200.

Figure 4. Lipid analyses by thin-layer chromatography. The extracts from tissues were normalized by wet tissue weight. One lal+/lal+ (lane 4), one lal+/lal- (lane 5) and four lal-/lal- (lanes 6-9) samples are shown. Cholesteryl oleate (CE), triolein (TG) and free cholesterol (C) were used as standard. (A) Liver; (B) spleen; (C) small intestine.


The plasma cholesterol and TG levels were evaluated in 4- and 8-week-old mice without high fat or high cholesterol challenge. No differences were detected among the various genotypes (Table 3).

Table 2. Cholesteryl ester and triglyceride contents in mouse livers and spleens
Genotype Liver Spleen Small intestine
CE TG CE TG CE TG
lal+/lal+ (n = 5) 1.2 ± 0.1 1.1 ± 0.3 2.6 ± 0.8 6.6 ± 1.3 18.8 ± 0.5 5.1 ± 0.3
lal+/lal- (n = 5) 1.7 ± 0.2 2.1 ± 0.1 2.7 ± 0.5 18.2 ± 6.8 13.5 ± 1.5 3.9 ± 0.3
lal-/lal- (n = 4) 38.7 ±11.4 38.7 ± 12.1 25.5 ± 5.4 1.8 ± 0.7 30.7 ± 4.0 6.4 ± 1.4
All values are given as µg/mg wet weight of tissue ± SD.

Table 3. Total cholesterol and triglyceride levels in plasma
Genotype Total cholesterol (mg/dl) Triglycerides (mg/dl)
lal+/lal+ (n = 5) 121.4 ± 16.3 92.6 ± 8.9
lal+/lal- (n = 5) 115.1 ± 12.1 101.4 ± 9.6
lal-/lal- (n = 5) 127.6 ± 26.4 91.1 ± 7.3

Histological analyses

The light microscopic examination of the affected mice was performed at 3 and 8 weeks of age. The pathological findings were evident in the liver, intestine and adrenal glands. The lobular architecture of the liver was intact, with no evidence of fibrosis or portal expansion. The hepatocytes were slightly enlarged, causing mild compression of the sinusoidal spaces. There was diffuse vacuolization of the hepatocytes in the lal-/lal- mice compared with the lal+/lal+ controls (Fig. 4B). In frozen section preparations, these hepatocytes showed prominent lipid droplets when stained with Oil red-O (Fig. 4C) or Sudan B black (data not shown). When viewed under a polarized light source, the hepatocytes showed numerous anisotropic crystals (Fig. 4D). Electron microscopic analyses of liver showed numerous membrane-bound lipid containing droplets (not shown). Occasional Kupffer cells also showed similar cytoplasmic inclusions (data not shown). The adrenal glands showed a pale staining expanded zone in the innermost portion of the cortex where large swollen vacuolated cells had accumulated (Fig. 4E). These vacuolated cells showed large Oil Red-O-staining droplets of lipids (Fig. 4F) and this zone also showed accumulations of numerous anisotropic crystals under polarized light (Fig. 4G). No microcalcifications were observed in the adrenal glands. The small intestinal villi showed mildly distended villi with small clusters of Oil Red-O lipid-stained (Fig. 4H) foamy cells in the lamina propria. The mucosal epithelium lining the villi also showed small amounts of small Oil Red-O-staining lipid droplets in the cytoplasm. Anisotropic crystals were also observed in the small bowel mucosa (data not shown). No significant lipid accumulation was observed in cytospin preparations of bone marrow cells or in sections of the spleen. No storage cells were observed in multiple brain tissue sections.

