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Human Molecular Genetics Advance Access originally published online on December 23, 2005
Human Molecular Genetics 2006 15(3):493-500; doi:10.1093/hmg/ddi465
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ß-Mannosidosis mice: a model for the human lysosomal storage disease

Mei Zhu1, Kathryn L. Lovell2, Jon S. Patterson3, Thomas L. Saunders5, Elizabeth D. Hughes5 and Karen H. Friderici1,4,*

1Department of Microbiology and Molecular Genetics, 2Department of Neurology and Ophthalmology and Neuroscience Program, 3Pathobiology and Diagnostic Investigation, College of Veterinary Medicine and 4Department of Pediatrics and Human Development, Michigan State University, East Lansing, Michigan, USA and 5Department of Internal Medicine and Transgenic Animal Model Core, University of Michigan, Ann Arbor, Michigan, USA

* To whom correspondence should be addressed at: Department of Microbiology and Molecular Genetics, 5163 Biomedical Physical Sciences Building, Michigan State University, East Lansing, MI 48824, USA. Tel: +1 5173556463 ext. 1558; Email: frideric{at}msu.edu

Received November 7, 2005; Accepted December 16, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ß-Mannosidase, a lysosomal enzyme which acts exclusively at the last step of oligosaccharide catabolism in glycoprotein degradation, functions to cleave the unique ß-linked mannose sugar found in all N-linked oligosaccharides of glycoproteins. Deficiency of this enzyme results in ß-mannosidosis, a lysosomal storage disease characterized by the cellular accumulation of small oligosaccharides. In human ß-mannosidosis, the clinical presentation is variable and can be mild, even when caused by functionally null mutations. In contrast, two existing ruminant animal models have disease that is consistent and severe. To further explore the molecular pathology of this disease and to investigate potential treatment strategies, we produced a ß-mannosidase knockout mouse. Homozygous mutant mice have undetectable ß-mannosidase activity. General appearance and growth of the knockout mice are similar to the wild-type littermates. At >1 year of age, these mice exhibit no dysmorphology or overt neurological problems. The mutant animals have consistent cytoplasmic vacuolation in the central nervous system and minimal vacuolation in most visceral organs. Thin-layer chromatography demonstrated an accumulation of disaccharide in epididymis and brain. This mouse model closely resembles human ß-mannosidosis and provides a useful tool for studying the phenotypic variation in different species and will facilitate the study of potential therapies for lysosomal storage diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Lysosomal storage diseases constitute a significant portion of genetic disease. Dysfunction of any one of the more than 40 enzymes that reside in the lysosome results in the accumulation of the associated uncatabolized substrate (1Go,2Go). Accumulation of this substrate or toxic intermediates may lead to cellular toxicity. Storage occurs in various cell types, but clinical symptoms characteristic of lysosomal storage disease are often referable to the nervous system and include progressive mental and motor retardation and impaired hearing, as well as dysostosis, and immune defects. Disease presentation can range from severe and consistent to mild and variable depending on the enzyme involved, the severity of the mutation and the species concerned.

ß-Mannosidosis (MIM no. 248510 [OMIM] ) is a rare lysosomal storage disease that was identified first in Nubian goats (3Go) and subsequently in humans (4Go,5Go). The Salers breed of cattle also has the disease (6Go). Affected goats and cattle have very similar clinical features that include inability to stand, facial dysmorphism (dome-shaped skulls, small palpebral fissures, depressed nasal bridge and elongated, narrow muzzle), intention tremors, multiple muscle contractures and digital joint hyperextension (6Go,7Go). Affected animals die in the neonatal period if intensive care is not provided. Gross neuropathological characteristics include ventricular dilation with a marked paucity of myelin in cerebral hemispheres and cerebellum. Microscopic examination reveals extensive cytoplasmic vacuolation in the nervous system and visceral organs, restricted to specific cell types. Regionally specific myelin deficiency is present in the central nervous system but not in peripheral nerves (7Go–9Go). Affected goats and calves are hypothyroid and thyroid follicular epithelium shows severe vacuolation (10Go,11Go). ß-Mannosidosis cases in the two ruminant species result from severe mutations that totally inactivate enzymatic activity of the protein (12Go,13Go).

