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Human Molecular Genetics, 2003, Vol. 12, No. 24 3349-3358
DOI: 10.1093/hmg/ddg366
© 2003 Oxford University Press

Mouse model of N-acetylgalactosamine-6-sulfate sulfatase deficiency (Galns-/-) produced by targeted disruption of the gene defective in Morquio A disease

Shunji Tomatsu1,3,*, Koji O. Orii1,4, Carole Vogler2, Jun Nakayama5, Beth Levy2, Jeffrey H. Grubb1, Monica A. Gutierrez1, Soomin Shim1, Seiji Yamaguchi3, Tatsuo Nishioka1, Adriana Maria Montaño6, Akihiko Noguchi7, Tadao Orii4, Naomi Kondo4 and William S. Sly1

1Edward A. Doisy Department of Biochemistry and Molecular Biology and 2Department of Pathology, Saint Louis University School of Medicine, St Louis, MO, USA, 3Shimane Medical University, Shimane, Japan, 4Department of Pediatrics, Gifu University School of Medicine, Gifu, Japan, 5Department of Pathology, Shinshu University School of Medicine, Matsumoto, Japan, 6Department of Pediatrics, Cardinal Glennon Children's Hospital, Saint Louis University, St Louis, MO, USA and 7Department of Biosystems Science, Graduate University for Advanced Studies, Kanagawa, Japan

Received August 13, 2003; Revised October 10, 2003; Accepted October 21, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mucopolysaccharidosis IVA is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), a lysosomal enzyme required for the stepwise degradation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). To generate a model for studies of the pathophysiology and of potential therapies, we disrupted exon 2 of Galns, the homologous murine gene. Homozygous Galns-/- mice have no detectable GALNS enzyme activity and show increased urinary glycosaminoglycan (GAGs) levels. These mice accumulate GAGs in multiple tissues including liver, kidney, spleen, heart, brain and bone marrow. At 2 months old, lysosomal storage is present primarily within reticuloendothelial cells such as Kupffer cells and cells of the sinusoidal lining of the spleen. Additionally, by 12 months old, vacuolar change is observed in the visceral epithelial cells of glomeruli and cells at the base of heart valves but it is not present in parenchymal cells such as hepatocytes and renal tubular epithelial cells. In the brain, hippocampal and neocortical neurons and meningeal cells had lysosomal storage. KS and C6S were more abundant in the cytoplasm of corneal epithelial cells of Galns-/- mice compared with wild-type mice by immunohistochemistry. Radiographs revealed no change in the skeletal bones of mice up to 12 months old. Thus, targeted disruption of the murine Galns gene has produced a murine model, which shows visceral storage of GAGs but lacks the skeletal features. The complete absence of GALNS in mutant mice makes them useful for studies of pharmacokinetics and tissue targeting of recombinant GALNS designed for enzyme replacement.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mucopolysaccharidosis type IVA (MPS IVA: Morquio type A) is an autosomal recessive disorder caused by the deficiency of lysosomal N-acetylgalactosamine-6-sulfate sulfatase (GALNS: E.C.3.1.6.4). GALNS is one of several sulfatases required to degrade glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate (C6S). As in other mucopolysaccharidoses, MPS IVA patients show a broad spectrum of clinical severity. Phenotypes vary from the classical form with severe bone dysplasia (spondyloepiphyseal dysplasia), short trunk dwarfism, hearing loss, heart valve involvement, corneal opacity, and a life span of 20–30 years, to milder forms with fewer manifestations. Patients with mild forms can have a close to normal quality of life with little bone and visceral organ involvement. Even in severe forms of the disease, there has been no evidence of mental retardation or description of lysosomal storage in brain.

The broad range of clinical phenotypes seen in MPS IVA is presumed to be the result of many different GALNS mutations. Purification of the enzyme, followed by isolation and characterization of the full-length cDNA encoding the human GALNS protein and genomic sequence (1–3) has facilitated investigations into the molecular genetic heterogeneity in MPS IVA.

We have carried out molecular analyses of over 200 MPS IVA patients from 25 ethnic or geographic origins (4–17). To date, over 100 different mutations have been identified. Genotype–phenotype correlation exists for many of these mutations. Two large deletions lead to the severe form of MPS IVA and many point mutations produce a broad range of phenotypes.

