242 HIGHLY EFFICIENT SLEEPING BEAUTY TRANSPOSON-MEDIATED TRANSGENESIS IN BOVINE FETAL FIBROBLASTS
Authors: Fili A. E. , Alessio A. P. , Garrels W. , Forcato D. O. , Olmos Nicotra M. F. , Liaudat A. C. , Bevacqua R. J. , Savy V. , Hiriart M. I. , Rodriguez N. , Talluri T. R. , Ivics Z. , Salamone D. F. , Kues W. A. , Bosch P. 
Published: November 2015

Active transposon-mediated transgenesis is an emerging tool for basic and applied research in livestock. We have demonstrated the effectiveness of a helper-independent piggyBac transposon (pGENIE-3) for gene transfer into the genome of bovine cells (Alessio et al. 2014 Reprod. Domest. Anim. 49, 8). Here, we extend our previous research by examining the suitability of a Sleeping Beauty (SB) transposon-based methodology to deliver transgenes into the genome of bovine fetal fibroblasts (BFF), and the ability of these cells to support in vitro embryo development upon somatic cell nuclear transfer (SCNT). In a first experiment, BFF were chemically cotransfected (JetPRIME®, Polyplus-transfection, Illkirch, France) with a helper plasmid (pCMV-SB100X), which carries an expression cassette for the SB transposase, and the donor vector (pT2/Venus/RMCE) harboring an expression cassette for a fluorescent protein (Venus) flanked by the SB inverted terminal repeats (ITR). Three different ratios of helper and donor plasmids were studied: 1 : 2, 1 : 1 and 2 : 1. After 15 days of culture, the number of fluorescent colonies was counted on an inverted microscope. When vectors were used at ratios of 1 : 1 and 2 : 1, a 78-fold and 88-fold increase (P ≤ 0.05) in the number of fluorescent colonies compared with that in the no-transposase control were calculated. In a second experiment, BFF were chemically cotransfected with the helper vector pCMV-SB100X, and 2 donor transposons: pT2/Venus/RMCE and pT2/SV40-Neo. The former harbors a neo resistance cassette framed by SB ITRs. Different ratios of helper:donors (1 : 1 : 1, 2 : 1 : 1 and 2 : 0.5 : 0.5) were studied, and each ratio compared with a no-transposase control. After 15 days of antibiotic selection, the number of G418-resistant colonies was determined. Every time a functional SB transposase vector was included, the number of fluorescent and G418-resistant colonies was markedly higher compared with that in the respective control without transposase (P ≤ 0.001). Interestingly, all G418-resistant colonies expressed Venus. Molecular characterisation of genomic insertions in 6 monoclonal cell lines was performed by PCR and splinkerette PCR. PCR analysis confirmed presence of the Venus transgene in all cell lines. Splinkerette PCR results revealed at least 15 transposase-catalyzed genomic insertions of the transgene. Individual cells from a polyclonal SB transgenic fibroblast culture were used as nuclear donors to produce zona-free SCNT embryos. Of the reconstructed embryos, 33% reached blastocyst stage and about half of them expressed Venus. In conclusion, SB transposase is able to actively transpose monomeric copies of transgenes into the genome of bovine cells, which can be reprogrammed upon nuclear transfer to generate morphologically normal embryos expressing the transgene of interest.