In an effort to develop a porcine model of Alzheimer's disease we used handmade cloning to produce seven transgenic Göttingen minipigs. The donor fibroblasts had been stably transfected with a plasmid cassette containing, as transgene, the cDNA of the neuronal variant of the human amyloid precursor protein gene with the Swedish mutation preceded by beta-globin sequences to induce splicing and a human PDGF beta promoter fragment to drive transcription. Transgene insertion had occurred only at the GLIS3 locus where a single complete copy of the transgene was identified in intronic sequences in opposite direction. Similar and robust levels of the transgene transcript were detected in skin biopsies from all piglets and the sequence of full-length transcript was verified. Consistent with PDGF beta promoter function, high levels of transgene expression, including high level of the corresponding protein, was observed in brain tissue and not in heart or liver tissues. A rough estimate predicts that accumulation of the A beta peptide in the brain may develop at the age of 1-2 years.

We describe a new human isoform, GFAP⑀, of the intermediary filament protein GFAP (glial fibrillary acidic protein). GFAP⑀ mRNA is the result of alternative splicing and a new polyadenylation signal, and thus GFAP⑀ has a new C-terminal protein sequence. This provides GFAP⑀ with the capacity for specific binding of presenilin proteins in yeast and in vitro. Our observations suggest a direct link between the presenilins and the cytoskeleton where GFAP⑀ is incorporated. Mutations in GFAP and presenilins are associated with Alexander disease and Alzheimer's disease, respectively. Accordingly, GFAP⑀ should be taken into consideration when studying neurodegenerative diseases.

Targeted transgenesis using site-specific recombinases is an attractive method to create genetically modified animals as it allows for integration of the transgene in a pre-selected transcriptionally active genomic site. Here we describe the application of recombinase-mediated cassette exchange (RMCE) in cells from a Göttingen minipig with four RMCE acceptor loci, each containing a green fluorescence protein (GFP) marker gene driven by a human UbiC promoter. The four RMCE acceptor loci segregated independent of each other, and expression profiles could be determined in various tissues. Using minicircles in RMCE in fibroblasts with all four acceptor loci and followed by SCNT, we produced piglets with a single copy of a transgene incorporated into one of the transcriptionally active acceptor loci. The transgene, consisting of a cDNA of the Alzheimer’s disease-causing gene PSEN1M146I driven by an enhanced human UbiC promoter, had an expression profile in various tissues similar to that of the GFP marker gene. The results show that RMCE can be done in a pre-selected transcriptionally active acceptor locus for targeted transgenesis in pigs.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-012-9671-6) contains supplementary material, which is available to authorized users.

Mutations in the amyloid-β protein precursor gene (AβPP), the presenilin 1 gene (PSEN1) or the presenilin 2 gene (PSEN2) that increase production of the AβPP-derived peptide Aβ42 cause early-onset Alzheimer's disease. Rodent models of the disease show that further increase in Aβ42 production and earlier brain pathology can be obtained by coexpressing AβPP and PSEN1 mutations. To generate such elevated Aβ42 level in a large animal model, we produced Göttingen minipigs carrying in their genome one copy of a human PSEN1 cDNA with the Met146Ile (PSEN1M146I) mutation and three copies of a human AβPP695 cDNA with the Lys670Asn/Met671Leu (AβPPsw) double-mutation. Both transgenes were expressed in fibroblasts and in the brain, and their respective proteins were processed normally. Immunohistochemical staining with Aβ42-specific antibodies detected intraneuronal accumulation of Aβ42 in brains from a 10- and an 18-month-old pig. Such accumulation may represent an early event in the pathogenesis of Alzheimer's disease.

This study examined how silages of different grass and clover species affect dry matter (DM) intake, milk production, and eating behavior in dairy cows. The primary growth of perennial ryegrass (early and late harvested), festulolium, tall fescue, red clover, and white clover swards were cut, wilted, and ensiled without additives. Thirty-six Danish Holstein cows were fed ad libitum with total mixed rations containing 70% forage on DM basis in an incomplete Latin square design. The forage source was either 1 of the 6 pure silages or late perennial ryegrass silage mixed (50:50 on DM basis) with either red clover or white clover silage. Intake of DM, milk yield, and milk lactose concentration were higher, whereas milk fat and protein concentrations were lower when cows were fed clover compared with grass. No differences in DM intake and milk composition were detected between cows fed red clover and white clover, but white clover resulted in higher milk yield than red clover. Lower body weight, probably caused by lower rumen fill, in cows fed pure white clover compared with the other treatments indicated that intake was regulated physiologically instead of physically. Cows fed early perennial ryegrass, which had the highest silage organic matter digestibility (OMD), did not produce the expected amount of energy-corrected milk (ECM) compared with the other treatments based on the amount of OM digested in the gastrointestinal tract, but the reason was unclear. Across all other grass species, ECM was related to OMD. Inclusion of 50% clover in the diet increased ECM with 2.3 kg/d, and the response to OMD was comparable to the response for the grass silages. In situ fiber degradation profile parameters indicated that fiber in festulolium differed compared with fiber in the other grass species and resembled fiber in clover. Drinking and eating behavior differed markedly in cows fed pure white clover compared with the other treatments. Water intake per drinking bout was comparable among treatments, but cows fed pure white clover had higher drinking bout duration and reduced drinking rate. Additionally, meal size was smaller for cows fed pure white clover compared with the other treatments, for which meal size was similar. In conclusion, differences in ECM between different grass species can be explained by differences in OMD, and at a given OMD level inclusion of clover in the diet increased ECM.

The aim of this meta-analysis was to compare feed intake, milk production, milk composition and organic matter (OM) digestibility in dairy cows fed different grass and legume species. Data from the literature was collected and different data sets were made to compare families (grasses v. legumes, Data set 1), different legume species and grass family (Data set 2), and different grass and legume species (Data set 3+4). The first three data sets included diets where single species or family were fed as the sole forage, whereas the approach in the last data set differed by taking the proportion of single species in the forage part into account allowing diets consisting of both grasses and legumes to be included. The grass species included were perennial ryegrass, annual ryegrass, orchardgrass, timothy, meadow fescue, tall fescue and festulolium, and the legume species included were white clover, red clover, lucerne and birdsfoot trefoil. Overall, dry matter intake (DMI) and milk production were 1.3 and 1.6 kg/day higher, respectively, whereas milk protein and milk fat concentration were 0.5 and 1.4 g/kg lower, respectively, for legume-based diets compared with grass-based diets. When comparing individual legume species with grasses, only red clover resulted in a lower milk protein concentration than grasses. Cows fed white clover and birdsfoot trefoil yielded more milk than cows fed red clover and lucerne, probably caused by a higher OM digestibility of white clover and activity of condensed tannins in birdsfoot trefoil. None of the included grass species differed in DMI, milk production, milk composition or OM digestibility, indicating that different grass species have the same value for milk production, if OM digestibility is comparable. However, the comparison of different grass species relied on few observations, indicating that knowledge regarding feed intake and milk production potential of different grass species is scarce in the literature. In conclusion, different species within family similar in OM digestibility resulted in comparable DMI and milk production, but legumes increased both DMI and milk yield compared with grasses.

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