GALNS is encoded by a member of the sulfatase gene family of which 10 different sulfatase human genes have been cloned. All of these sulfatase genes and their gene products are closely related, showing 20–35% similarity at the amino acid level. The amino acid residue C79 in exon 2 of human GALNS gene was identified as a catalytic site conserved among all known sulfatases (18).

Although deficiency of GALNS has been described in humans, it has never been reported in other species. The lack of an animal model has limited the development and testing of novel enzyme replacement and/or gene therapy regimes for MPS IVA. The development of a murine model for MPS IVA could potentially serve as an important experimental system for the development of both enzyme replacement therapy (ERT) and gene therapy regimens. Information obtained from treating the mouse model could be important for developing treatments for the human disease.

This report describes the targeted disruption of murine Galns gene, characterization of the resulting deficiency of GALNS, and pathological findings. Furthermore, the initial phenotypic observations of our model are compared with the other murine models of MPS disorders.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Targeting of Galns in ES cells and generation of Galns knock-out mice
GALNS-deficient mice with a deletion of portions of intron 1 and exon 2 were generated by gene targeting in murine embryonic stem cells as described in Methods and shown schematically in Figure 1. This strategy generated mice heterozygous for the mutation that no longer contained the neor marker, with the expected Mendelian segregation at birth (19). Combined data from crosses between these heterozygous Galns+/- mice showed a normal distribution of 25% (+/+), 53% (+/-), and 22% (-/-), in over 100 offspring analyzed, suggesting that the survival rate of Galns-/- offspring is not reduced. Total cellular RNA was isolated from tissues of homozygous Galns-/-, heterozygotes and wild-type mice using a guanidinium/phenol solution. No Galns transcript was observed in Galns-/- mice by Northern blot (Fig. 2) and RT–PCR before or after removal of the neor gene by Cre (data not shown).



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  		Figure 1. Targeted disruption of the Galns gene. (A) The structure of the endogenous wild-type gene, the targeting construct, the disrupted allele, and the neor-excised allele are presented schematically on successive lines. The shadow bars above lines represent the 5' probe used for Southern blots. The large arrows show the loxP sequences. Abbreviations for restriction enzymes are: RI, EcoRI; S, SacI; X, XbaI; B, BamHI; H, HindIII; K, KpnI. (B) Southern blot identification of the disrupted gene is shown (bottom): the wild-type and targeted mutant alleles were identified by hybridization of the 5' probe to KpnI restriction fragments. The hybridized fragment is smaller in the disrupted allele than in the normal allele (7.1 versus 12.0 kb) because of the presence of the additional KpnI site in the multicloning site of targeting vector. Lanes 1 and 6, wild-type mouse (normal alleles); lane 2, targeted ES cell line (one wild-type, one mutant allele); lanes 3–5, Galns+/- mice produced from initial chimeras (both bands present). (C) Detection of the knockout allele at the murine Galns gene by genomic PCR amplification. Two sets of primers produce a single 1.2 kb fragment for homozygotes, two fragments of 1.2 and 1.8 kb for heterozygotes, and a single 1.8 kb fragment for wild-type mice. Lane 1, Galns-/- (1.2 kb); lane 2, Galns+/- (1.2 and 1.8 kb); lane 3, a normal control, Galns+/+ (1.8 kb).

 


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  		Figure 2. Expression of murine Galns mRNA. (A) Northern blot analysis of murine Galns mRNA from the livers (A), spleens (B), kidney (C), and lungs (D) of wild type (+/+) and homozygote (-/-) mice. Twenty micrograms of total RNA from each tissue were analyzed by northern blotting with murine Galns cDNA 32P-labeled probe. The 2.3 kb murine Galns mRNA transcript was expressed in wild-type tissues (lanes 1 and 2) and was not detected in Galns+/+ tissues (lane 3).

 
Phenotype of the GALNS-deficient mice
Galns-/- mice were normal in appearance and were generally healthy. Both males and females were fertile at least up to 12 months of age, as indicated by the normal litter sizes produced from mating Galns-/- males to Galns+/- females or vice versa. No obvious difference in weight or mortality rate was seen in the first 12 months of life. At 12 months of age, radiographic analysis of the axial and appendicular skeleton of Galns-/- mice did not reveal any obvious abnormalities of long bones, thorax, or calvaria. Thus, homozygous mice carrying Galns-/- have no obvious phenotype and lack the skeletal features of human MPS IVA patients.

