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Development of transgenic rats producing human beta-amyloid precursor protein as a model for Alzheimer's disease: transgene and endogenous APP genes are regulated tissue-specifically.

Agca C, Fritz JJ, Walker LC, Levey AI, Chan AW, Lah JJ, Agca Y - BMC Neurosci (2008)

Bottom Line: Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver.The APP21 rat line expresses high levels of human APP and could be a useful model for AD.Determination of the elements that are responsible for tissue-specific expression of APP may enable new treatment options for AD.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Missouri College of Veterinary Medicine, Department of Veterinary Pathobiology Columbia, MO 65211, USA. agcac@missouri.edu

ABSTRACT

Background: Alzheimer's disease (AD) is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. In addition to idiopathic disease, AD is also associated with autosomal dominant inheritance, which causes a familial form of AD (FAD). Some instances of FAD have been linked to mutations in the beta-amyloid protein precursor (APP). Although there are numerous mouse AD models available, few rat AD models, which have several advantages over mice, have been generated.

Results: Fischer 344 rats expressing human APP driven by the ubiquitin-C promoter were generated via lentiviral vector infection of Fischer 344 zygotes. We generated two separate APP-transgenic rat lines, APP21 and APP31. Serum levels of human amyloid-beta (Abeta)40 were 298 pg/ml for hemizygous and 486 pg/ml for homozygous APP21 animals. Serum Abeta42 levels in APP21 homozygous rats were 135 pg/ml. Immunohistochemistry in brain showed that the human APP transgene was expressed in neurons, but not in glial cells. These findings were consistent with independent examination of enhanced green fluorescent protein (eGFP) in the brains of eGFP-transgenic rats. APP21 and APP31 rats expressed 7.5- and 3-times more APP mRNA, respectively, than did wild-type rats. Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. A similar expression pattern was also seen for the endogenous rat APP. The unexpected similarity in the tissue-specific expression patterns of endogenous rat APP and transgenic human APP mRNAs suggests regulatory elements within the cDNA sequence of APP.

Conclusion: This manuscript describes the generation of APP-transgenic inbred Fischer 344 rats. These are the first human AD model rat lines generated by lentiviral infection. The APP21 rat line expresses high levels of human APP and could be a useful model for AD. Tissue-specific expression in the two transgenic rat lines and in wild-type rats contradicts our current understanding of APP gene regulation. Determination of the elements that are responsible for tissue-specific expression of APP may enable new treatment options for AD.

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Northern blot hybridization of total RNA from tissues of the APP31 and APP21 lines as well as WT rats. Total RNA hybridized with the human APP probe (upper autoradiogram) prior to hybridization with 18S rRNA (lower autoradiogram). B: Brain, H: Heart, K: Kidney, Li: Liver, Lu: Lung.
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Figure 4: Northern blot hybridization of total RNA from tissues of the APP31 and APP21 lines as well as WT rats. Total RNA hybridized with the human APP probe (upper autoradiogram) prior to hybridization with 18S rRNA (lower autoradiogram). B: Brain, H: Heart, K: Kidney, Li: Liver, Lu: Lung.

Mentions: Expression of the APP transgene was determined by Northern blot analysis in homozygous rats (Fig. 4). The APP probe used for Northern blot analysis hybridizes both the human APP transgene as well as native rat APP. The BLAST score (NCBI, Bethesda, MD) of the 773 bp human APP probe template to rat APP mRNA was 547. The molecular weight of the APP mRNA was approximately 2.5 kb. Northern blot analysis showed that the APP21 line expressed the highest levels of APP mRNA, the APP31 line expressed intermediate levels (Fig. 4 and 5A), and the native rat APP mRNA expression was the lowest. Expression of APP showed a tissue-dependent variation. The transgene was highly expressed in kidney and lung of both the APP21 and APP31 lines. Expression in brain was intermediate, and expression in liver and heart was the lowest for both Tg lines (Fig. 4 and 5A). Similar expression patterns for rat APP mRNA were observed in non-transgenic rats, such that liver and heart contained lower APP compared to brain, kidney and lung. The average expression among the organs analyzed was 4.3 times lower in WT rats than in APP Tg rats. Brain APP expression in the APP21 line was 1.7 and 2.9 times greater than in APP31 and WT rats, respectively (Fig. 5B).


