Four children with severe mycobacterial infections had a mutation in the gene for interferon-gamma receptor 1 that leads to the absence of receptors on cell surfaces and a functional defect in the up-regulation of tumor necrosis factor alpha by macrophages in response to interferon-gamma. The interferon-gamma pathway is important in the response to intracellular pathogens such as mycobacteria.

Machado-Joseph disease (MJD; MIM 109150) is a late-onset neurodegenerative disorder caused by the expansion of a polyglutamine tract within the MJD1 gene. We have previously reported the generation of human yeast artificial chromosome (YAC) constructs encompassing the MJD1 locus into which expanded (CAG)(76) and (CAG)(84) repeat motifs have been introduced by homologous recombination. Transgenic mice containing pathological alleles with polyglutamine tract lengths of 64, 67, 72, 76 and 84 repeats, as well as the wild type with 15 repeats, have now been generated using these YAC constructs. The mice with expanded alleles demonstrate a mild and slowly progressive cerebellar deficit, manifesting as early as 4 weeks of age. As the disease progresses, pelvic elevation becomes markedly flattened, accompanied by hypotonia, and motor and sensory loss. Neuronal intranuclear inclusion (NII) formation and cell loss is prominent in the pontine and dentate nuclei, with variable cell loss in other regions of the cerebellum from 4 weeks of age. Interestingly, peripheral nerve demyelination and axonal loss is detected in symptomatic mice from 26 weeks of age. In contrast, transgenic mice carrying the wild-type (CAG)(15) allele of the MJD1 locus appear completely normal at 20 months. Disease severity increases with the level of expression of the expanded protein and the size of the repeat. These mice are representative of MJD and will be a valuable resource for the detailed analysis of the roles of repeat length, tissue specificity and level of expression in the neurodegenerative processes underlying MJD pathogenesis.

Transgenic mice were generated using a purified 248-kb yeast artificial chromosome (YAC) bearing an intact 82-kb human (-globin locus and 148 kb of fanking sequence. Seventeen of 148 Fo pups were transgenic. RNase protection analysis of RNA isolated from the blood of 13 r and 13-globin-positive founders showed that only the human 3-globin gene was expressed in the adult founders. Studies of F1 and F2 fetuses demonstrated that the genes of the f-locus YAC displayed the proper developmental switches in 3-like globin gene expression. Expression of e-and -globin, but not (-globin, was observed in the yolk sac, there was only minor y and mostly 13 expression in the 14-day liver, and only (3 mRNA in the blood of the adult animals. Structural data showed that the locus was intact. These results indicate that it is now possible to dissect regulatory mechanisms within the context of an entire locus in vivo by using the ability to perform mutagenesis efficiently in yeast via homologous recombination, followed by purification ofthe altered YAC

Construction of animal models of human inherited diseases is particularly important for testing gene therapy approaches. Towards this end, we constructed a mouse model for Charcot-Marie-Tooth disease type 1A by pronuclear injection of a YAC containing the human PMP22 gene. In one transgenic line, the YAC DNA is integrated in about eight copies and the PMP22 gene is strongly expressed to give a peripheral neuropathy closely resembling the human pathology. The disorder is dominant, causes progressive weakness of the hind legs, and there is severe demyelination in the peripheral nervous system including the presence of onion bulb formations. This approach will be valuable for pathologies produced by over-expression of a gene including trisomy and amplification in cancer. Such models will be particularly useful for testing gene therapy approaches if the transgene is human.

Charcot-Marie-Tooth disease type 1A is most commonly caused by a duplication of a 1.5 Mb region of chromosome 17 which includes the peripheral myelin protein 22 gene (PMP22). Over-expression of this gene leads to a hypomyelinating/demyelinating neuropathy and to severely reduced nerve conduction velocity. Previous mouse and rat models have had relatively high levels of expression of the mouse or human PMP22 gene leading to severe demyelination. Here we describe five lines of transgenic mice carrying increasing copies of the human PMP22 gene (one to seven) and expressing increasing levels of the transgene. From histological and electrophysiological observations there appears to be a threshold below which expression of PMP22 has virtually no effect; below a ratio of human/mouse mRNA expression of approximately 0.8, little effect is observed. Between a ratio of 0.8 and 1.5, histological and nerve conduction velocity abnormalities are observed, but there are no behavioural signs of neuropathy. An expression ratio >1.5 leads to a severe neuropathy. A second observation concerns the histology of the different lines; the level of expression does not affect the type of demyelination, but influences the severity of involvement.