Efficient piggyBac transposon-mediated transgene integration into bovine fetal fibroblast genome

ALESSIO, ANA PAULA; FILI, ALEJANDRO; FORCATO, DIEGO; OLMOS NICOTRA, MARÍA FLORENCIA; ALUSTIZA, FABRISIO; RODRIGUEZ, NANCY; OWENS, JESSE B.; MOISYADI, STEFAN; KUES, WILFRIED A.; BOSCH, PABLO

Giessen

Conferencia; 7th Annual Conference of Physiology and Pathology of Reproduction, 39th Joint Conference on Veterinary and Human Reproductive Medicine; 2014

Efficient piggyBac transposon-mediated transgene integration into bovine fetal fibroblast genome A. Alessio1, A. Fili1, D. Forcato1, F. Olmos-Nicotra1, F. Alustiza F1, N. Rodriguez1, J. Owens2, S. Moisyadi2, W.A. Kues3, P. Bosch1 1Department of Molecular Biology, FCEFQyN, Universidad Nacional de Río Cuarto, Córdoba, Argentina 2Department of Anatomy, Biochemistry and Physiology; John A. Burns School of Medicine; Honolulu, HI USA. 3Friedrich-Loeffler-Institut, Institut für Nutztiergenetik, Mariensee, Germany. Enzyme mediated transgene insertion into the genome, referred to as active transgenesis, is gaining attention because its effectiveness and long-term transgene expression. The aim of this study was to assess a piggyBac transposon system to mediate stable transgene integrations into the bovine genome. To this end, bovine fetal fibroblasts were transfected with either pmGENIE-3 (a helper-independent piggyBac transposon (PB) conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); (Urschitz et al. PNAS USA 2010; 107:8117?22) or pmGENIE-3∕Δ (a control plasmid in which PB transposase sequence is truncated). After 14 days of hygromycin selection, the number of antibiotic resistant and EGFP positive colonies was recorded for both experimental groups and expressed as mean±SEM. Individual colonies were isolated (cloning rings) and expanded for further molecular analysis. The number of resistant and EGFP positive colonies was significantly higher in the pmGENIE-3 group compared with the control (126±68 vs 3±1.4 respectively, n=3; p≤0.05). Genomic DNA from five monoclonal transgenic cell lines transfected with pmGENIE-3 was subjected to nonrestrictive Linear Amplification-Mediated PCR (nrLAM-PCR) to determine the exact genomic insertion sites into the bovine genome. A total of seventeen transgene insertions were determined by nrLAM-PCR, which were preferentially located in intergenic regions of the genome. Analysis of the genomic sequences flanking the transgene revealed unequivocally that all transgene insertions occurred by transposition. These results demonstrate that PB transposase is active in primary bovine cells and greatly enhances transgene integration by transposition. Selected primary fibroblast cultures will be employed in somatic cell nuclear transfer (SCNT) to produce transgenic offspring. The financial support of CONICET and Agencia Nacional de Promoción Científica y Tecnológica de la Argentina is gratefully acknowledged.