In contrast with ruminant ß-mannosidosis, human cases have a milder and heterogeneous clinical expression (14Go). The most severe cases are associated with mental retardation, developmental delay, dysmorphology, frequent infections and hearing loss (4Go,5Go,15Go–18Go). Siblings with the disease may have differing levels of severity (19Go). Onset can be in the neonatal period, with hypotonia, swallowing difficulties or seizures as presenting symptoms (20Go,21Go). In contrast to ruminant ß-mannosidosis, many of the affected individuals, including those with null mutations, achieve maturity and can have comparatively mild disease (22Go–24Go). None of the human cases had clinical signs indicative of central nervous system hypomyelination. No human ß-mannosidosis cases have been submitted to autopsy, so little is known about the distribution of storage material or severity of tissue vacuolation.

In the lysosome, N-linked glycoproteins are degraded sequentially from the non-reducing end of the molecule. ß-Mannosidase (MANBA; 3.2.1.25 [EC] ) is required for the final degradative cleavage of the glycan moiety of glycoproteins in humans, rodents and lagomorphs. In these species, the non-sequential cleavage of the GlcNAc(ß1–4) GlcNAc bond can be accomplished by chitobiase, an enzyme that is not present in ruminants and carnivores (25Go). Therefore, lack of ß-mannosidase in humans results in storage of the disaccharide Man(ß1–4)GlcNAc (26Go,27Go), whereas in ruminants, storage of the trisaccharide Man(ß1–4)GlcNAc(ß1–4)GlcNAc predominates (28Go,29Go). The ß-mannoside linkage is unique to N-linked glycoproteins; therefore, unlike most other lysosomal enzymes, ß-mannosidase has only a single substrate in the cell. In addition, the accumulated substrate that is stored in ß-mannosidosis is an unusually small molecule.

Reasons for the phenotypic differences between human and ruminant ß-mannosidosis are unknown but speculation has centered on differences in the size and nature of the storage product and in the different developmental programs of the species (14Go). To address these aspects of the molecular pathology, we developed and characterized a mouse model of ß-mannosidosis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of ß-mannosidase-deficient mice
A targeting vector was constructed to disrupt the murine ß-mannosidase gene (Manba) (Fig. 1A). Following drug selection, 480 ES cell clones were screened by PCR and 108 correctly targeted clones (22.5% recombinant efficiency) were identified. Four euploid clones were injected into blastocysts. In total, 31 chimeric mice (24 males and seven females) were identified by coat color. Three chimeric males with germline transmission from two independent ES clones were backcrossed to C57BL/6J females. Thirty-nine F1 heterozygous mice (17 males and 22 females) were generated and 22 of them (four males and 18 females) were intercrossed for experimental use. A total of 199 F2 mice from two independent ES lines were born, and the offspring showed expected Mendelian ratios: 20.1% for wild-types, 29.1% for homozygous mutants and 50.8% for heterozygotes. RT–PCR (data not shown) demonstrated the deletion of exon 5. Homozygous mutant, heterozygous and wild-type mice did not exhibit differences in general appearance, growth or weight. Both males and females were fertile. No obvious mortality and behavior changes were observed to over 12 months of age. There were no phenotypic and biological differences between mutant mice from two independent ES clones.


Figure 4651
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  		Figure 1. Targeted disruption of the murine ß-mannosidase gene. (A) The structure of the endogenous ß-mannosidase gene, the targeting construct and the disrupted allele. The targeting construct contains 1.4 kb of 5'-homologous sequence and 5.8 kb of 3'homologous sequence (bold lines). The 5'probe (bar) and PCR primers (arrows) used to screen for recombinant ES clones (c, b, d) and to screen mouse litters (a, b, d) are indicated. Exons are shown as filled boxes. (B) Southern blot analysis of tail DNA. The 5'probe was hybridized to BciVI-digested genomic DNA: wild-type allele, 19 kb; mutant allele, 6.4 kb. (C) PCR-based screening for the homologous recombinant ES clones. The primer pairs were c+b for wild-type allele (1.5 kb) and c+d for disrupted allele (1.8 kb). Lanes 1 and 2 are wild-type ES lines; lanes 3 and 4 are targeted ES cell lines. (D) PCR analysis of tail genomic DNA with primer pairs of a+b for wild-type mouse (210 bp) and a+d for the disrupted allele (470 bp).

 
Biochemical findings
Enzyme activity
ß-Mannosidase activity was determined in plasma and tissue homogenates from kidney, liver, spleen, pancreas, brain, testis, epididymis, heart, lung and skeletal muscle. Homozygous mutant mice had no detectable activity in any tissue, confirming the targeted allele was not functional. Heterozygotes had approximately half the enzyme activity of the wild-type (Fig. 2A). {alpha}-Mannosidase and acid phosphatase were assayed at the ages of 6 and 36 weeks for comparison. The {alpha}-mannosidase activity was increased in the mutant when compared with the wild-type, especially at older ages (Fig. 2B), whereas the acid phosphatase activity measured at the two ages was not different between mutant and wild-type (Fig. 2C).