Biochemical findings
Enzyme activity.
GALNS enzyme activity was determined in homogenates of liver, kidney, brain, spleen, lung, heart, muscle, bone, bone marrow cells from femur, and serum. In wild-type mice, the activity was highest in the kidney, bone marrow, spleen and liver and lowest in brain, skeletal muscle and heart. Homozygous mutant mice had no activity, confirming that the targeted allele was null. Heterozygotes had half the normal activity (Table 1). The gene dosage response was seen in all tissues tested.


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  		Table 1. Tissue levels of GALNS activity (units/mg)
 
Secondary elevation.
Several other lysosomal enzymes including {alpha}-galactosidase and ß-galactosidase exhibited slight but not statistically significant secondary elevations in several tissues including liver, spleen, brain and serum (Fig. 3). Elevations of ß-glucuronidase and ß-hexosaminidase activities (data not shown) were even less pronounced, but the trend was the same. In contrast, {alpha}-mannosidase activity was not elevated in the GALNS-deficient mice.



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  		Figure 3. Secondary elevation of {alpha}-galactosidase and ß-galactosidase activities in mutant mice compared with heterozygotes and control littermates. Enzyme activities were assayed in tissues of homozygous mutant, heterozygous and wild-type mice. Enzyme activities were expressed as nmol/h/mg (units/mg) protein from tissue homogenates prepared from Galns+/+, Galns+/- and Galns-/- control mice. Six mice were analyzed for each group. Activities are expressed as the mean with observed range. The average ß-galactosidase levels of normal control in serum, kidney, spleen, liver, lung and brain are 94 units/ml, 339, 614, 117, 72 and 86 units/mg protein, respectively. The average {alpha}-galactosidase levels of normal control in serum, kidney, spleen, liver, lung and brain are 13.8 units/ml, 24, 71, 37, 14 and 20 units/mg protein, respectively.

 
Urinary GAG excretion (Fig. 4).
Urine was collected from Galns-/- (n=10), +/+ and +/- littermates (mixed background; n=12), and C57BL/6 mice (n=16). Additionally, urine was collected from four different MPS VII mouse strains; gusmps/mps (n=7), Gustm(E536A)Sly (n=8), Gustm(L175F)Sly (n=7), and Gustm(E536Q)Sly (n=5) (20). Total urinary GAG excretion expressed as a ratio of milligrams GAGs to grams creatinine excretion was elevated in Galns-/- mice when compared with B6 mice (P=0.034) and their littermates (P=0.063). Galns-/- mice at 2–6 months of age excreted 387±275 mg GAG/g Cre, which was on average 2- and 6-fold greater than their littermates (142±142 mg GAG/g Cre) and normal control B6 mice (60±55 mg GAG/g Cre), respectively. Severe murine MPS VII models (gusmps/mps and Gustm(E536A)Sly) excreted 5-fold greater levels of GAGs than control littermates. The level of increase in Galns-/- mice was more comparable to that of milder murine MPS VII models (Gustm(L175F)Sly and Gustm(E536Q)Sly) (20). Urinary KS was not detectable in Galns-/- mice or normal control mice.



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  		Figure 4. Urinary glycosaminoglycan excretion compared with C57BL/6 mice and four different MPS VII mouse models. The abbreviation of mouse below each bar describes as follows: B6, C57BL/6; mixed, mixed strain normal control litter mates; MPS IVA, Galns-/-; E536Q, Gustm(E536Q)Sly (a mild form); L175F, Gustm(L175F)Sly (a mild form); E536A, Gustm(E536A)Sly (a severe form), and gusmps/mps, the original MPS VII mouse (a severe form). We compared the value of each genotype with the value of control B6 mice by one-way ANOVA analysis. Results are expressed as mean±SD and P values are indicated if there is statistical significance.

 
Corneal GAGs and KS.
The level of total GAGs in mutant cornea (extracted from 10 mice and pooled) was 2.3-fold higher than that in littermate corneas (2.94 versus 1.23 mg GAG/g wet tissue weight) while the corneal KS level per mg protein in pooled extracts of corneal tissue from 10 mutant mice was 1.7 fold of littermates (176 versus 109 ng KS/mg protein).

