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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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Comparison of promoters following lentivirus injection in rat brain. Lentiviruses were stereotaxically injected into rat hippocampus and examined after three months. A. eGFP expression driven by the ubiquitin-C promoter (Ubi-C) was consistently superior to that of other promoters, including the platelet-derived growth factor promoter (PDGF) and cytomegalovirus. Lower panels show representative higher power images of eGFP driven by Ubi-C- (B), cytomegalovirus- (C), and PDGF- (D) promoters in lentivirus/eGFP-injected rats.
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Figure 1: Comparison of promoters following lentivirus injection in rat brain. Lentiviruses were stereotaxically injected into rat hippocampus and examined after three months. A. eGFP expression driven by the ubiquitin-C promoter (Ubi-C) was consistently superior to that of other promoters, including the platelet-derived growth factor promoter (PDGF) and cytomegalovirus. Lower panels show representative higher power images of eGFP driven by Ubi-C- (B), cytomegalovirus- (C), and PDGF- (D) promoters in lentivirus/eGFP-injected rats.

Mentions: Comparison of enhanced green fluorescent protein (eGFP) expression driven from ubiquitin-C (Ubi-C), cytomegalovirus (CMV), or platelet-derived growth factor (PDGF) promoters in SD rat brains after stereotaxic injection of lentivirus showed that the Ubi-C promoter yielded consistently superior eGFP expression than did the CMV-eGFP and PDGF-eGFP viruses. Figure 1 shows eGFP expression in rat brains three weeks post-injection of Ubi-C-eGFP and CMV-eGFP lentiviruses. Enhanced GFP expression driven by CMV and PDGF promoters appeared to decrease dramatically over time. In contrast, strong Ubi-C- eGFP expression persisted without apparent diminution at the longest time-point examined (13 months, data not shown). These results with Ubi-C are consistent with previous findings using the Ubi-C promoter in driving transgene expression [16].


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)

Comparison of promoters following lentivirus injection in rat brain. Lentiviruses were stereotaxically injected into rat hippocampus and examined after three months. A. eGFP expression driven by the ubiquitin-C promoter (Ubi-C) was consistently superior to that of other promoters, including the platelet-derived growth factor promoter (PDGF) and cytomegalovirus. Lower panels show representative higher power images of eGFP driven by Ubi-C- (B), cytomegalovirus- (C), and PDGF- (D) promoters in lentivirus/eGFP-injected rats.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Comparison of promoters following lentivirus injection in rat brain. Lentiviruses were stereotaxically injected into rat hippocampus and examined after three months. A. eGFP expression driven by the ubiquitin-C promoter (Ubi-C) was consistently superior to that of other promoters, including the platelet-derived growth factor promoter (PDGF) and cytomegalovirus. Lower panels show representative higher power images of eGFP driven by Ubi-C- (B), cytomegalovirus- (C), and PDGF- (D) promoters in lentivirus/eGFP-injected rats.
Mentions: Comparison of enhanced green fluorescent protein (eGFP) expression driven from ubiquitin-C (Ubi-C), cytomegalovirus (CMV), or platelet-derived growth factor (PDGF) promoters in SD rat brains after stereotaxic injection of lentivirus showed that the Ubi-C promoter yielded consistently superior eGFP expression than did the CMV-eGFP and PDGF-eGFP viruses. Figure 1 shows eGFP expression in rat brains three weeks post-injection of Ubi-C-eGFP and CMV-eGFP lentiviruses. Enhanced GFP expression driven by CMV and PDGF promoters appeared to decrease dramatically over time. In contrast, strong Ubi-C- eGFP expression persisted without apparent diminution at the longest time-point examined (13 months, data not shown). These results with Ubi-C are consistent with previous findings using the Ubi-C promoter in driving transgene expression [16].

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