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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.

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Immunoblot analysis of ataxin-3 expression in tissues of K300 mice. Total protein was extracted from brain, lung, heart, spleen, testis, skeletal muscle, and liver samples collected from transgenic (mut/mut) and wild-type (wt/wt) animals as well as from a primary culture of human SCA3 fibroblasts (GM06153), which was used as a positive control. The extracts were resolved by 12% SDS/PAGE, and the blot was stained with a polyclonal antibody for ataxin-3. In wt/wt animals, the analysis revealed a mouse ataxin-3 41-kDa band present at relatively high concentrations in testis, cerebral cortex, lungs, liver, and spleen that was hardly detectable in skeletal muscles and heart. A similar 41-kDa band and the human mutant ataxin-3 57-kDa band were not detected in any tissues from mut/mut animals. Additionally, we detected a 34-kDa band of the ataxin-3 isoform in testis from wt/wt animals. This isoform was not present in the testis of mut/mut animals (a). Upon increasing the total protein load to 200 μg per lane, we were still unable to detect a human mutant ataxin-3 57-kDa band, but we did detect two faint immunoreactive bands of 33 and 26 kDa, only in testis of mut/mut animals. The apparent 7-kDa molecular weight difference between the 33- and 26-kDa bands is the same as that between mouse ataxin-3 (41 kDa) and the 34-kDa ataxin-3 isoform detected in wt/wt testis (b)
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Fig3: Immunoblot analysis of ataxin-3 expression in tissues of K300 mice. Total protein was extracted from brain, lung, heart, spleen, testis, skeletal muscle, and liver samples collected from transgenic (mut/mut) and wild-type (wt/wt) animals as well as from a primary culture of human SCA3 fibroblasts (GM06153), which was used as a positive control. The extracts were resolved by 12% SDS/PAGE, and the blot was stained with a polyclonal antibody for ataxin-3. In wt/wt animals, the analysis revealed a mouse ataxin-3 41-kDa band present at relatively high concentrations in testis, cerebral cortex, lungs, liver, and spleen that was hardly detectable in skeletal muscles and heart. A similar 41-kDa band and the human mutant ataxin-3 57-kDa band were not detected in any tissues from mut/mut animals. Additionally, we detected a 34-kDa band of the ataxin-3 isoform in testis from wt/wt animals. This isoform was not present in the testis of mut/mut animals (a). Upon increasing the total protein load to 200 μg per lane, we were still unable to detect a human mutant ataxin-3 57-kDa band, but we did detect two faint immunoreactive bands of 33 and 26 kDa, only in testis of mut/mut animals. The apparent 7-kDa molecular weight difference between the 33- and 26-kDa bands is the same as that between mouse ataxin-3 (41 kDa) and the 34-kDa ataxin-3 isoform detected in wt/wt testis (b)

Mentions: To investigate the expression of the ataxin-3 protein in the transgenic mice, total protein was extracted from the cerebral cortex, skeletal muscles (quadriceps), heart, lungs, liver, spleen, testis, and GM06153 human fibroblasts as a positive control and resolved by 12% SDS–PAGE. In wt/wt animals, the immunoblot analyses revealed a specific band representing mouse ataxin-3 with an estimated molecular weight of 41 kDa that was present at relatively high concentrations in testis, cerebral cortex, lungs, liver, and spleen and was hardly detectable in skeletal muscles and the heart (Fig. 3a). The band detected in wt/wt mice had a lower molecular weight than the positive control band of wt human ataxin-3 (48 kDa). The similar mouse ataxin-3 41-kDa band and the expected human mutant 57-kDa band were not detected in mut/mut animals in any of the tissues analyzed (Fig. 3a). Moreover, none of these immunoreactive bands was observed upon increasing the protein load to 200 μg of mut/mut protein extract from cerebral cortex and testis per lane (Fig. 3b). Additionally, analysis of the ataxin-3 protein in testis from wt/wt animals revealed an additional strongly immunoreactive band of 34 kDa that we describe as ataxin-3 isoform, since it was not present in testis of mut/mut animals (Fig. 3a, b). Upon increasing the total protein extract content to the large amount of 200 μg per lane, we were able to observe two faint immunoreactive bands of 33 and 26 kDa, respectively, that only appeared in mut/mut animals, but exclusively in testis and not in cerebral cortex (Fig. 3b). Note that the apparent 7-kDa molecular weight difference was also present between mouse ataxin-3 (41 kDa) and the 34-kDa ataxin-3 isoform detected in wt/wt testis.Fig. 3


Mouse ataxin-3 functional knock-out model.