Pathological features
The Galns-/- mice evaluated were 2 months (n=3), 7 months (n=1) and 12 months (n=3) of age. Histological evidence of storage was apparent in most of the affected mice (Fig. 5) although there was variability from mouse to mouse that did not always correlate with the animal's age. In the liver (Fig. 5B) and the spleen (not shown), storage was restricted to the sinus lining cells. There was no storage in the hepatic parenchymal cells. In the kidney (Fig. 5D), particularly in the 12-month-old mice, there was a vacuolar change affecting the visceral epithelial cells of the glomeruli. Tubular epithelial cells had no evidence of storage. Cells at the base of the heart valves (Fig. 5H) and interstitial cells in the heart had vacuolar distention. Three of seven mice had slight distention of the sinus lining cells in the bone marrow (Fig. 5F). There was no apparent storage in the chondrocytes or in the osteoblasts. In the brain, hippocampal neurons (Fig. 5J) and neocortical glial cells from five Galns-/- mice had storage. In four, neocortical neurons also had minimal evidence of lysosomal storage. There was a small amount of storage in meningeal cells (Fig. 5L) in six Galns-/- mice. The cornea from three mice had very rare stromal fibroblasts containing cytoplasmic vacuoles (data not shown). The long bones from the Galns-/- showed no morphological alteration when compared as a group with the bones from control mice.



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  		Figure 5. Morphological alteration in tissues of Galns-/- mice. Galns+/- mice had no evidence of lysosomal storage in liver (A), kidney (C), bone marrow (E), heart (G), hippocampus (I) and meninges (K). (B) Liver from a 2-month-old Galns-/- mouse has Kupffer cells that are distended with lysosomal storage (arrow). The hepatocytes have no storage. A similar amount and distribution of storage was present in 12 month old Galns-/- mice. (D) Kidney from a 12-month-old Galns-/- mouse has vacuolar change affecting the visceral epithelial cells of the glomeruli (arrow). Tubular epithelial cells had no evidence of storage. (F) The sinus lining cells in bone marrow of a 12-month-old Galns-/- mouse (arrow) contain a small amount of storage. (H) In the heart valve of the 12-month-old Galns-/- mouse, cells at the base of the heart valves (arrow) and interstitial cells in the heart had vacuolar distention. (J) The hippocampal neurons (arrow) in a 12-month-old Galns-/- mouse have lysosomal storage. (L) The meninges covering the brain contain cells distended with lysosomal storage (arrow) in a 12-month-old Galns-/- mouse. (A–L, toluidine blue, 1 cm=27 µm).

 
In order to examine the distribution patterns of KS and C6S in the corneas of wild-type and Galns-/- mice, immunohistochemistry was performed using specific antibodies against KS and C6S (Fig. 6). In the wild-type mice, KS was expressed in the corneal epithelium, stroma and endothelium as shown previously (21) (Fig. 6A). In Galns-/- mice, by contrast, KS was more abundant in the perinuclear cytoplasm of corneal epithelial cells (Fig. 6D). Although the amount of KS staining appeared slightly increased in the stroma of Galns-/- mice, no increase in KS was apparent in corneal endothelial cells (Fig. 6D). C6S was less abundant than KS in the cornea of the wild-type mice (Fig. 6B). However, in the Galns-/- mice, C6S was also increased in the cytoplasm of corneal epithelial cells (Fig. 6E). These results suggest that both KS and C6S are increased in the cytoplasm, possibly in lysosomes in the corneal epithelium of Galns-/- mice. Immunofluorescence studies were also carried out on sections of costal cartilage and the growth plate in the femur. KS and C6S were detected in some chondrocytes of costal cartilage and growth plate in the femur, but no significant differences were seen between the wild-type mice and Galns-/- mice (data not shown).



