The ob gene product, leptin, is an important circulating signal for the regulation of body weight. To identify high affinity leptin-binding sites, we generated a series of leptin-alkaline phosphatase (AP) fusion proteins as well as [125I]leptin. After a binding survey of cell lines and tissues, we identified leptin-binding sites in the mouse choroid plexus. A cDNA expression library was prepared from mouse choroid plexus and screened with a leptin-AP fusion protein to identify a leptin receptor (OB-R). OB-R is a single membrane-spanning receptor most related to the gp130 signal-transducing component of the IL-6 receptor, the G-CSF receptor, and the LIF receptor. OB-R mRNA is expressed not only in choroid plexus, but also in several other tissues, including hypothalamus. Genetic mapping of the gene encoding OB-R shows that it is within the 5.1 cM interval of mouse chromosome 4 that contains the db locus.

An X chromosome-linked mouse mutant (gene symbol, mdx) has been found that has elevated plasma levels of muscle creatine kinase and pyruvate kinase and exhibits histological lesions characteristic of muscular dystrophy. The mutants show mild clinical symptoms and are viable and fertile. Linkage analysis with four X chromosome loci indicates that mdx maps in the Hq Bpa region of the mouse X chromosome. This gives a gene order of mdx-Tfm-Pgk-1-Ags, the same as for the equivalent genes on the human X chromosome.There are two major forms of X chromosome-linked muscular dystrophy in man: Duchenne (McKusick number, 31020) and Becker (McKusick number, 31010) (1-3); recent linkage analyses with restriction fragment-length polymorphisms suggests that they could be allelic (4, 5). For neither of these syndromes is the primary lesion or a homologous animal model known. Several mouse mutants exhibit myopathies (6, 7); the most investigated of these are the autosomal dy and dy2 mutants (6). There is, however, controversy over whether the dy mutants are myogenic or neurogenic in origin (8-10) and, therefore, their suitability as animal models of Duchenne/Becker muscular dystrophy (11).Therefore, it is of considerable interest that a spontaneous mutation (mdx) arose in our inbred C57BL/10 colony of mice that is X chromosome-linked and produces viable homozygous animals that have high serum levels of muscle enzymes and exhibit histological lesions similar to human muscular dystrophy.MATERIALS AND METHODS Animals. C57BL/lOScSn inbred mice were originally obtained from M. Festing (MRC Laboratory Animals Centre, Carshalton, Surrey, U.K.) and further inbred for five generations prior to the spontaneous appearance of the X chromosome-linked muscular dystrophy (mdx) mutation. Segregation analysis was performed on animals within this stock. The position of mdx on the X chromosome was located by crossing to four mutants: Mottled-blotchy (Moblo), Tabby (Ta), sparse-fur (spf) and Hypophosphataemia (Hyp) (all the gift of M. Lyon, MRC Radiobiology Unit, Harwell, Oxon, U.K.).Enzyme Assays. Pyruvate kinase (PK; EC 2.7.1.40) activity was determined by a semiautomated enzyme assay (12) and expressed as urmol/min per ml of whole blood ± SEM. In some experiments the blood was fractionated into plasma and erythrocytes after withdrawal by heart puncture into heparinized tubes. The blood was centrifuged at 1,000 x gavg for 15 min, and the plasma was withdrawn; the leukocyte and interface layers were discarded, and the erythrocytes were resuspended twice in 0.15 M NaCl and recentrifuged and then were lysed in 0.05 M Tris, 7.4/1 mM EDTA/1 mM dithiothreitol/0.1% Triton X-100. Cellulose acetate electrophoresis and staining of creatine kinase (EC 2.7.3.2) was performed according to the protocol provided by Helena Laboratories (Beaumont, TX).Histology. All mice were examined clinically before being killed, and as many muscles as possible were surveyed. Ether-killed mice were skinned, eviscerated, fixed in 10% Formal/saline, halved in the median...

Bacterial lipopolysaccharide (LPS) provokes a vigorous, generalized proinflammatory state in the infected host. Genetic regulation of this response has been localized to the Lps locus on mouse chromosome 4, through study of the C3H/HeJ and C57BL/10ScCr inbred strains. Both C3H/HeJ and C57BL/10ScCr mice are homozygous for a mutant Lps allele (Lpsd/d) that confers hyporesponsiveness to LPS challenge, and therefore exhibit natural tolerance to its lethal effects. Genetic and physical mapping of 1,345 backcross progeny segregating this mutant phenotype confined Lps to a 0.9-cM interval spanning 1.7 Mb. Three transcription units were identified within the candidate interval, including Toll-like receptor 4 (Tlr4), part of a protein family with members that have been implicated in LPS-induced cell signaling. C3H/HeJ mice have a point mutation within the coding region of the Tlr4 gene, resulting in a nonconservative substitution of a highly conserved proline by histidine at codon 712, whereas C57BL/ 10ScCr mice exhibit a deletion of Tlr4. Identification of distinct mutations involving the same gene at the Lps locus in two different hyporesponsive inbred mouse strains strongly supports the hypothesis that altered Tlr4 function is responsible for endotoxin tolerance.

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Mutations in the mouse microphthalmia (mi) gene affect the development of a number of cell types including melanocytes, osteoclasts and mast cells. Recently, mutations in the human mi gene (MITF) were found in patients with Waardenburg Syndrome type 2 (WS2), a dominantly inherited syndrome associated with hearing loss and pigmentary disturbances. We have characterized the molecular defects associated with eight murine mi mutations, which vary in both their mode of inheritance and in the cell types they affect. These molecular data, combined with the extensive body of genetic data accumulated for murine mi, shed light on the phenotypic and developmental consequences of mi mutations and offer a mouse model for WS2.

Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive disorder characterized by hypopigmentation, severe immunologic deficiency with neutropenia and lack of natural killer (NK) cells, a bleeding tendency and neurologic abnormalities. Most patients die in childhood. The CHS hallmark is the occurrence of giant inclusion bodies and organelles in a variety of cell types, and protein sorting defects into these organelles. Similar abnormalities occur in the beige mouse, the proposed model for human CHS. Two groups have recently reported the identification of the beige gene, however the two cDNAs were not at all similar. Here we describe the sequence of a human cDNA homologous to mouse beige, identify pathologic mutations and clarify the discrepancies of the previous reports. Analysis of the CHS polypeptide demonstrates that its modular architecture is similar to the yeast vacuolar sorting protein, VPS15.

Congenic breeding strategies are becoming increasingly important as a greater number of complex trait linkages are identified. Traditionally, the development of a congenic strain has been a time-consuming endeavour, requiring ten generations of backcrosses. The recent advent of a dense molecular genetic map of the mouse permits methods that can reduce the time needed for congenic-strain production by 18-24 months. We present a theoretical evaluation of marker-assisted congenic production and provide the empirical data that support it. We present this 'speed congenic' method in a user-friendly manner to encourage other investigators to pursue this or similar methods of congenic production.

The mutated gene responsible for the tubby obesity phenotype has been identified by positional cloning. A single base change within a splice donor site results in the incorrect retention of a single intron in the mature tub mRNA transcript. The consequence of this mutation is the substitution of the carboxy-terminal 44 amino acids with 24 intron-encoded amino acids. The normal transcript appears to be abundantly expressed in the hypothalamus, a region of the brain involved in body weight regulation. Variation in the relative abundance of alternative splice products is observed between inbred mouse strains and appears to correlate with an intron length polymorphism. This allele of tub is a candidate for a previously reported diet-induced obesity quantitative trait locus on mouse chromosome 7.