title: Identification and re-addressing of a transcriptionally permissive locus in the porcine genome.
abstract: Recently, we established the Sleeping Beauty transposon system for germ line competent transgenesis in the pig. Here, we extend this approach to re-target a transposon-tagged locus for a site-specific gene knock-in, and generated a syngeneic cohort of piglets carrying either the original transposon or the re-targeted event. A Cre-loxP-mediated cassette exchange of the tagging transposon with a different reporter gene was performed, followed by flow cytometric sorting and somatic cell nuclear transfer of recombined cells. In parallel, the original cells were employed in somatic cell nuclear transfer to generate clone siblings, thereby resulting in a clone cohort of piglets carrying different reporter transposons at an identical chromosomal location. Importantly, this strategy supersedes the need for an antibiotic selection marker. This approach expands the arsenal of genome engineering technologies in domestic animals, and will facilitate the development of large animal models for human diseases. Potentially, the syngeneic cohort of pigs will be instrumental for vital tracking of transplanted cells in pre-clinical assessments of novel cell therapies.
authors.affiliation: Friedrich-Loeffler-Institut (FLI), 31535, Mariensee, Neustadt, Germany.|Medical School Hannover, Hannover, Germany.
authors.forename: Wiebke
authors.initials: W
authors.lastname: Garrels
authors.affiliation: Friedrich-Loeffler-Institut (FLI), 31535, Mariensee, Neustadt, Germany.
authors.forename: Ayan
authors.initials: A
authors.lastname: Mukherjee
authors.affiliation: Friedrich-Loeffler-Institut (FLI), 31535, Mariensee, Neustadt, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Holler
authors.affiliation: Friedrich-Loeffler-Institut (FLI), 31535, Mariensee, Neustadt, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Cleve
authors.affiliation: Friedrich-Loeffler-Institut (FLI), 31535, Mariensee, Neustadt, Germany.
authors.forename: Thirumala R
authors.initials: TR
authors.lastname: Talluri
authors.affiliation: Friedrich-Loeffler-Institut (FLI), 31535, Mariensee, Neustadt, Germany.|Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Brigitte
authors.initials: B
authors.lastname: Barg-Kues
authors.affiliation: Friedrich-Loeffler-Institut (FLI), 31535, Mariensee, Neustadt, Germany.|Weser-Ems-Union, Nordenham, Germany.
authors.forename: Mike
authors.initials: M
authors.lastname: Diederich
authors.affiliation: Friedrich-Loeffler-Institut (FLI), 31535, Mariensee, Neustadt, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Köhler
authors.affiliation: Friedrich-Loeffler-Institut (FLI), 31535, Mariensee, Neustadt, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Petersen
authors.affiliation: Friedrich-Loeffler-Institut (FLI), 31535, Mariensee, Neustadt, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Lucas-Hahn
authors.affiliation: Friedrich-Loeffler-Institut (FLI), 31535, Mariensee, Neustadt, Germany.
authors.forename: Heiner
authors.initials: H
authors.lastname: Niemann
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Izsvák
authors.affiliation: Paul-Ehrlich-Institut (PEI), Langen, Germany.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Ivics
authors.affiliation: Friedrich-Loeffler-Institut (FLI), 31535, Mariensee, Neustadt, Germany. wilfried.kues@fli.bund.de.
authors.forename: Wilfried A
authors.initials: WA
authors.lastname: Kues
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004251:DNA Transposable Elements; D005260:Female; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci; D016678:Genome; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D018895:Microsatellite Repeats; D053652:Nuclear Transfer Techniques; D034421:Sus scrofa / Q000235:genetics*; D019076:Transgenes
chemical_list: D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins
keywords: Active transgenesis; Large animal model; Livestock; Somatic cell nuclear transfer; Targeted integration