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  		Figure 6. Detection of KS and C6S in the cornea of 2-month-old wild-type and age-matched Galns-/- mice. Distribution patterns of KS and C6S in the cornea of wild-type and Galns-/- mice were analyzed by immunohistochemistry. Immunostaining for both KS and C6S is more prominent in the cytoplasm of corneal epithelial cells of Galns-/- mice (D and E) than those of wild-type mice (A and B). C and F indicate control experiments using second antibody alone for wild-type and Galns-/- mice, respectively. The clefts in the stroma are artifacts from tissue processing. Immunostaining was performed using 5D4 antibody (A and D) and MC21C antibody (B and E). The corneal epithelium, stroma, and endothelium are indicated as (Ep), (St) and (En) in the figure. Bar=100 µm. The corneal stroma fibroblasts in the mutant mice contained a small amount of vacuolar storage. Neither corneal epithelial nor endothelial cells had any lysosomal distention apparent by light microscopy.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We describe here the generation of a GALNS-deficient mouse produced by targeted disruption of murine Galns gene using homologous recombination in embryonic stem cells. Disruption of Galns gene was complete, as shown by the lack of expression of Galns mRNA and the absence of detectable GALNS enzyme activity in Galns-/- mice. Clinical and pathological characterization of Galns-/- mice revealed evidence of lysosomal storage in visceral organs, brain and the cornea and increased excretion of urinary GAGs as is found in MPS IVA in humans. It will ultimately be important for the Galns-/- mutation to be bred onto a uniform genetic background and possibly even evaluate clinical phenotypes on different mouse strain backgrounds since strain specific modifier genes could influence the phenotype and contribute to mouse-to-mouse variability in mice on a mixed strain background.

Between 2 and 12 months of age in MPS IVA mice, we observed storage with involvement of reticuloendothelial cells in spleen and liver, epithelial cells in kidney, cells at the base of the heart valves, neurons and meningeal cells. Despite the histological and histochemical evidence of storage, the mice had a normal phenotype, appeared healthy and were fertile up to an age of 12 months. They had neither clinical nor radiologic evidence of the skeletal abnormalities characteristic of severe human MPS IVA patients.

It is not uncommon for mouse models of human lysosomal storage diseases to have a milder phenotype than their human counterpart. Examples include murine GM1 gangliosidosis (22), {alpha}-mannosidosis (23), Tay–Sachs disease (24), Sanfilippo syndrome type B (25). In murine Tay–Sachs disease a second degradative pathway has been found to be the basis of the very mild phenotype (24). Although some ancillary pathway of degradation may exist in the Galns-/- mouse, a more likely explanation for the milder phenotype than that found in Morquio A patients lies in differences in the amount of synthesis and distribution of KS between murine and human tissue. Specifically, two different types of KS are found in the cornea (KS I) and skeleton (KS II) and the synthesis of KS II in the mouse and rat is much reduced compared with other animal species in most tissues except the cornea (26). The cartilage cells in rodents do not have KS chains in their aggrecan, which is a KS proteoglycan in other mammals (26). We report here that KS in urine and serum was actually undetectable in normal and MPS IVA mice, while KS in other species like human, dog and cat was easily detectable (data not shown). Histopathological examinations in MPS IVA mice also failed to show progressive GAG storage in cartilage cells and the bones. Taken together, these findings suggest that KS II and KS turnover is less important in skeletal tissues of mouse possibly because KS can be replaced by other GAGs such as chondroitin sulfate. In this view, the absence of KS II in murine aggrecan explains why MPS IVA mice have no skeletal phenotype. On the other hand, the expression of KS I in the cornea may account for the modest amount of storage detected in this tissue.

Storage material was found mainly in cells of the macrophage lineage in liver, heart, bone marrow and spleen, suggesting that these cells may act as a clearing house for circulating KS and/or C6S. These KS and/or C6S fragments would be taken up through the macrophage mannose receptor, which is known to also recognize terminal fucose and N-acetylglucosamine (27). Specifically, vacuoles were seen in sinusoidal cells but few if any were seen in hepatocytes in MPS IVA patients. This predominant deposition of GAG storage in reticuloendothelial cells has also been noted in human MPS IVA patients (personal communication, Dr T. Orii, Gifu University, Japan).

In humans, KS and C6S are abundant in bones and cornea but less prominent in visceral organs, brain, vessels and muscles. The dysfunction in the heart valves in MPS IVA patients can lead to death. The deposition of storage materials at the base of heart valves of MPS IVA mice is thus of interest. MPS IVA patients retain normal intelligence throughout their lives. There has been no description of storage material in brain. In fact, to our knowledge, no studies of brain in MPS IVA patients have been reported. The novel finding that MPS IVA mice have vacuoles in parts of the brain needs further investigation regarding its relevance to human MPS IVA disease.

