Limits...
Mouse ataxin-3 functional knock-out model.

Switonski PM, Fiszer A, Kazmierska K, Kurpisz M, Krzyzosiak WJ, Figiel M - Neuromolecular Med. (2010)

Bottom Line: Although the human transgene was inserted correctly, the resulting mice acquired the knock-out properties and did not express ataxin-3 protein in any analyzed tissues, as confirmed by western blot and immunohistochemistry.After applying 37 PCR cycles, we also detected a very low level of the correct exon 1/exon 2 isoform.We hypothesized that these splicing aberrations result from the deletion of further splicing sites and the presence of a strong splicing site in exon 4, which was confirmed by bioinformatic analysis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland.

ABSTRACT
Spinocerebellar ataxia 3 (SCA3) is a genetic disorder resulting from the expansion of the CAG repeats in the ATXN3 gene. The pathogenesis of SCA3 is based on the toxic function of the mutant ataxin-3 protein, but the exact mechanism of the disease remains elusive. Various types of transgenic mouse models explore different aspects of SCA3 pathogenesis, but a knock-in humanized mouse has not yet been created. The initial aim of this study was to generate an ataxin-3 humanized mouse model using a knock-in strategy. The human cDNA for ataxin-3 containing 69 CAG repeats was cloned from SCA3 patient and introduced into the mouse ataxin-3 locus at exon 2, deleting it along with exon 3 and intron 2. Although the human transgene was inserted correctly, the resulting mice acquired the knock-out properties and did not express ataxin-3 protein in any analyzed tissues, as confirmed by western blot and immunohistochemistry. Analyses of RNA expression revealed that the entire locus consisting of human and mouse exons was expressed and alternatively spliced. We detected mRNA isoforms composed of exon 1 spliced with mouse exon 4 or with human exon 7. After applying 37 PCR cycles, we also detected a very low level of the correct exon 1/exon 2 isoform. Additionally, we confirmed by bioinformatic analysis that the structure and power of the splicing site between mouse intron 1 and human exon 2 (the targeted locus) was not changed compared with the native mouse locus. We hypothesized that these splicing aberrations result from the deletion of further splicing sites and the presence of a strong splicing site in exon 4, which was confirmed by bioinformatic analysis. In summary, we created a functional ataxin-3 knock-out mouse model that is viable and fertile and does not present a reduced life span. Our work provides new insights into the splicing characteristics of the Atxn3 gene and provides useful information for future attempts to create knock-in SCA3 models.

Show MeSH

Related in: MedlinePlus

The cellular phenotype of the K300 mouse model. The cryosections prepared from cerebellum and cerebral cortex were stained using anti-ubiquitin Ab, and confocal images were acquired. The intranuclear inclusions were not detected in cryosections from K300 mut/mut mouse brain (b, d). The positive control brains from N171/82Q HD animals revealed a large number of intranuclear inclusions positive for ubiquitin in both cerebellum and cerebral cortex (a, c). Additionally, the cryosections from testis of K300 mut/mut and wt/wt animals were stained using anti-ataxin-3 antibodies and the DAB chromogen, and the nuclei were counterstained with hematoxylin dye. Testis from K300 mut/mut animals did not stain positively for ataxin-3, and testis of wt/wt animals exhibited specific, positive brown staining for ataxin-3 in a subset of cells in testis (e, f). The immunoblotting of total proteins with anti-ubiquitin antibodies revealed no difference in the levels of total ubiquitin in mut/mut compared with wt/wt animals (g)
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3044828&req=5

Fig4: The cellular phenotype of the K300 mouse model. The cryosections prepared from cerebellum and cerebral cortex were stained using anti-ubiquitin Ab, and confocal images were acquired. The intranuclear inclusions were not detected in cryosections from K300 mut/mut mouse brain (b, d). The positive control brains from N171/82Q HD animals revealed a large number of intranuclear inclusions positive for ubiquitin in both cerebellum and cerebral cortex (a, c). Additionally, the cryosections from testis of K300 mut/mut and wt/wt animals were stained using anti-ataxin-3 antibodies and the DAB chromogen, and the nuclei were counterstained with hematoxylin dye. Testis from K300 mut/mut animals did not stain positively for ataxin-3, and testis of wt/wt animals exhibited specific, positive brown staining for ataxin-3 in a subset of cells in testis (e, f). The immunoblotting of total proteins with anti-ubiquitin antibodies revealed no difference in the levels of total ubiquitin in mut/mut compared with wt/wt animals (g)

Mentions: Nuclear inclusions are the hallmark of polyglutamine disorders and are present in both patients and mouse models. Therefore, we examined the brains of K300 animals for the presence of intranuclear inclusions by immunofluorochemistry using anti-ubiquitin antibodies in cerebral cortex and cerebellum of the K300 mut/mut and using N171/82Q animals as positive controls (Huntington’s disease mouse model; Schilling et al. 1999). As expected, the cortex and cerebellum of N171/82Q mice revealed intranuclear inclusions positive for ubiquitin (Fig. 4a, c). Similar structures were not detected in cells from cortex or cerebellum of K300 mut/mut mice stained with anti-ubiquitin antibodies (Fig. 4b, d). To demonstrate the loss of ataxin-3 expression, we cryosectioned the testis from K300 mut/mut animals and wt/wt animals and stained the tissue using anti-ataxin-3 antibodies and the DAB chromogen. The cells in testis of wt/wt animals were positively stained for ataxin-3 (Fig. 4e), and no staining was detected in mut/mut animals (Fig. 4f).Fig. 4


Mouse ataxin-3 functional knock-out model.