DISCUSSION

The creation of mouse models of human disease by gene targeting has provided an essential tool for the evaluation of pathophysiology and development of therapeutic approaches to genetically caused and influenced states in man. Many of these models provide phenocopies of the human diseases, although rarely are the phenotypes precisely matched. We have created a disruption of the LAL gene that creates a lal-/lal- genotype and a phenotype that has characteristics of the two human disorders associated with LAL deficiency, WD and CESD. Clinically, the mice resemble CESD, i.e. the lal-/lal- mice survive to reproductive age with few phenotypic manifestations. Lower than normal weight is the primary manifestation at a gross level. In comparison, the lal-/lal- mice biochemically resemble WD with the large accumulations of CEs and TGs in lysosomes of various organs. The absence of mLAL mRNA and protein indicates that these are null homozygotes for LAL. The demonstration of total deficiency of enzymatic activity is more difficult. There are other cellular lipases that have activity in the pH range of lysosomal lipase and interfere with assays using the fluorogenic oleate substrate (26). In addition, since the animals are born in the expected Mendelian genotypic ratios, this ensures the lack of a complex phenotype encompassing more than what is observable following birth, i.e. there are no embryonic lethal variants in the strains analyzed. Furthermore, the phenotype appears to be homogeneous, and lal-/lal- × lal-/lal- crosses lead to a phenotype that is indistinguishable from that in the parent. The major variation in phenotype is the progressivity of the organ involvement with age.

The spectrum of tissue involvement closely mimics that in human WD. Although in human CESD and lal-/lal- mouse, livers are orange-yellow, the storage of CEs and TGs in this organ more closely resembles human WD. Sacrificing lal-/lal- mice from 4 to 10 weeks of postnatal life shows a progressivity of hepatic color from a grayish-white to an orange-yellow, and an increasing deposition of CEs and TGs. These progress with age. Also, at this age, liver involvement did not include portal fibrosis as observed in WD and CESD. The storage does not appear to interfere with normal functioning, survival or procreation of the lal-/lal- mice.

Involvement of the intestine is highly similar to that described in CESD patients by Partin and Schubert (27). The accumulation of CE crystals in the intestinal mucosa and the distribution of lipid deposition is essentially identical to that seen in human CESD. In comparison, the human small intestine in WD is heavily infiltrated and a pale yellow color (28). It will be important to analyze the lal-/lal- mice on diets of varying lipid content to determine how these effect intestinal pathology. Such dietary changes might promote the steatorrhea common in human WD, but absent in the lal-/lal- mice.

Involvement of the adrenal gland is histologically identical to that observed in early WD in humans (2). We did not observe, even microscopically, adrenal calcification in the lal-/lal- mice, although this is very common, albeit not universal, in human WD. Involvement of the zona fasciculata and reticulis of foam cells is highly similar to that in human WD. Fitoussi et al. (29) proposed a pathophysiology of WD adrenal calcification based upon differential toxicity of mildly oxidized LDL. This mechanism was based on the poor down-regulation of the LDL receptor pathway in WD leading to higher uptake of mildly oxidized LDL and a sustained rise in intracellular Ca2+. This was proposed to lead to cell death and calcium deposition (30). This hypothesis may be directly testable in the lal-/lal- mice. Interestingly, the Yoshida rats, spontaneous LAL deletion mutant rat models of WD, also had no detectable adrenal calcification (31,32).

CEs and TGs are the main lipids that accumulate in the tissues of patients with WD. The level of free cholesterol usually is also increased (2). The lal-/lal- mice have accumulation of CEs in liver, spleen and small intestine. The TG levels in lal-/lal- mice were increased 35-fold in liver, which is higher than that reported in human WD or in the rat model of WD. In comparison, TGs are decreased in affected spleen, which is similar to that observed in the LAL-deficient rats (32,33). Curiously, the TGs are increased (3-fold) in heterozygous mice. Free cholesterol levels are increased in lal-/lal- mouse tissues, including 2- to 3-fold in liver and small intestine, and ~1.5-fold in spleen. These results suggest that the intracellular cholesterol synthesis is enhanced in tissues from lal-/lal- mice due to disruption of the LDL receptor-mediated endocytosis of CEs and resultant HMG-CoA reductase and LDL receptor gene up-regulation (34,35). This mechanism may also account for the normal total cholesterol levels in plasma of lal-/lal- mice since plasma LDL-CE uptake would be promoted.

