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. 2008 Feb 26:9:28.
doi: 10.1186/1471-2202-9-28.

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

Cansu Agca  1 , Jason J Fritz, Lary C Walker, Allan I Levey, Anthony Ws Chan, James J Lah, Yuksel Agca
Affiliations

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

Cansu Agca et al. BMC Neurosci. .

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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Figures

Figure 1
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.
Figure 2
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.
Figure 3
Figure 3
Southern blot hybridization of genomic DNA obtained from APP21 and APP31 rats. A. Genomic DNA from APP21 (116, 117, 118, 121, 124, 125) and APP31 (31, 86, 88) animals were digested with BamHI and hybridized with a human APP probe. B. Genomic DNA from APP31 (31, 86, 88) and APP21 (116, 117, 118, 121, 124, 125) were digested with EcoRI and hybridized with a human APP probe.
Figure 4
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.
Figure 5
Figure 5
Gene expression differences among organs obtained from APP21, APP31 and WT rats. A. The expression of APP genes is normalized by dividing the net intensity of APP bands by the 18S rRNA bands. ** represent significantly greater APP expression in APP21 compared to APP31 and WT rats (P < 0.05); * represents significantly greater APP expression in APP31 compared to WT rats (P < 0.05). B. APP expression-difference between APP21 and APP31 rats compared to WT animals. The values were obtained by dividing the APP expression in each organ (B: Brain, H: Heart, K: Kidney, Li: Liver, Lu: Lung) by APP expression in WT rats.
Figure 6
Figure 6
Human APPSw/Ind expression in transgenic rat brain. A. A low power (2×) micrograph demonstrates widespread expression of human APP in the neocortex and hippocampus. Higher power (20×) images show punctate cytoplasmic staining in large cortical pyramidal neurons (B) and hippocampal pyramidal neurons (C). APP staining appears to demarcate the margins of CA2 pyramidal neurons, which were largely devoid of staining (C). Low power (2×) micrograph of an adjacent section stained with an anti-GFAP antibody. GFAP staining reveals the presence of glia and astrocytes within the neocortex and hippocampus (D). Higher power (20×) images from the cortex (E) and hippocampal dentate gyrus (F) are shown for comparison to (B-C) and illustrate that human APPSw/Ind expression in occurring predominately in neurons.
Figure 7
Figure 7
Gene expression analysis of APP-transgenic mice. A. Northern blot hybridization of total RNA from C3-3 APP-transgenic mouse tissues. Total RNA hybridized with a human APP probe (upper autoradiogram) prior to hybridization with 18S rRNA (lower autoradiogram). B. Gene expression differences among organs obtained from C3-3 mice. The expression of APP genes was normalized by dividing the net intensity of APP bands by the 18S rRNA bands. Different letters above the bars represent statistically significant (P < 0.05) expression levels. B: Brain, H: Heart, K: Kidney, Li: Liver, Lu: Lung.
Figure 8
Figure 8
DNA construct of pLVU-APPSw/Ind. LTR: long terminal repeat; UB: ubiquitin-C promoter; APP: human amyloid precursor protein; WRE: woodchuck hepatitis virus post-transcriptional regulatory element.
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