The deficiency of one enzyme involved in degradation of GAG is often accompanied by increases in other lysosomal enzymes. This had been reported in the MPS I (28), II, IIIA (29), IIIB (25), VI (30) and VII murine models (20,31) but the elevations are very modest in most tissues in the MPS VIA mouse. Such increases may reflect an increased number of lysosomes, increased biosynthesis, and/or stabilization of the enzymes by storage material. The reason the secondary elevation of other lysosomal enzymes is less prominent in MPS IVA mice compared with other MPS murine models could be due to the lower amount of GAG storage in this disorder or the milder secondary effect of the KS/C6S stored. Declines in the level of secondary elevations of lysosomal enzyme activities have been proposed as indicators of effectiveness of treatments such as ERT and gene therapy (20). The very modest secondary elevations of other lysosomal enzymes in this model will make this a less useful marker of therapeutic response.

In other murine models, the level of urinary GAG excretion correlates with severity of visceral and bone involvement. For example, the level of GAG was in proportion with the clinical severity in the murine MPS VII model [this study, Fig. 6, and (20,31)]. MPS I mice have a mild phenotype and have about the same level of GAG excretion in urine (3-fold that of control mice) as mild MPS VII mice (28). MPS IVA mice showed only a 2-fold increase in total GAG excretion compared with normal control mice, which is consistent with their mild phenotype and apparent absence of bone involvement.

Potential therapies for lysosomal storage disorders include bone marrow transplantation, somatic gene therapy and ERT. These therapeutic approaches depend in part at least on the expectation that lysosomal enzymes can be secreted and taken up through a mannose-6-phosphate receptor mediated mechanism (32,33). The reversibility of some of the features of human MPS IVA was suggested by the results of bone marrow transplantation (unpublished data, Dr T. Orii, Gifu University, Japan). Factors that influence the responsiveness to these forms of therapy relate to the age of the patient and the degree of progression prior to the commencement of therapy or to efficiency of enzyme delivery to the skeleton. Until now, there has been no animal model for MPS IVA reported which would permit evaluation of experimental therapies for this disease. The GALNS deficient mouse described here, even though it lacks the skeletal phenotype of the human disease, should prove useful for evaluation of pharmacokinetics and targeting of various forms of GALNS in experimental ERT protocols.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Construction of the Galns gene targeting vector
The Galns gene was cloned from a 129/Sv mouse genomic library (Stratagene), as previously described (34). The cysteine residue found in the catalytic domain of all sulfatases is critical for the enzyme activity (18), and thus exon 2 encoding the Cys79 residue of human GALNS was selected for replacement in the gene disruption construct. After digestion with the restriction endonuclease HindIII, the 1.8 kb fragment containing part of intron 1 and exon 2 was replaced by a cassette containing the neor gene under control of the mouse phosphoglycerate kinase promoter (20). A cassette containing the Herpes simplex virus thymidine kinase gene under control of the mouse phosphoglycerate kinase promoter was cloned into the vector upstream of the Galns gene (Fig. 1). The loxP sequences are positioned at both ends of the neor gene. The continued presence of neomycin-resistant gene (neor) in the construct may affect the clinical phenotype in the mouse model (35). This lox–neo–lox cassette can be eliminated by mating the heterozygotes with transgenic mice expressing the Cre recombinase enzyme. After neor is removed, this deletion eliminates exon 2, causing a frame shift. The final construct contains 4.2 and 4.5 kb of 5' and 3' homology, respectively.