Switonski PM, Fiszer A, Kazmierska K, Kurpisz M, Krzyzosiak WJ, Figiel M - Neuromolecular Med. (2010)

The cellular phenotype of the K300 mouse model. The cryosections prepared from cerebellum and cerebral cortex were stained using anti-ubiquitin Ab, and confocal images were acquired. The intranuclear inclusions were not detected in cryosections from K300 mut/mut mouse brain (b, d). The positive control brains from N171/82Q HD animals revealed a large number of intranuclear inclusions positive for ubiquitin in both cerebellum and cerebral cortex (a, c). Additionally, the cryosections from testis of K300 mut/mut and wt/wt animals were stained using anti-ataxin-3 antibodies and the DAB chromogen, and the nuclei were counterstained with hematoxylin dye. Testis from K300 mut/mut animals did not stain positively for ataxin-3, and testis of wt/wt animals exhibited specific, positive brown staining for ataxin-3 in a subset of cells in testis (e, f). The immunoblotting of total proteins with anti-ubiquitin antibodies revealed no difference in the levels of total ubiquitin in mut/mut compared with wt/wt animals (g)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044828&req=5

Fig4: The cellular phenotype of the K300 mouse model. The cryosections prepared from cerebellum and cerebral cortex were stained using anti-ubiquitin Ab, and confocal images were acquired. The intranuclear inclusions were not detected in cryosections from K300 mut/mut mouse brain (b, d). The positive control brains from N171/82Q HD animals revealed a large number of intranuclear inclusions positive for ubiquitin in both cerebellum and cerebral cortex (a, c). Additionally, the cryosections from testis of K300 mut/mut and wt/wt animals were stained using anti-ataxin-3 antibodies and the DAB chromogen, and the nuclei were counterstained with hematoxylin dye. Testis from K300 mut/mut animals did not stain positively for ataxin-3, and testis of wt/wt animals exhibited specific, positive brown staining for ataxin-3 in a subset of cells in testis (e, f). The immunoblotting of total proteins with anti-ubiquitin antibodies revealed no difference in the levels of total ubiquitin in mut/mut compared with wt/wt animals (g)
Mentions: Nuclear inclusions are the hallmark of polyglutamine disorders and are present in both patients and mouse models. Therefore, we examined the brains of K300 animals for the presence of intranuclear inclusions by immunofluorochemistry using anti-ubiquitin antibodies in cerebral cortex and cerebellum of the K300 mut/mut and using N171/82Q animals as positive controls (Huntington’s disease mouse model; Schilling et al. 1999). As expected, the cortex and cerebellum of N171/82Q mice revealed intranuclear inclusions positive for ubiquitin (Fig. 4a, c). Similar structures were not detected in cells from cortex or cerebellum of K300 mut/mut mice stained with anti-ubiquitin antibodies (Fig. 4b, d). To demonstrate the loss of ataxin-3 expression, we cryosectioned the testis from K300 mut/mut animals and wt/wt animals and stained the tissue using anti-ataxin-3 antibodies and the DAB chromogen. The cells in testis of wt/wt animals were positively stained for ataxin-3 (Fig. 4e), and no staining was detected in mut/mut animals (Fig. 4f).Fig. 4

Bottom Line: Although the human transgene was inserted correctly, the resulting mice acquired the knock-out properties and did not express ataxin-3 protein in any analyzed tissues, as confirmed by western blot and immunohistochemistry.After applying 37 PCR cycles, we also detected a very low level of the correct exon 1/exon 2 isoform.We hypothesized that these splicing aberrations result from the deletion of further splicing sites and the presence of a strong splicing site in exon 4, which was confirmed by bioinformatic analysis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland.

ABSTRACT
Spinocerebellar ataxia 3 (SCA3) is a genetic disorder resulting from the expansion of the CAG repeats in the ATXN3 gene. The pathogenesis of SCA3 is based on the toxic function of the mutant ataxin-3 protein, but the exact mechanism of the disease remains elusive. Various types of transgenic mouse models explore different aspects of SCA3 pathogenesis, but a knock-in humanized mouse has not yet been created. The initial aim of this study was to generate an ataxin-3 humanized mouse model using a knock-in strategy. The human cDNA for ataxin-3 containing 69 CAG repeats was cloned from SCA3 patient and introduced into the mouse ataxin-3 locus at exon 2, deleting it along with exon 3 and intron 2. Although the human transgene was inserted correctly, the resulting mice acquired the knock-out properties and did not express ataxin-3 protein in any analyzed tissues, as confirmed by western blot and immunohistochemistry. Analyses of RNA expression revealed that the entire locus consisting of human and mouse exons was expressed and alternatively spliced. We detected mRNA isoforms composed of exon 1 spliced with mouse exon 4 or with human exon 7. After applying 37 PCR cycles, we also detected a very low level of the correct exon 1/exon 2 isoform. Additionally, we confirmed by bioinformatic analysis that the structure and power of the splicing site between mouse intron 1 and human exon 2 (the targeted locus) was not changed compared with the native mouse locus. We hypothesized that these splicing aberrations result from the deletion of further splicing sites and the presence of a strong splicing site in exon 4, which was confirmed by bioinformatic analysis. In summary, we created a functional ataxin-3 knock-out mouse model that is viable and fertile and does not present a reduced life span. Our work provides new insights into the splicing characteristics of the Atxn3 gene and provides useful information for future attempts to create knock-in SCA3 models.

Show MeSH
Related in: MedlinePlus