Figure 4652
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  		Figure 2. ß-Mannosidase activity and other lysosomal enzyme activity assay. For each group, four mice were analyzed. Error bars represent one standard deviation. Enzyme activities are expressed as nmol/h/mg protein. (A) ß-Mannosidase activities were assayed in tissues of homozygous mutant (–/–), heterozygous (+/–) and wild-type mice (+/+). The enzyme activity of the homozygous mutant was non-detectable. (B) Activities of {alpha}-mannosidase and acid phosphatase were assayed in tissues of homozygous mutant and wild-type mice at two different ages. {alpha}-Mannosidase activities increased in the tissues of homozygous mutants when compared with wild-type, and especially at the older age. Significant differences (P>0.005) between mutant and wild-type are marked with asterisks (*) (C) The enzyme activity of acid phosphatase showed no difference between mutant and wild-type mice.

 
Pathological characterizations of organs
Tissues from control and mutant mice between the ages of 4 and 37 weeks were analyzed in H&E-stained 6 µm paraffin sections and, for some tissues, in Toluidine blue-stained, 1 µm sections. Histological evidence of storage was apparent in selected organs of all mutant mice, although there was variation that did not correlate consistently with age.

Epididymis
Small numbers of epithelial cells lining the ducts in the body of the epididymis contained fine, clear vacuoles in the apical cytoplasm. In a few cells, the cytoplasm was filled diffusely with fine to coarse vacuoles; electron microscopy revealed these vacuoles to be electron-lucent and membrane-bound (Fig. 3B+inset). Vacuolation in the epithelium lining the head and tail of the epididymis was not apparent.


Figure 4653
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  		Figure 3. (A–F) Organ histopathology of ß-mannosidase –/– mice compared with wild-type mice. All light microscopic sections were from 18-week-old mice, stained with Toluidine blue. Magnification 50x. (A and B) Body of epididymis of control (A) and mutant (B) mice. Mutant mice have scattered cells with fine, intracytoplasmic vacuoles; the inset shows an electron micrograph of a diffusely vacuolated cell. (C and D) Kidneys of control (C) and mutant (D) mice. Occasional tubular epithelial cells in kidneys from mutant mice contained fine, intracytoplasmic vacuoles. (E and F) Thyroid glands of control (E) and mutant (F) mice. Follicular epithelial cells from mutant mice did not show the level of vacuolation seen in ruminants (G and H), although they occassionally contained fine, intracytoplasmic vacuoles not evident here. Thyroid glands of control (G) and mutant (H) newborn goats for comparison.

 
Liver
In both control and mutant animals, most hepatocytes showed some degree of indistinctly and finely vacuolated, granular cytoplasm which represented glycogen storage, as confirmed by periodic acid Schiff (PAS) stain. However, in mutant mice only, very small numbers of hepatocytes contained discrete, fine, clear intracytoplasmic vacuoles even in PAS-stained sections. Occasional Kupffer cells and sinusoidal endothelial cells also contained fine intracytoplasmic vacuoles.

Kidney
In the kidney, fine to coarse vacuoles in the apical cytoplasm of both proximal and distal tubular epithelial cells were present in Toluidine blue-stained, 1 µm sections, although the number of affected cells was small (Fig. 3D).

Thyroid gland
Fine, intracytoplasmic vacuoles were present in small numbers of follicular epithelial cells from the thyroid gland, and most cells showed no vacuolation (Fig. 3F).

Other organs
In the duodenum, intracytoplasmic vacuoles were observed in some mutant animals in scattered smooth muscle cells of the outer longitudinal muscular layer and in occasional myenteric plexus neurons. Routine light microscopic examination revealed no significant histological findings in skin, heart, spleen, skeletal muscles (thigh), lung, pancreas, adrenal gland, testis, uterus and bone.

