Jakobsen, Jannik E.2; Johansen, Marianne Gregers2; Schmidt, Mette9; Dagnæs-Hansen, Frederik3; Dam, Karen4; Gunnarsson, Anders5; Liu, Ying6; Kragh, Peter Michael7; Li, Rong6; Holm, Ida Elisabeth8; Callesen, Henrik6; Mikkelsen, Jacob Giehm2; Nielsen, Anders Lade2; Jørgensen, Arne Lund2
1 Veterinary Reproduction & Obstetrics, Department of Large Animal Sciences, Faculty of Life Sciences, Københavns Universitet2 Institut for Biomedicin - Human Genetik3 Health4 Institut for Medicinsk Mikrobiologi og Immunologi5 Department of Biomedicine, Aarhus University6 Institut for Husdyrvidenskab - Forplantningsbiologi7 Institut for Human Genetik8 Institut for Klinisk Medicin - Patologisk Institut, Aalborg Sygehus Nord9 Veterinary Reproduction & Obstetrics, Department of Large Animal Sciences, Faculty of Life Sciences, Københavns Universitet
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.