Early-Onset and Robust Amyloid Pathology in a New Homozygous Mouse Model of Alzheimer's Disease

BACKGROUND: Transgenic mice expressing mutated amyloid precursor protein (APP) and presenilin (PS)-1 or -2 have been successfully used to model cerebral beta-amyloidosis, one of the characteristic hallmarks of Alzheimer's disease (AD) pathology. However, the use of many transgenic lines is limited by premature death, low breeding efficiencies and late onset and high inter-animal variability of the pathology, creating a need for improved animal models. Here we describe the detailed characterization of a new homozygous double-transgenic mouse line that addresses most of these issues. METHODOLOGY/PRINCIPAL FINDINGS: The transgenic mouse line (ARTE10) was generated by co-integration of two transgenes carrying the K670N/M671L mutated amyloid precursor protein (APP(swe)) and the M146V mutated presenilin 1 (PS1) both under control of a neuron-specific promoter. Mice, hemi- as well as homozygous for both transgenes, are viable and fertile with good breeding capabilities and a low rate of premature death. They develop robust AD-like cerebral beta-amyloid plaque pathology with glial inflammation, signs of neuritic dystrophy and cerebral amyloid angiopathy. Using our novel image analysis algorithm for semi-automatic quantification of plaque burden, we demonstrate an early onset and progressive plaque deposition starting at 3 months of age in homozygous mice with low inter-animal variability and 100%-penetrance of the phenotype. The plaques are readily detected in vivo by PiB, the standard human PET tracer for AD. In addition, ARTE10 mice display early loss of synaptic markers and age-related cognitive deficits. By applying a gamma-secretase inhibitor we show a dose dependent reduction of soluble amyloid beta levels in the brain. CONCLUSIONS: ARTE10 mice develop a cerebral beta-amyloidosis closely resembling the beta-amyloid-related aspects of human AD neuropathology. Unifying several advantages of previous transgenic models, this line particularly qualifies for the use in target validation and for evaluating potential diagnostic or therapeutic agents targeting the amyloid pathology of AD

C57BL/6N albino/agouti mutant mice as embryo donors for efficient germline transmission of C57BL/6 ES cells.

We generated C57BL/6NTac mice carrying a tyrosinase loss-of function mutation and a reversion of the nonagouti locus to agouti. This strain has a high superovulation response, allows visual detection of chimeric coat color contribution of C57BL/6 ES-cells and provides a simplified breeding format that generates black G1 offspring of pure inbred C57BL/6 background in one step, providing the ideal host for genetically manipulated C57BL/6 ES cells

Hybrid Embryonic Stem Cell-Derived Tetraploid Mice Show Apparently Normal Morphological, Physiological, and Neurological Characteristics

ES cell-tetraploid (ES) mice are completely derived from embryonic stem cells and can be obtained at high efficiency upon injection of hybrid ES cells into tetraploid blastocysts. This method allows the immediate generation of targeted mouse mutants from genetically modified ES cell clones, in contrast to the standard protocol, which involves the production of chimeras and several breeding steps. To provide a baseline for the analysis of ES mouse mutants, we performed a phenotypic characterization of wild-type B6129S6F(1) ES mice in relation to controls of the same age, sex, and genotype raised from normal matings. The comparison of 90 morphological, physiological, and behavioral parameters revealed elevated body weight and hematocrit as the only major difference of ES mice, which exhibited an otherwise normal phenotype. We further demonstrate that ES mouse mutants can be produced from mutant hybrid ES cells and analyzed within a period of only 4 months. Thus, ES mouse technology is a valid research tool for rapidly elucidating gene function in vivo

Generation of chimeras and germline transmission of C57BL/6NTac targeted ES clones injected into Albino A++ and Albino host embryos.

<p>* GLT indicates germline Transmission.</p

Restoration of the agouti locus in C57BL/6NTac ES cells and mice.

