RESUMO
Mice representing precise genetic replicas of Huntington's disease (HD) were made using gene targeting to replace the short CAG repeat of the mouse Huntington's disease gene homolog (HDH:) with CAG repeats within the length range found to cause HD in humans. Mice with alleles of approximately 150 units in length exhibit late-onset behavioral and neuroanatomic abnormalities consistent with HD. These symptoms include a motor task deficit, gait abnormalities, reactive gliosis and the formation of neuronal intranuclear inclusions predominating in the striatum. This model differs from previously described HDH: knock-ins by its method of construction, longer repeat length and more severe phenotype. To our knowledge, this is the first knock-in mouse model of HD to show increased glial fibrillary acidic protein immunoreactivity in the striatum, suggesting that these mice have neuronal injury similar to that found early in the course of HD. These mice will serve as useful reagents in experiments designed to reveal the molecular nature of neuronal dysfunction underlying HD.
Assuntos
Modelos Animais de Doenças , Doença de Huntington/genética , Doença de Huntington/fisiopatologia , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Expansão das Repetições de Trinucleotídeos , Alelos , Animais , Comportamento Animal , Encéfalo/metabolismo , Encéfalo/patologia , Núcleo Celular/ultraestrutura , Marcação de Genes , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/genética , Gliose/metabolismo , Transtornos do Crescimento/genética , Homozigoto , Proteína Huntingtina , Doença de Huntington/patologia , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Coxeadura Animal/genética , Coxeadura Animal/fisiopatologia , Camundongos , Camundongos Mutantes Neurológicos , Mutação , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Peptídeos/genéticaRESUMO
The introduction of subtle mutations to predetermined locations in the mouse genome has aided in the assessment of gene function and the precise modeling of inherited disorders. Subtle mutations can be engineered into the mouse genome by the tag and exchange gene targeting strategy (Askew et al., 1993; Stacey et al., 1994; Wu et al., 1994). This two-step method involves both a positive and a negative selection. The negative selection step typically generates a large amount of undesired background that may prevent the practical recovery of gene targeted clones (Vazquez et al., 1998). In this work we describe a strategy to effectively manage this background by calculation of a tolerable level of background for a specific targeting event, pre-screening for clones with low background, subcloning and growth of cell lines under selection. This strategy was used to repeatedly and efficiently alter the mouse Huntington's disease homologue (Hdh) resulting in an average of 15 percent of the clones having the desired modification. Analysis of the remaining background clones showed they arose de novo by a mechanism that involved physical loss of the marker rather than mutation or inactivation. We calculated the rate of loss of this marker as 8.3 x 10(-6) events/cell/generation. We further show that the exchanged clones retained the capacity to contribute to the mouse germline demonstrating the utility of this strategy in the production of mouse lines with Hdh variants.
Assuntos
Marcação de Genes , Técnicas de Transferência de Genes , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Animais , Southern Blotting , Éxons , Proteína Huntingtina , Íntrons , Camundongos , Modelos Genéticos , Mutagênese , MutaçãoRESUMO
Several neurological disorders have been attributed to the inheritance of long CAG-polyglutamine repeats. Unlike classical mutations, whose deleterious effects are totally dependent on the context of the gene in which they reside, these translated CAG repeat mutations have been shown to cause neurotoxicity and neuronal intranuclear inclusions when expressed outside their natural gene context. We provide a description of mice with different lengths of repeat in the foreign context of the murine Hprt locus, focusing on aspects of the phenotype that provide an insight into the mechanism by which this unusual mutation might cause toxicity.
Assuntos
Encefalopatias/genética , Hipoxantina Fosforribosiltransferase/genética , Mutação , Doenças do Sistema Nervoso/genética , Peptídeos/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Humanos , Camundongos , Camundongos Mutantes Neurológicos , Cromossomo XRESUMO
The mutations responsible for several human neurodegenerative disorders are expansions of translated CAG repeats beyond a normal size range. To address the role of repeat context, we have introduced a 146-unit CAG repeat into the mouse hypoxanthine phosphoribosyltransferase gene (Hprt). Mutant mice express a form of the HPRT protein that contains a long polyglutamine repeat. These mice develop a phenotype similar to the human translated CAG repeat disorders. Repeat containing mice show a late onset neurological phenotype that progresses to premature death. Neuronal intranuclear inclusions are present in affected mice. Our results show that CAG repeats do not need to be located within one of the classic repeat disorder genes to have a neurotoxic effect.