Homologous recombination in ES cells and generation of germline chimeras
The vector (25 µg) was linearized with NotI restriction endonuclease before electroporation into embryonic stem (ES) cells (Incyte Genome Systems, St Louis, MO, USA), which are derived from mouse strain 129SvJ. The ES cells, 107, were electroporated in a 1 ml cuvette at 240 V and 500 µF by a Bio-Rad gene pulser. After 24 h, the cells were placed under selection with 200 µg/ml G418 (GIBCO/BRL, Gaithersburgh, MD, USA) and 2 µM gangciclovir (Syntex, Palo Alto, CA, USA) for 6 days. ES cell colonies resistant to double selection were isolated and subjected to PCR, followed by Southern blot analysis. Surviving colonies were subjected to PCR analyses to identify clones that had undergone homologous recombination. The PCR method utilizes a forward primer in the loxP–neor–loxP cassette sequence [LoxL(2)R: 5'-CTTCGTATAGCATACATTACGAAGTTATGCGA-3']. and a reverse primer in intron 4 outside the targeting sequences [MOex5-1R: 5'-ACAGGGATGTTGGGCTTGGCCTTGTTGTCATACGG-3']. PCR amplification from the disrupted allele produces a 4.5 kb fragment. Positive clones identified by PCR were then confirmed by Southern analysis. Genomic DNAs of the positive clones by PCR screening were digested with KpnI, transferred to nylon membranes and hybridized with the 5' probe (a 1.3 kb SacI–BamHI genomic fragment) generating 7.1 kb fragment in the recombinants instead of 12.0 kb fragment in the normal allele shown in Figure 1. About 10% of the survivors of the drug selection were found to have a disrupted Galns gene.

Two independent targeted ES clones were injected into C57BL/6J blastocysts and three chimeric mice were generated. Chimeric males were back-crossed for germline transmission to C57BL/6J females. Germline transmission was achieved from all three chimeric mice. The resultant F1 mice transmitted to the germline from all three chimeric mice were crossed with mice expressing Cre enzyme to remove the neor gene, and the resultant neo-excised heterozygous mice were mated to produce homozygous mutant mice. This excision leaves only 34 bp of one loxP site in intron 1. The removal of neor was diagnosed by PCR of tail DNA using a forward primer in the loxP–neor–loxP cassette sequence (VDE: 5'-GGATCCTCTAGAAATGCCATTTCATTACCTCTTT-3') and a reverse primer in intron 3 (mGMO4R: 5'-GGCTTAGGAAAGCATCTTTCCTAGGCTAGA-3'), which amplified a 1.3 kb fragment while non neor-excised allele did not reveal a detectable fragment under these conditions. After excision of neor, we performed multiple PCR using two sets of primers to distinguish Galns-/-, Galns+/- and wild-type (Galns+/+) mice. A combination of a forward primer in the deleted portion of intron 1 (mGMO1': 5'-GCTCTTCCTTGTCACAGAGGAACC-3') and a reverse primer in exon 4 (MOex4-1R: 5'-CTTGTTGGTATAGCCTTCTTCAGGAGCTC-3') shows 1.8 kb fragment for normal allele but no amplification for mutant allele. PCR using another set of primers, mGALNS2 (5'-CCCCTGGAGTGTAGTCACAGACAGTTGCAAGC-3') and VDE 1 vector sequence (5'-GGAGAAAGAGGTAATGAAATGGCATTTCTAGA-3'), reveals a 1.2 kb fragment for the mutant allele but no amplification for normal allele (Fig. 1). Heterozygotes were intercrossed for experimental use and backcrossed to C57BL/6 mice to put the recombinant alleles on a congenic background. Genotyping was performed by PCR analysis of DNA obtained by tail biopsies at 10 days, confirmed by assay of GALNS activity in these tails. The resultant homozygous mice with partial deletion of intron 1 and exon 2 were named Galns-/-. Two independent lines were analyzed further and results were no different between those two lines, one of which was chosen studies reported here.

Enzyme assays
GALNS and other lysosomal enzymes, ß-glucuronidase, {alpha}-galactosidase, {alpha}-mannosidase, ß-galactosidase and ß-hexosaminidase, were assayed fluorometrically using 4-methylumbelliferyl substrates, as described previously (36–38). Tissues were dissected and immediately homogenized (by Brinkmann Polytron homogenizer for 30 s on ice) in 5 vos of homogenization buffer (25 mM Tris–HCl, pH 7.2, 1 mM PMSF). The homogenate was centrifuged to remove debris. If necessary, centrifuged homogenate was diluted in homogenization buffer prior to assay. Briefly, GALNS assays on dilutions of wild-type and MPS IVA (Galns-/-) tissue extracts were as follows: homogenate (2 µl) was incubated for 24 h at 37°C in a reaction mixture of 30 µl of 22 mM 4-methylumbelliferyl-ß-galactopyranoside-6-sulfate (Melford Laboratories Ltd, Suffolk, UK) in 0.1 M NaCl, 0.1 M Na-acetate, pH 4.3. After incubation for 24 h at 37°C, 2 µl of a solution containing 10 mg/ml of ß-galactosidase in 0.1 M NaCl, 0.1 M sodium acetate, pH 4.3, were added and the samples were incubated for an additional 30 min at 37°C. The reaction was stopped with 2 ml of 1 M glycine NaOH buffer, pH 10.5. Assays of other lysosomal enzymes were incubated for 1 h, followed by adding the glycine stop buffer. Activity was expressed as nanomoles of 4-methylumbelliferone released per milligram of protein per hour. Proteins were determined by the micro-Lowry assay.

