EARLY FETAL DEVELOPMENT OF NUCLEAR TRANSFER BOVINE EMBRYOS GENERATED FROM FIBROBLASTS GENETICALLY MODIFIED BY PIGGYBAC TRANSPOSITION

ALESSIO AP; FILI AE; FORCATO DO; OLMOS NICOTRA MF; ALUSTIZA FE; RODRIGUEZ N; LIAUDAT AC; VILAR SAMPAIO R; SANGALLI J; BRESSAN F; FANTINATO-NETO P; MEIRELLES F; OWENS J; MOISYADI S; KUES WA; BOSCH P.

Versailles

Congreso; 41st Annual Conference of the IETS; 2015

International Embryo Transfer Society

Transposon-mediated transgene integration is a well-established transgenic tool for genome manipulation in small animal models. However, translation of this active transgenic method to the large animal setting requires further investigation. We have previously demonstrated that a helper-independent piggyBac transposon system can efficiently transpose transgenes into the bovine genome (Alessio et al., Reprod Dom Anim, 49 (Suppl.1):8, 2014). The aims of this study were: a) to study the effectiveness of a hyperactive version of the piggyBac transposase to mediate chromosomal integrations of transgenes in cultured fibroblast, and b) to determine the ability of the genetically modified cells to support early embryo and fetal development upon somatic cell nuclear transfer (SCNT). Bovine fetal fibroblasts (BFFs) were chemically transfected with either pmGENIE-3 (a helper-independent piggyback (PB) transposon conferring genes for hygromycin resistance and enhanced green fluorescent protein (EGFP); Urschitz et al. PNAS USA 2010; 107:8117-22), hypmGENIE-3 (carrying an hyperactive version of the piggyBac transposase; Yusa et al. PNAS USA 2011; 108:1531-1536) or pmGENIE-3/ΔpiggyBac (a control plasmid lacking a functional PB transposase). Upon transfection, cell cultures were subjected to 14 days of hygromycin selection. Antibiotic resistant and EGFP positive colonies were counted and data analyzed by ANOVA and Tukey´s test. For SCNT, hypmGENIE-3 and pmGENIE-3 polyclonal cell lines were selected by FACS and individual cells used as nuclear donors. Day 7 blastocysts were non-surgically transferred to synchronized recipients. Conceptuses were recovered by day 30 of gestation, observed under UV light and processed to obtain genomic DNA for molecular characterization of integrated transgenes. The mean number of colonies in hypmGENIE-3 group was significantly higher than those in pmGENIE-3 and the control groups (324±17.82 vs 100±16.1 and 2.78±0.81 respectively, n=4-7; p