Development of transgenic rats producing human beta-amyloid precursor protein as a model for Alzheimer's disease: transgene and endogenous APP genes are regulated tissue-specifically.

Agca C, Fritz JJ, Walker LC, Levey AI, Chan AW, Lah JJ, Agca Y - BMC Neurosci (2008)

Northern blot hybridization of total RNA from tissues of the APP31 and APP21 lines as well as WT rats. Total RNA hybridized with the human APP probe (upper autoradiogram) prior to hybridization with 18S rRNA (lower autoradiogram). B: Brain, H: Heart, K: Kidney, Li: Liver, Lu: Lung.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2268936&req=5

Figure 4: Northern blot hybridization of total RNA from tissues of the APP31 and APP21 lines as well as WT rats. Total RNA hybridized with the human APP probe (upper autoradiogram) prior to hybridization with 18S rRNA (lower autoradiogram). B: Brain, H: Heart, K: Kidney, Li: Liver, Lu: Lung.
Mentions: Expression of the APP transgene was determined by Northern blot analysis in homozygous rats (Fig. 4). The APP probe used for Northern blot analysis hybridizes both the human APP transgene as well as native rat APP. The BLAST score (NCBI, Bethesda, MD) of the 773 bp human APP probe template to rat APP mRNA was 547. The molecular weight of the APP mRNA was approximately 2.5 kb. Northern blot analysis showed that the APP21 line expressed the highest levels of APP mRNA, the APP31 line expressed intermediate levels (Fig. 4 and 5A), and the native rat APP mRNA expression was the lowest. Expression of APP showed a tissue-dependent variation. The transgene was highly expressed in kidney and lung of both the APP21 and APP31 lines. Expression in brain was intermediate, and expression in liver and heart was the lowest for both Tg lines (Fig. 4 and 5A). Similar expression patterns for rat APP mRNA were observed in non-transgenic rats, such that liver and heart contained lower APP compared to brain, kidney and lung. The average expression among the organs analyzed was 4.3 times lower in WT rats than in APP Tg rats. Brain APP expression in the APP21 line was 1.7 and 2.9 times greater than in APP31 and WT rats, respectively (Fig. 5B).

Bottom Line: Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver.The APP21 rat line expresses high levels of human APP and could be a useful model for AD.Determination of the elements that are responsible for tissue-specific expression of APP may enable new treatment options for AD.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Missouri College of Veterinary Medicine, Department of Veterinary Pathobiology Columbia, MO 65211, USA. agcac@missouri.edu

ABSTRACT

Background: Alzheimer's disease (AD) is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. In addition to idiopathic disease, AD is also associated with autosomal dominant inheritance, which causes a familial form of AD (FAD). Some instances of FAD have been linked to mutations in the beta-amyloid protein precursor (APP). Although there are numerous mouse AD models available, few rat AD models, which have several advantages over mice, have been generated.

Results: Fischer 344 rats expressing human APP driven by the ubiquitin-C promoter were generated via lentiviral vector infection of Fischer 344 zygotes. We generated two separate APP-transgenic rat lines, APP21 and APP31. Serum levels of human amyloid-beta (Abeta)40 were 298 pg/ml for hemizygous and 486 pg/ml for homozygous APP21 animals. Serum Abeta42 levels in APP21 homozygous rats were 135 pg/ml. Immunohistochemistry in brain showed that the human APP transgene was expressed in neurons, but not in glial cells. These findings were consistent with independent examination of enhanced green fluorescent protein (eGFP) in the brains of eGFP-transgenic rats. APP21 and APP31 rats expressed 7.5- and 3-times more APP mRNA, respectively, than did wild-type rats. Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. A similar expression pattern was also seen for the endogenous rat APP. The unexpected similarity in the tissue-specific expression patterns of endogenous rat APP and transgenic human APP mRNAs suggests regulatory elements within the cDNA sequence of APP.

Conclusion: This manuscript describes the generation of APP-transgenic inbred Fischer 344 rats. These are the first human AD model rat lines generated by lentiviral infection. The APP21 rat line expresses high levels of human APP and could be a useful model for AD. Tissue-specific expression in the two transgenic rat lines and in wild-type rats contradicts our current understanding of APP gene regulation. Determination of the elements that are responsible for tissue-specific expression of APP may enable new treatment options for AD.

Show MeSH
Related in: MedlinePlus