<p>(A) Gene targeting strategy. The Neomycin (NeoR)/Puromycin (PuroR) selection marker is displayed as a grey box (neoR and puroR). Numbered dashes indicate external probes used for Southern Blot analysis of ES cell clones, lettered arrows indicate oligos used for genotyping of mice. Deletion of the FRT flanked selection was obtained in vivo simultaneously with germline transmission. (<b>B</b>) Southern Blot validation with genomic DNA isolated from 6 ES cell clones and wildtype C57BL/6 genomic DNA as a control (W) using external probe 1 in combination with BglI restriction digest (upper panel, Probe 1, B) leading to a wildtype allele of 13,9 kb (Wt) and a targeted allele of 9,3 kb (tm) and confirmatory Southern Blot validation with external probe 2 in combination with PspOMI restriction digest (lower panel, Probe 2, P), leading to a wildtype allele of 16,2 kb (Wt) and a targeted allele of 14,8 kb (tm). (<b>C</b>) PCR verification. Clone B-B10 was selected for chimera generation and germline transmission. Primer combinations a + b (upper panel) resulted in amplification of a 290 bp wildtype <i>A</i> allele in BALB/c control mice (lane 1, BALB/c) and a 387 bp restored <i>A^tm1.1</i> allele in homozygous Albino A++ mice (lane 3,A++) and in chimeras generated with C57BL/6 ES cells injected in homozyogus A++ host embryos (lane 4, A++/B6 Ch). Primer combinations c + b (lower panel) amplified a 280 bp C57BL/6 wildtype <i>a</i> allele in C57BL/6 control mice (lane 2, C57BL/6) and in A++/B6 Ch (lane 4, A++/B6 Ch). Note: PCR amplicons are of different size for the BALB/c <i>A</i> and the A++ restored <i>A^tm1.1</i> alleles. Also, in contrast to homozygous A++ mice, A++/B6 Ch amplify both, the ES cell derived <i>a</i> and the A++ derived <i>A^tm1.1</i> alleles.</p

Inactivation of the tyrosinase locus in C57BL/6NTac ES cells and mice.

<p>(A) Gene targeting scheme for the tyrosinase locus. A point mutation was introduced in exon 1 (C103S) of the tyrosinase allele. The Puromycin selection marker (PuroR) is shown as a grey box. Numbered dashes indicate external probes used for Southern Blot analysis of ES cell clones, lettered arrows indicate oligos used for genotyping of mice. Deletion of the F3 flanked selection marker was achieved in vitro in targeted ES cell clone A-B6. (<b>B</b>) Southern Blot validation with genomic DNA isolated from 6 ES cell clones and wildtype C57BL/6 genomic DNA as a control (W) using external probe 1 in combination with EcoRI restriction digest (upper panel, Probe 1, E) leading to a wildtype allele of 4,8 kb (Wt) and a targeted allele of 7,7 kb (tm) and confirmatory Southern Blot validation with external probe 2 in combination with BglI restriction digest (lower panel, Probe 2, B), leading to a wildtype allele of 12,7 kb (Wt) and a targeted allele of 17,7 kb (tm). (<b>C</b>) PCR verification. Clone A-B6 was selected for chimera generation and germline transmission. Primer combinations f + g (upper panel) applied on genomic DNA displayed a 151 bp wildtype <i>Tyr</i> allele in BALB/c control mice (lane 1, Balb/c), C57BL/6 control mice (lane 2, C57BL/6) and in chimeras generated with C57BL/6 ES cells injected in homozyogus A++ host embryos (lane 4, A++/B6 Ch). In addition the modified <i>Tyr^tm1</i>Arte allele was detected in homozygous Albino A++ mice (lane 3, A++) and in chimeras generated with C57BL/6 ES cells injected in homozyogus A++ host embryos (lane 4, A++/B6 Ch). Lower panel: the introduced point mutation was verified by sequencing both strands of the PCR fragment amplified with primers d + e.</p