Pathological characterization of central nervous system
Examination of central nervous system of 11 mutant mice from 4 to 37 weeks of age showed cytoplasmic vacuolation in all animals. There was substantial variation in severity of vacuolation among regions examined and variation among animals. Variation occurred with respect to density of vacuoles within a neuron, size of vacuoles and the number of neurons affected. There was no consistent difference between mice generated from the two ES cell clones and no consistent progression of pathology with age. Pyramidal cells in the dorsolateral cerebral cortex most consistently showed severe vacuolation (Fig. 4A and B). The choroid plexus also consistently showed conspicuous vacuolation (Fig. 4D). Within the hippocampal cornu ammonis, specific segments of Ammon's horn showed differences in severity of vacuolation (Fig. 4E and F), with substantial variation among animals. Other regions with neuronal vacuolation to various extents included striatum, amygdala and deep cerebellar nuclei (Fig. 4G). Hypomyelination in optic nerve or corpus callosum was not apparent in Toluidine blue-stained, 1 µm sections. In spinal cord, neuronal cell bodies at all levels (cervical, thoracic, lumbar) contained fine, intracytoplasmic vacuoles (Fig. 4H).


Figure 4654
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  		Figure 4. Neuropathology of ß-mannosidase –/– mice compared with wild-type mice. (A) Dorsal cortex neurons of a 6-week-old mutant show severe vacuolation, 300x. (B) Electron micrograph of a cortical neuron from a 19-week-old mutant showing intracytoplasmic vacuolation, 2200x. (C and D) Choroid plexus from control (C) and mutant (D) mice, showing severe vacuolation in the mutant mouse, 250x. (E and F) Sections from the hippocampus of a 37-week-old mutant mouse showing absence of vacuolation in CA4 (E) and large vacuoles in pyramidal cells in CA1 (F), 300x. (G) Vacuolated neurons in deep cerebellar nucleus of a 37-week-old mutant mouse, 250x. (H) Vacuolated spinal cord motor neuron from an 18-week-old mutant mouse, 250x.

 
Accumulation of oligosaccharides in tissues
In agreement with the histological findings in mutant mice, accumulation of oligosaccharide was found primarily in brain (frontal cortex) and epididymis by thin-layer chromatography (Fig. 5). In contrast to the storage seen in goats, the oligosaccharide found in the mutant mice is exclusively disaccharide, and there is little accumulation in the kidney.


Figure 4655
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  		Figure 5. Thin-layer of oligosaccharides in ß-mannosidosis mice. Equal amounts of extract from kidney (kid), frontal cortex (brn) and epididymis (epi) from 9-month-old control (+/+) and homozygous mutant (–/–) mice were applied to silica gel 60 TLC plates. Chromatography controls, not shown here, were 2 µg monosaccharide (glucose), disaccharide (sucrose) and trisaccharide (raffinose). Authentic storage material from affected goat kidney (GK) is shown. Monosaccharide (MS), disaccharide Manß(1Go–4Go)GlcNAc (DS) and trisaccharide Manß(1Go–4Go)GlcNAc(ß1–4) GlcNAc (TS) are indicated.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have generated ß-mannosidase-deficient mouse lines by gene targeting. There was no detectable ß-mannosidase activity in the plasma (data not shown) and tissue homogenates from homozygous mutant mice. Heterozygous mice showed approximately half the enzyme activity of wild-type mice (Fig. 2). Deficiency of one lysosomal enzyme often causes the elevation of other lysosomal enzyme activities (30Go), especially those in the same degradation pathway. Tissues from human cases of ß-mannosidosis have not been evaluated, but goat tissues have elevated levels of {alpha}-mannosidase, {alpha}-fucosidase, ß-hexosaminidase A and ß-glucuronidase in kidney and brain (3Go,31Go). We examined {alpha}-mannosidase activity (Fig. 2) and found elevation of activity in brain, spleen and liver of the mutant mice at young age (6 weeks) and statistically significant increases in all tested tissues at older age (36 weeks). Acid phosphatase, an enzyme not in the N-linked glycoprotein degradation pathway, was assayed for comparison. As expected, acid phosphatase activity (Fig. 2) was not different between the mutant and wild-type mice. These enzymatic findings coupled with the genomic findings indicate successful disruption of the ß-mannosidase gene.

The homozygous mutant mice are fertile when young and have a normal clinical appearance to >1 year of age. However, microscopic analysis revealed cytoplasmic vacuolation in central nervous system and visceral organs. The null mutant mice did not show the severe neonatal neurological impairment or the substantial myelin abnormalities seen in ß-mannosidase-deficient goats and cattle (32Go–34Go). The dearth of clinical abnormalities and the signs of lysosomal storage in tissues suggest our mouse model more closely resembles the phenotypic expression seen in human ß-mannosidosis (4Go,5Go,15Go–19Go,21Go,24Go,35Go). No differences in storage material distribution or phenotype were observed when the effect of the mutation was examined in mice congenic for the 129X1/SvJ background (data not shown).

