title: Germline transgenic pigs by Sleeping Beauty transposition in porcine zygotes and targeted integration in the pig genome.
abstract: Genetic engineering can expand the utility of pigs for modeling human diseases, and for developing advanced therapeutic approaches. However, the inefficient production of transgenic pigs represents a technological bottleneck. Here, we assessed the hyperactive Sleeping Beauty (SB100X) transposon system for enzyme-catalyzed transgene integration into the embryonic porcine genome. The components of the transposon vector system were microinjected as circular plasmids into the cytoplasm of porcine zygotes, resulting in high frequencies of transgenic fetuses and piglets. The transgenic animals showed normal development and persistent reporter gene expression for >12 months. Molecular hallmarks of transposition were confirmed by analysis of 25 genomic insertion sites. We demonstrate germ-line transmission, segregation of individual transposons, and continued, copy number-dependent transgene expression in F1-offspring. In addition, we demonstrate target-selected gene insertion into transposon-tagged genomic loci by Cre-loxP-based cassette exchange in somatic cells followed by nuclear transfer. Transposase-catalyzed transgenesis in a large mammalian species expands the arsenal of transgenic technologies for use in domestic animals and will facilitate the development of large animal models for human diseases.
authors: Institut für Nutztiergenetik, Friedrich-Loeffler-Institut, Neustadt, Germany.
authors: Wiebke
authors: W
authors: Garrels
authors: Lajos
authors: L
authors: Mátés
authors: Stephanie
authors: S
authors: Holler
authors: Anna
authors: A
authors: Dalda
authors: Ulrike
authors: U
authors: Taylor
authors: Björn
authors: B
authors: Petersen
authors: Heiner
authors: H
authors: Niemann
authors: Zsuzsanna
authors: Z
authors: Izsvák
authors: Zoltán
authors: Z
authors: Ivics
authors: Wilfried A
authors: WA
authors: Kues
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D003593:Cytoplasm / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D005260:Female; D018014:Gene Transfer Techniques; D016678:Genome / Q000235:genetics*; D007267:Injections; D008297:Male; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism*; D013552:Swine / Q000235:genetics*; D019076:Transgenes / Q000235:genetics*; D019895:Transposases / Q000235:genetics / Q000378:metabolism; D015053:Zygote / Q000166:cytology / Q000378:metabolism*
chemical_list: D004251:DNA Transposable Elements; D019895:Transposases