title: Generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (RMCE) and somatic cell nuclear transfer (SCNT).
abstract: 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.
authors.affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle' 4, building 1240, 8000, Aarhus C, Denmark. jannikhumgen@hotmail.com
authors.forename: Jannik Ejnar
authors.initials: JE
authors.lastname: Jakobsen
authors.forename: Marianne G
authors.initials: MG
authors.lastname: Johansen
authors.forename: Mette
authors.initials: M
authors.lastname: Schmidt
authors.forename: Frederik
authors.initials: F
authors.lastname: Dagnæs-Hansen
authors.forename: Karen
authors.initials: K
authors.lastname: Dam
authors.forename: Anders
authors.initials: A
authors.lastname: Gunnarsson
authors.forename: Ying
authors.initials: Y
authors.lastname: Liu
authors.forename: Peter M
authors.initials: PM
authors.lastname: Kragh
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.forename: Ida E
authors.initials: IE
authors.lastname: Holm
authors.forename: Henrik
authors.initials: H
authors.lastname: Callesen
authors.forename: Jacob Giehm
authors.initials: JG
authors.lastname: Mikkelsen
authors.forename: Anders Lade
authors.initials: AL
authors.lastname: Nielsen
authors.forename: Arne Lund
authors.initials: AL
authors.lastname: Jørgensen
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D018076:DNA, Complementary / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D016678:Genome; D006801:Humans; D053652:Nuclear Transfer Techniques; D053764:Presenilin-1 / Q000235:genetics*; D045522:Recombinases / Q000235:genetics; D013552:Swine; D013556:Swine, Miniature / Q000235:genetics*; D019076:Transgenes
chemical_list: D018076:DNA, Complementary; C508853:PSEN1 protein, human; D053764:Presenilin-1; D045522:Recombinases