The lal-/lal- model has several advantages for exploring the physiological role of acid lipase and its deficiency states. These include a biochemical phenotype that resembles the more severe WD, but with the survivability of human CESD. Thus, the full WD phenotype may not be expressible in the mouse due to species difference in lipid metabolism and/or in lipoproteins as a reflection of the genetic backgound (42). In addition, the ability to make lal-/lal- crosses provides the opportunity to explore the pathogenesis of this disease in utero and to initiate investigations into the early pathology and therapy of this disease. In addition, the complete absence of LAL activity provides a tool to evaluate the specificity of acid lipase for cleavage of CEs and/or TGs in vivo. Thus, the recent site-directed mutagenesis and in vitro analyses can be confirmed or refuted in a physiological setting, at least in mice, and there is potential for dissecting the basis of the differential phenotypes of WD and CESD.

MATERIALS AND METHODS

Targeting vector (pKO-LAL) construction

The isolation and characterization of the mouse LAL gene have been described (8). A P1 LAL genomic clone from the 129SV strain was used to construct the targeting vector (Fig. 1). A 1.1 kb AciI-MspI fragment, covering part of exon 4 and intron 4, was amplified by PCR and cloned into the ClaI site of pKO as a short arm, 5[prime] to HPRT and 3[prime] to Herpes simplex virus thymidine kinase (HSV-TK). A 5.5 kb fragment, covering exons 6-7, was inserted into the BamHI and NotI sites of pKO 3[prime] to HPRT (36). The HPRT mini-gene and the HSV-TK gene were used for positive and negative selection, respectively. Orientation was determined by restriction enzyme digestion. The complete vector is shown in Figure 1A.

Gene targeting of embryonic stem cells

pKO-LAL was linearized by NotI, extracted with phenol/chloroform, diluted in 1× phosphate-buffered saline (PBS) and filter sterilized (Millipore). This linearized DNA (20 µg/ml in PBS) was then used for electroporation into hprt- E14tg2a cells [30 × 106 (36,37)] in PBS at 900 V and 14 µF [IBI gene zapper (36)]. The ES cells were plated on dishes containing a mitomycin C-treated mouse embryonic fibroblast feeder layer. After electroporation (24 h), the ES cells were subjected to double selection with 1× HAT (BRL, Life Technology, Gaithersburg, MD) and gancyclovir (2 µM; Syntex). The HAT- and gancyclovir-resistant colonies were picked and transferred to 24-well plates. After expansion and trypsinization, one-third of each clone was stored under liquid N2, used for PCR and Southern blots, and for expansion.

Analysis of DNA

Genomic DNA was isolated from ES cells and mouse tails as described (38). The genomic DNA was spooled, after precipitation in 2 vols of 100% ethanol. ES cells and mice were genotyped by allele-specific PCR and confirmed by Southern blot analysis. PCR primers for the targeted allele were m1 (5[prime]-TGAGGAGGTTGGAGCTAACAGCT-3[prime]), and hp (5[prime]-GCAGTGTTGGCTGTATTTTCCCA-3[prime]), located in the HPRT cassette, giving a 1.6 kb amplification product. The primers for the wild-type lal allele were m1 and r5 (5[prime]-GAAAATCCCTTGCCCTATTCATG-3[prime]), giving a 2.1 kb amplification product. To validate these results, the primer set, m2 (5[prime]-ATGTGTGGATGGGAAACAGCAGA-3[prime]) and hp, giving a 1.5 kb amplification product, was used for each clone (Fig. 1A and B). Paired primers (10 pmol) and ES cell or mice tail genomic DNA (1 µg) were used for PCR (1 min at 94°C, 1 min at 63°C and 2 min at 72°C for 35 cycles with hot start). Amplified DNAs were analyzed on 1.2% agarose gel. For Southern blot analysis, ES cell or mouse tail DNA (10 µg) was digested with BglII or BamHI and analyzed as described (8). Two external mLAL probes, 5[prime] to the small arm and 3[prime] to the long arm, were used for confirmation of LAL gene targeted in ES cells and mice. Signals were detected by phosphorimaging.