Analysis of GAGs and KS
Urine samples were collected on Saran Wrap from individual mouse stimulated by gently massaging the lower abdomen. GAGs were measured using 1,9-dimethylmethylene blue in a quantitative assay (38,39). Heparan sulfate from porcine intestinal mucosa (Sigma H9902) was used as standard. Creatinine was measured by mixing 10 µl of a urine sample with 500 µl saturated picric acid (Sigma) and 500 µl 0.2 M NaOH. Absorbance at 490 nm was measured after 20 min and compared with a standard. The GAG/creatinine ratio (µg of GAGs per mg of urinary creatinine) was used to normalize the urinary excretion of GAG. Corneal GAGs were determined as previously described (39). Briefly, the 20 corneas were collected from 10 mutant and 10 normal mice, and digested with papain for 4 h. After papain digestion, protein was measured in the digest. Then, chloroform extraction was done followed by centrifugation. The GAG concentration was determined on the clear supernatant. GAG content was expressed as µg of GAGs per mg of protein.

KS in urine, serum and cornea was also measured using the same materials by ELISA-Sandwich method. The KS standards for ELISA calibration and the anti-keratan sulfate monoclonal antibody (5-D-4) (40) were obtained from Seikagaku (Tokyo, Japan). The plate-washing buffer (PBS–0.05% Tween-20) and sample diluent (PBS including BSA) were brought to room temperature before use. The washing buffer (200 µl/well) was added to the microplate by multi-pipette then discarded. This washing procedure was repeated three times. Samples (50 µl/well) of KS standards and diluted unknown samples were added to the well and incubated at 37°C for 60 min. The plate wells were then washed four times as described above. Next, 25 µl/well of horseradish peroxidase conjugated streptavidin and 25 µl/well of biotinylated antibody were added to the plate and the plate again incubated for 60 min at 37°C. After washing the plate four times, 50 µl/well of substrate solution (tetramethylbenzidine) was is added to each well and the plate incubated for 10 min at room temperature. Finally, the reaction is stopped with 50 µl of stop solution (1 N HCl). The absorbance was measured at 450 nm with microplate spectrophotometer reference to 630–650 nm. The KS concentration was read by applying the absorbances of each sample to the calibration curve.

Pathological examinations
Liver, spleen, kidney, heart, rib, eye and brain were examined from seven Galns-/- mice from 2 to 12 months of age. Tissues fixed in 4% paraformaldehyde/2% glutaraldehyde, and embedded in Spurr's resin were sectioned, stained with Toluidine blue, evaluated by light microscopy, and compared with age matched control mice. The long bones were fixed in formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin and examined by light microscopy. Skeletons from 7- and 12-month-old Galns-/- mice were radiographed and compared to those of normal mice, as previously described (41).

Immunohistochemical detection of KS and C6S in the cornea of 2-month-old Galns-/- mice as well as the age-matched wild-type mice was carried out using monoclonal antibodies, 5D4 and MC21C specific for KS and C6S, respectively, as described previously (21). These primary antibodies were purchased from Seikagaku (Tokyo, Japan), and horseradish peroxidase-labeled anti-mouse IgG was used for the secondary antibody. The peroxidase reaction was developed with a diaminobenzidine/H2O2 solution, and counter-staining was performed with hematoxylin. A control experiment was done by omitting the primary antibody from the staining procedure and no specific staining was found.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Austrian MPS, the German MPS, the International Morquio Organization and the Genzyme Corporation.


   FOOTNOTES
 
* To whom correspondence should be addressed at: E.A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 S. Grand Blvd, St Louis, MO 63104, USA. Tel: +1 3145778131, Fax: +1 3147761183; Email: tomatsus{at}slu.edu Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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