Little is known about the distribution of storage material or severity of cytoplasmic vacuolation in human cases. Only blood and skin biopsy specimens have been examined and these showed cytoplasmic vacuolation of cells in blood and lymph vessels, endothelial cells, fibroblasts, secretary portions of eccrine sweat glands, neural cells and basal keratinocytes in the epidermis in angiokeratoma lesions (24Go,35Go,36Go). Light and electronic microscopic examination of the homozygous mutant mice revealed cytoplasmic vacuolation in the central nervous system and some visceral organs but not in all the organs examined. Many of the affected humans show mental retardation and emotional changes (4Go,5Go,15Go–17Go,19Go,36Go,37Go). We did not see obvious physical or behavioral abnormalities in the ß-mannosidase-deficient mice, including preliminary tests such as rotarod, open field and hanging wire (data not shown). Taken together, further behavioral testing in the mice to more specifically analyze learning, working memory, long-term memory and fear conditioning will be necessary to investigate functional effects.

An unusual feature of ß-mannosidosis in humans and mice is the nature of the lysosomal storage product. The ß-mannoside linkage is found only in the N-glycosyl moiety of proteins synthesized in the endoplasmic reticulum. Lack of ß-mannosidase activity is expected to result in a trisaccharide, Manß(1Go–4Go)GlcNAcß(1Go–4Go)GlcNAc, produced in the stepwise degradation of glycoprotein glycosyl groups. Humans and mice, but not ruminants, have an additional lysosomal enzyme, chitobiase, that cleaves between the GlcNAc–GlcNAc, which predicts the accumulation of Manß(1Go–4Go)GlcNAc, an unusually small storage product of only ~400 molecular weight. We show that the storage product in mice is indeed a disaccharide, similar to that found in humans and different from the ruminant storage products. The smaller sized storage product may contribute to the phenotypic differences between species. In contrast to our ß-mannosidosis mice, {alpha}-mannosidosis knockout mice display prominent lysosomal storage in a variety of visceral organs and in both central and peripheral nervous systems (38Go). This may well be due to the larger and more heterogeneous nature of the stored oligosaccharides found in {alpha}-mannosidosis.

Our mouse model will be a valuable tool to better understand the mechanisms of pathogenesis for lysosomal storage disease. The substrates for the enzyme are ubiquitous, the storage product is small and uniform and the storage in the nervous system is consistent. This will be an important model for evaluation of various treatment options, including enzyme-replacement and gene-therapy approaches.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Construction of the targeting vector
We cloned and sequenced the full-length cDNA of the murine ß-mannosidase gene (GenBank accession no. AF306557, and data not shown). Using mouse-specific sequence information and the genomic structure of the human ß-mannosidase gene, a portion of the ß-mannosidase gene bearing genomic sequences from intron 3 to intron 7 was cloned from a 129X1/SvJ mouse genomic library (Stratagene) by using a PCR-based screening (39Go). We chose exon 5 for disruption because it is an out-of-frame exon; the splicing of exon 4 to exon 6 would result in the introduction of a premature translational stop codon. A 1.4 kb PCR fragment containing a part of intron 4 and 20 bp of exon 5 was cloned into XhoI sites in front of the neo cassette of the pPNT vector (40Go) (Fig. 1A). The downstream 5.7 kb segment of the homologous region was cloned into the vector in two steps. First, an XbaI/EcoRI fragment containing 1.6 kb of intron 5 was inserted into the XbaI/EcoRI digested vector. Then, a SalI/EcoRI adaptor was added to the 4.1 kb EcoRI/SalI fragment bearing exons 6 and 7 and subsequently was inserted into the EcoRI site of the construct. The final construct was sequenced to confirm the boundaries of the homologous region.

