Generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (RMCE) and somatic cell nuclear transfer (SCNT).

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.

Reelin expression during embryonic development of the pig brain.

BACKGROUND: Reelin is an extracellular glycoprotein of crucial importance in the developmental organisation of neurons in the mammalian cerebral cortex and other laminated brain regions. The pig possesses a gyrencephalic brain that bears resemblance to the human brain. In order to establish an animal model for neuronal migration disorders in the pig, we have studied the expression pattern and structure of Reelin during pig brain development. RESULTS: We determined the sequence of pig Reelin mRNA and protein and identified a high degree of homology to human Reelin. A peak in Reelin mRNA and protein expression is present during the period of major neurogenesis and neuronal migration. This resembles observations for human brain development. Immunohistochemical analysis showed the highest expression of Reelin in the Cajal-Reztius cells of the marginal zone, in resemblance with observations for the developing brain in humans and other mammalian species. CONCLUSIONS: We conclude that the pig might serve as an alternative animal model to study Reelin functions and that manipulation of the pig Reelin could allow the establishment of an animal model for human neuronal migration disorders.

Determination of odor detection threshold in the Gottingen minipig.

The aim of the study was to examine the ability of Göttingen minipigs to acquire an olfaction-based operant conditioning task and to determine the detection threshold for ethyl acetate and ethanol. We used an automated olfactometer developed for rodents to train and test 14 pigs. Odor sampling and reliable responding were obtained after three to fifteen 160-trial sessions. Successful transfer of the task from ethyl acetate to ethanol was achieved in 1-4 sessions. Detection threshold for ethyl acetate varied between 10(-2)% and 10(-6)% v/v and for ethanol between 0.1% and 5 × 10(-6)% v/v. The results provide evidence that minipigs can successfully acquire 2-odorant discrimination using a food-rewarded instrumental conditioning paradigm for testing olfactory function. This olfactory discrimination paradigm provides reliable measures of olfactory sensitivity and thereby enables detection of changes in olfaction in a porcine model of Alzheimer's disease currently being developed.

Hemizygous minipigs produced by random gene insertion and handmade cloning express the Alzheimer's disease-causing dominant mutation APPsw.

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.

Expression of the Alzheimer's Disease Mutations AßPP695sw and PSEN1M146I in Double-Transgenic Göttingen Minipigs.

Mutations in the amyloid-ß protein precursor gene (AßPP), the presenilin 1 gene (PSEN1) or the presenilin 2 gene (PSEN2) that increase production of the AßPP-derived peptide Aß42 cause early-onset Alzheimer's disease. Rodent models of the disease show that further increase in Aß42 production and earlier brain pathology can be obtained by coexpressing AßPP and PSEN1 mutations. To generate such elevated Aß42 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 AßPP695 cDNA with the Lys670Asn/Met671Leu (AßPPsw) double-mutation. Both transgenes were expressed in fibroblasts and in the brain, and their respective proteins were processed normally. Immunohistochemical staining with Aß42-specific antibodies detected intraneuronal accumulation of Aß42 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.

Aromatic l-amino acid decarboxylase expression profiling and isoform detection in the developing porcine brain.

Aromatic l-amino acid decarboxylase (AADC) enzymatic activity is essential for the biosynthesis of the serotonin and dopamine neurotransmitters, and AADC activity is functionally associated with a number of human neuronal disorders. Here we describe the molecular characterization of AADC from the pig. Pig AADC shows a high degree of similarity to human and rodent AADC at the cDNA and protein level. Exon position shuffling has exchanged the location of the stop codon in pig AADC to the last exon 15 instead for the exon 14 position in the human, the rat, and the mouse AADC. Several pig AADC isoforms were identified, including the also in human described extraneuronal and neuronal isoforms generated by alternative splicing and alternative promoter usage. The AADC expression in the developing pig brain is highly expressed in the basal ganglia and the brain stem regions, and also significantly expressed in the cortex, the hippocampus and the cerebellum. Moreover, we observe that both the neuronal and the extraneuronal AADC mRNA isoforms were present at early brain developmental stages in the brain stem and the basal ganglia. This presents the first evidence that the non-neuronal AADC isoform also is expressed in the brain. Together our results propose that the porcine model is useful for future functional delineations of the AADC gene at the molecular level.

Genetic polymorphism and sequence evolution of an alternatively spliced exon of the glial fibrillary acidic protein gene, GFAP.

Isoform GFAPepsilon of the human cytoskeletal protein GFAP carries, as the result of alternative splicing of exon 7a of GFAP, a novel 42-amino-acid-long C-terminal region with binding capacity for the presenilin proteins. Here we show that exon 7a is present in a variety of mammals but absent from GFAP of chicken and fish. Comparison of the mouse and human GFAP exons showed an increased rate of nonsynonymous nucleotide substitutions in exon 7a compared to the other exons. This resulted in 10 nonconservative and 2 conservative amino acid substitutions and suggests that exon 7a has evolved under different functional constraints. Exons 7a of humans and higher primates are 100% identical apart from alanine codon 426, which is conserved in only 9% of the human alleles, while 21 and 70% of the alleles, respectively, have a valine or a threonine codon at that position. Threonine represents a potential phosphorylation site, and positive selection of that effect could explain the high allele frequency.