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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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eGFP expression in the brain of a transgenic rat (Ubi-C promoter). Upper panel: eGFP expression is restricted to neurons in transgenic rat brain. A. Confocal microscopy shows extensive eGFP expression in dentate granule cells. B. GFAP immunoreactivity reveals astroglial cells. C. Merged images show a lack of colocalization of eGFP and GFAP signals. Middle panel: confocal images reveal robust eGFP expression in neuronal cell bodies. D. Cortex. E. Striatum. F. CA1 region of hippocampus. G. Hippocampal dentate gyrus. Lower panel: Mixed primary cultures from E18 embryos were stained with anti-beta III tubulin to identify neurons (I; red) and GFAP to label glial cells (J; purple). eGFP expression in cultured neurons was concentrated in the nucleus as well as the cytoplasm (H; green). Arrowheads denote individual neurons in all panels, and the merged image (K) shows eGFP in neurons but not in glial cells.
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Figure 2: eGFP expression in the brain of a transgenic rat (Ubi-C promoter). Upper panel: eGFP expression is restricted to neurons in transgenic rat brain. A. Confocal microscopy shows extensive eGFP expression in dentate granule cells. B. GFAP immunoreactivity reveals astroglial cells. C. Merged images show a lack of colocalization of eGFP and GFAP signals. Middle panel: confocal images reveal robust eGFP expression in neuronal cell bodies. D. Cortex. E. Striatum. F. CA1 region of hippocampus. G. Hippocampal dentate gyrus. Lower panel: Mixed primary cultures from E18 embryos were stained with anti-beta III tubulin to identify neurons (I; red) and GFAP to label glial cells (J; purple). eGFP expression in cultured neurons was concentrated in the nucleus as well as the cytoplasm (H; green). Arrowheads denote individual neurons in all panels, and the merged image (K) shows eGFP in neurons but not in glial cells.

Mentions: While stable, high-level transgene expression is highly desirable in Tg animals, neuron-selective expression is also an important consideration in selecting an optimal promoter. Brain expression of the eGFP transgene driven by a Ubi-C promoter was examined in Tg SD rats created by injection of eGFP-lentivirus into fertilized zygotes. Strong eGFP expression was seen in adult rats; by confocal microscopy, eGFP expression appeared to be restricted to neurons (Fig. 2). When eGFP distribution was directly compared to immunostaining with glial fibrillary acidic protein (GFAP) to label astrocytes, there was strong divergence in the staining patterns. To confirm the apparent restriction of eGFP driven by the Ubi-C promoter to neurons, primary cultures were established from E18 Tg SD embryos and employed for colocalization studies using markers for neurons and glial cells. Confocal images of mixed primary cultures stained with neuron-specific beta tubulin and GFAP is shown in Figure 2J. Enhanced GFP is expressed in neurons, but not in glial cells, confirming the neuronal specificity of eGFP expression driven by the Ubi-C promoter.


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)

eGFP expression in the brain of a transgenic rat (Ubi-C promoter). Upper panel: eGFP expression is restricted to neurons in transgenic rat brain. A. Confocal microscopy shows extensive eGFP expression in dentate granule cells. B. GFAP immunoreactivity reveals astroglial cells. C. Merged images show a lack of colocalization of eGFP and GFAP signals. Middle panel: confocal images reveal robust eGFP expression in neuronal cell bodies. D. Cortex. E. Striatum. F. CA1 region of hippocampus. G. Hippocampal dentate gyrus. Lower panel: Mixed primary cultures from E18 embryos were stained with anti-beta III tubulin to identify neurons (I; red) and GFAP to label glial cells (J; purple). eGFP expression in cultured neurons was concentrated in the nucleus as well as the cytoplasm (H; green). Arrowheads denote individual neurons in all panels, and the merged image (K) shows eGFP in neurons but not in glial cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: eGFP expression in the brain of a transgenic rat (Ubi-C promoter). Upper panel: eGFP expression is restricted to neurons in transgenic rat brain. A. Confocal microscopy shows extensive eGFP expression in dentate granule cells. B. GFAP immunoreactivity reveals astroglial cells. C. Merged images show a lack of colocalization of eGFP and GFAP signals. Middle panel: confocal images reveal robust eGFP expression in neuronal cell bodies. D. Cortex. E. Striatum. F. CA1 region of hippocampus. G. Hippocampal dentate gyrus. Lower panel: Mixed primary cultures from E18 embryos were stained with anti-beta III tubulin to identify neurons (I; red) and GFAP to label glial cells (J; purple). eGFP expression in cultured neurons was concentrated in the nucleus as well as the cytoplasm (H; green). Arrowheads denote individual neurons in all panels, and the merged image (K) shows eGFP in neurons but not in glial cells.
Mentions: While stable, high-level transgene expression is highly desirable in Tg animals, neuron-selective expression is also an important consideration in selecting an optimal promoter. Brain expression of the eGFP transgene driven by a Ubi-C promoter was examined in Tg SD rats created by injection of eGFP-lentivirus into fertilized zygotes. Strong eGFP expression was seen in adult rats; by confocal microscopy, eGFP expression appeared to be restricted to neurons (Fig. 2). When eGFP distribution was directly compared to immunostaining with glial fibrillary acidic protein (GFAP) to label astrocytes, there was strong divergence in the staining patterns. To confirm the apparent restriction of eGFP driven by the Ubi-C promoter to neurons, primary cultures were established from E18 Tg SD embryos and employed for colocalization studies using markers for neurons and glial cells. Confocal images of mixed primary cultures stained with neuron-specific beta tubulin and GFAP is shown in Figure 2J. Enhanced GFP is expressed in neurons, but not in glial cells, confirming the neuronal specificity of eGFP expression driven by the Ubi-C promoter.

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