Gene targeting in ES cells and generation of null mutant mice
The vector was linearized with NotI restriction endonuclease before electroporation into R1 ES cells (41Go). After positive (G418 for neo gene) and negative (gangciclovir for TK gene) drug selection, we screened five 96-well culture plates by PCR analysis using primers c, b and d (Fig. 1A). The primer sequences were: c, 5'-ACAGGAGTGGTTAGGCATCG-3'; b, 5'-AGATTCCCTGAGAGGGGAAG-3' and d, 5'-CTGTCCATCTGCACGAGACT-3'. Five correctly targeted ES clones were expanded and confirmed by PCR and Southern blot analysis using the 5'-probe and were subjected to a chromosome count. Eventually, four ES clones were injected into C57BL/6J blastocysts. Chimeric males with 90% or more agouti coat color were back-crossed to C57BL/6J females and 129X1/SvJ females (data not shown). The heterozygous F1 mice were intercrossed for experimental use and back-crossed to C57BL/6J mice to generate a congenic background. The F1 and F2 mice were genotyped by PCR analysis (3 min denaturation at 94°C, then 35 cycles of 30 s at 94°C, 30 s at 60°C and 30 s at 72°C) on tail DNA. Primers for genotyping were a, 5'-CTCAGGACCTTCGGAAGAAC-3', b and d (Fig. 1A). The genotypes of F2 mice were further confirmed for gene disruption by Southern blot analysis using the 5'-probe (Fig. 1A and B). The 5'-probe is a 781 bp PCR product from intron sequence outside of the homologous region. Total RNA was isolated from tissues (kidney and testis) of homozygous mutant, heterozygote and wild-type mice using TRIzol reagent (cat. no. 15 596, Invitrogen). RT–PCR was performed for detection of transcripts. Mice from two independent lines were analyzed, and there was no phenotypic difference between them.

Enzyme assays
Tissue extracts from 6- and 36-week-old mice were prepared as previously described (Lovell et al. (33)). Enzyme activity was determined using the fluorogenic substrate, 4-methyl-umbelliferyl-ß-D-mannopyranoside (M0905, Sigma). For comparison, {alpha}-mannosidase and acid phosphatase were also assayed fluorometrically using substrates 4-methyl-umbelliferyl-{alpha}-D-mannopyranoside (M4383, Sigma) and 4-methylumbelliferyl phosphate (M8883, Sigma), respectively. For enzyme assays, 20 µl of substrate was added to 10 µl of the tissue extract and incubated at 37°C for 30 min (ß-mannosidase) or 10 min for other enzymes. Each tissue extract was run in duplicate. Reactions were stopped by adding 170 µl 0.1 M glycine (pH>10.8). Fluorescence of the liberated 4-MU was measured using a luminescence spectrometer (LS50B, Perkin Elmer). Proteins were determined by the BCA method (no. 23225, Pierce). A one-tailed t-test for equality of the mean was used to determine the statistical significance of the difference in {alpha}-mannosidase activity between mutant and wild-type mice.

Pathological examinations
Tissue samples (including brain, optic nerve, spinal cord, thyroid gland, kidney, heart, spleen, liver, pancreas, adrenal gland, lung, skin, bone, skeletal muscle, epididymis, peripheral nerve, testes, duodenum, uterus) were removed immediately after euthanasia with carbon dioxide and fixed in 10% formalin for paraffin embedding, 4% paraformaldehyde for Immunobed embedding or 4% glutaraldehyde for Epon embedding. Tissues were examined from 11 mutant animals (one at 4 weeks, two at 6 weeks, four at 18 weeks and four at 37 weeks of age) and six control animals (at least one at each age). H&E-stained 6 µm paraffin sections and/or Toluidine blue-stained, 1 µm sections were examined under light microscopy for all tissues. For some animals, 3 µm Immunobed sections showing one coronal hemisphere were stained with H&E or Toluidine blue. Selected tissues that showed pathological changes were examined by electron microscopy.

Oligosaccharide analysis
Tissue from control and homozygous mutant mice was minced on ice and sonicated for 30 s in 5 ml distilled deionized water per gram of tissue. After centrifugation for 10 min at 15 000g, the supernatant was transferred to a fresh tube (42Go). Lipids and proteins were removed by extraction with 4 vol of CHCl3/methanol (3:1). The aqueous supernatant was evaporated and the residue resuspended in H20 at 1/10 the original volume. Two microliter samples were applied to silica gel 60 plates (5721-7, EM Science® Merk®) and developed for 1 h in 4:1 acetonitrile/H2O (43Go). Oligosaccharides were visualized with 0.2% orcinal in 20% sulfuric acid.


   ACKNOWLEDGEMENTS
 
The authors thank Dr John Fyfe for helpful discussion and critical reading of the article and Dr Robert Lovell for statistical analysis. We thank Dr Richard Mulligan for use of the pPNT targeting vector and Drs Andras Nagy, Reka Nagy and Abramow-Newerly for R1 ES cells. This work was supported by funds from the MSU foundation and NIDDK grant DK49782 to K.H.F. Grant support also came from the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000815).

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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