Northern blot analysis

Total RNA from mouse liver was isolated from 0.2 g of frozen tissue using a Qiagen RNeasy kit. Total RNA (10 µg) was run on a 1% agarose gel containing formaldehyde (39) and transferred to a nylon membrane (Hybond N+; Amersham). The membrane was hybridized with 32P-labeled mLAL cDNA probe (5). The integrity of the RNA was checked by ethidium bromide staining of 18S and 28S rRNAs. The filter was also stripped and rehybridized with a human [beta]-actin cDNA probe to demonstrate the integrity of this mRNA (40).

Lysosomal acid lipase activity assays

mLAL activities were estimated with the fluorogenic substrate, 4-methylumbelliferyl-oleate and the natural substrates, 14C-labeled cholesteryl oleate and triolein (11). All assays were conducted in duplicate and the results are the means of three experiments. Assays were linear within the time frame used, and <10% of substrates were cleaved.

Western blot analysis

Tissues from liver, spleen, intestine and kidney were harvested and homogenized in lysis buffer (10 mM Na2PO4, pH 6.8, 1 mM EDTA, 10 mM [beta]-mercaptoethanol, 0.25% Triton X-100 and 0.02% Na azide) using an electric homogenizer. Homogenates were sonicated further for 5 min on ice (11). The supernatant was harvested after centrifugation (10 000 g) at 4°C for 10 min. The protein concentration was determined by the protein assay (Bio-Rad). The proteins were resolved on 12.5% SDS-PAGE and transferred into PVDF membrane using the Phast system (Pharmacia). The western blot analyses were as described (11).

Light and electron microscopic analyses of mouse tissues

Light microscopic examination of the livers, spleen, adrenal glands, intestine, kidneys, reproductive tract, skeletal muscle, heart, thymus, pancreas and brain was performed. The paraffin sections were stained with hematoxylin/eosin and frozen sections with Oil Red-O for light microscopic analysis. Anisotropic cholesterol crystals were detected in unstained sections of tissue. The tissue was fixed in 4% paraformaldehyde, saturated with 30% sucrose and cryostat sections cut at 10 µm and examined under a polarized light source. Liver tissue samples for electron microscopy were fixed in 3% gluteraldehyde in cacodylate buffer, post-fixed in 1% osmic acid, embedded in LX resin (Ladd Research Industries), and ultrathin sections stained with lead uranyl acetate were examined on a Zeiss EM912 electron microscope.

Free cholesterol, CE and TG analyses

Total tissue lipids were extracted by the procedure developed by Folch (41). Briefly, tissues pulverized in liquid N2 were extracted with chloroform:methanol (2:1 v/v) [1:20; w/v], and then with 0.2 vols of water. The phases were clarified by adding a few drops of 2 M KCl. The lower organic phase was collected through a separation funnel, and the upper phase was re-extracted with chloroform:methanol (2:1). The organic phases were pooled, dried under N2 and then under vacuum. The dried white films were solubilized in small volumes of chloroform:methanol (2:1) and aliquots were applied to TLC plates (Whatman PE Sil G, 250 µm). The lipids were resolved in n-hexane:ethyl ether:acetic acid (80:20:2 by vol) and visualized by exposure to I2 vapors. Cholesteryl oleate, free cholesterol and triolein (Nu-chek) were used as standards. The resultant TLC plates were scanned with a Nikon Scantouch and the levels of lipids were quantified against standards on the same plate with ImageQuant 2.1 program (Molecular Dynamics).

ACKNOWLEDGEMENTS

The authors thank Dr Hung Li for the pKO vector, Drs John Duffy and Karen Yager for their help with ES cells, the Children's Hospital Research Foundation Transgenic Core Facility (Dr S.S. Potter, Director) for its invaluable assistance with blastocyst injection, Lisa Artmayer and Jaya Mishra for their expert technical assistance, Alica Emley for the color photograph, Dr David Hui for conducting the plasma lipid analyses, and Maryann Koenig for her expert clerical assistant. This work was supported grants from the American Heart Association, National Center (9501340; H.D.) and NIH (DK 36729; G.A.G.). Additional support was from the Lucille P. Markey Charitable Trust and the Children's Hospital Research Foundation.

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*To whom correspondence should be addressed. Tel: +1 513 636 7290; Fax: +1 513 636 7297; Email: grabg0@chmcc.org

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