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

Immunoblot analysis of ataxin-3 expression in tissues of K300 mice. Total protein was extracted from brain, lung, heart, spleen, testis, skeletal muscle, and liver samples collected from transgenic (mut/mut) and wild-type (wt/wt) animals as well as from a primary culture of human SCA3 fibroblasts (GM06153), which was used as a positive control. The extracts were resolved by 12% SDS/PAGE, and the blot was stained with a polyclonal antibody for ataxin-3. In wt/wt animals, the analysis revealed a mouse ataxin-3 41-kDa band present at relatively high concentrations in testis, cerebral cortex, lungs, liver, and spleen that was hardly detectable in skeletal muscles and heart. A similar 41-kDa band and the human mutant ataxin-3 57-kDa band were not detected in any tissues from mut/mut animals. Additionally, we detected a 34-kDa band of the ataxin-3 isoform in testis from wt/wt animals. This isoform was not present in the testis of mut/mut animals (a). Upon increasing the total protein load to 200 μg per lane, we were still unable to detect a human mutant ataxin-3 57-kDa band, but we did detect two faint immunoreactive bands of 33 and 26 kDa, only in testis of mut/mut animals. The apparent 7-kDa molecular weight difference between the 33- and 26-kDa bands is the same as that between mouse ataxin-3 (41 kDa) and the 34-kDa ataxin-3 isoform detected in wt/wt testis (b)
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Fig3: Immunoblot analysis of ataxin-3 expression in tissues of K300 mice. Total protein was extracted from brain, lung, heart, spleen, testis, skeletal muscle, and liver samples collected from transgenic (mut/mut) and wild-type (wt/wt) animals as well as from a primary culture of human SCA3 fibroblasts (GM06153), which was used as a positive control. The extracts were resolved by 12% SDS/PAGE, and the blot was stained with a polyclonal antibody for ataxin-3. In wt/wt animals, the analysis revealed a mouse ataxin-3 41-kDa band present at relatively high concentrations in testis, cerebral cortex, lungs, liver, and spleen that was hardly detectable in skeletal muscles and heart. A similar 41-kDa band and the human mutant ataxin-3 57-kDa band were not detected in any tissues from mut/mut animals. Additionally, we detected a 34-kDa band of the ataxin-3 isoform in testis from wt/wt animals. This isoform was not present in the testis of mut/mut animals (a). Upon increasing the total protein load to 200 μg per lane, we were still unable to detect a human mutant ataxin-3 57-kDa band, but we did detect two faint immunoreactive bands of 33 and 26 kDa, only in testis of mut/mut animals. The apparent 7-kDa molecular weight difference between the 33- and 26-kDa bands is the same as that between mouse ataxin-3 (41 kDa) and the 34-kDa ataxin-3 isoform detected in wt/wt testis (b)
Mentions: To investigate the expression of the ataxin-3 protein in the transgenic mice, total protein was extracted from the cerebral cortex, skeletal muscles (quadriceps), heart, lungs, liver, spleen, testis, and GM06153 human fibroblasts as a positive control and resolved by 12% SDS–PAGE. In wt/wt animals, the immunoblot analyses revealed a specific band representing mouse ataxin-3 with an estimated molecular weight of 41 kDa that was present at relatively high concentrations in testis, cerebral cortex, lungs, liver, and spleen and was hardly detectable in skeletal muscles and the heart (Fig. 3a). The band detected in wt/wt mice had a lower molecular weight than the positive control band of wt human ataxin-3 (48 kDa). The similar mouse ataxin-3 41-kDa band and the expected human mutant 57-kDa band were not detected in mut/mut animals in any of the tissues analyzed (Fig. 3a). Moreover, none of these immunoreactive bands was observed upon increasing the protein load to 200 μg of mut/mut protein extract from cerebral cortex and testis per lane (Fig. 3b). Additionally, analysis of the ataxin-3 protein in testis from wt/wt animals revealed an additional strongly immunoreactive band of 34 kDa that we describe as ataxin-3 isoform, since it was not present in testis of mut/mut animals (Fig. 3a, b). Upon increasing the total protein extract content to the large amount of 200 μg per lane, we were able to observe two faint immunoreactive bands of 33 and 26 kDa, respectively, that only appeared in mut/mut animals, but exclusively in testis and not in cerebral cortex (Fig. 3b). Note that the apparent 7-kDa molecular weight difference was also present between mouse ataxin-3 (41 kDa) and the 34-kDa ataxin-3 isoform detected in wt/wt testis.Fig. 3

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