Targeted RMCE-live piglets generated by SCNT following sequential double site-specific gene modifications of a porcine EGFP line

Site-specific nucleases (ZFN, Tal effector nucleases and CRISPRs) boosted the genome editing of different species, and EGFP-specific ZFNs were successfully used in rat (Geurtz et al. 2010) and in pig (Watanabe et al. 2010, Whyte et al. 2010). Previously (Brunetti et al., 2008) we generated an EGFP transgenic porcine line (V2G) characterized by a single integration of pCAGGS-EGFP cassette, high ubiquitous EGFP expression, Mendelian transgene transmission and expression in F1. The aim of this work was to generate live cloned piglets using modified cells after two site-specific modifications (ZFNs and RMCE) on the transcriptionally active GFP-locus (V2G), avoiding any cell rejuvenation by SCNT. Homology arms for promoterless targeting vector were derived from pCAGGSEGFP vector (promoter fragment = Left-Homology-Arm = LHA; polyA sequence = Right-Homology-Arm = RHA). Cloning floxed (lox2272/lox5171) puromycin and hygromycin resistance coding sequences between LHA and RHA sequences, and we generated the targeting/RMCE vectors (pB50 30 Puro-PL and pB50 30 Hygro-PL) and theirs positive controls (C+) for PCR set-up (100–1,000 plasmid copies). V2G fibroblasts cultured in DMEM + M199(1:1) +10 %FCS, bFGF in 5 %CO2, 5 %O2, were transfected using Nucleofector (V-024 program). In ZFN-mediated gene targeting, 2 lg of each ZFNs coding vectors (Sigma-CompoZr ) and 2 lg of pB50 30 Puro-PL/KpnI vector, were used to ‘nucleofect’ 0.9 9 106 Verro2GFP fibroblasts. Transfected cells were cultured under puromycin selection (2 lg/ml) for 8 days, and 0.45 9 106 cells were used for the Cre-mediated cassette exchange (1 lg pB50 30 Hygro-PL/KpnI + 2 lg pCAG:CreEGFP). Transfected cells were plated in 10 Petri dishes (Ø = 150 mm) and cultured under hygromycin selection (200 lg/ml) for 12 days, picked up and expanded in 24 multi-well plates for SCNT. Thirty-one colonies were PCR screened for hygromycin and 16 (51.6 %) colonies were positive for Cre-mediated targeted insertion. Four colonies were used in zona-free SCNT experiments. On Day6, 172 compacted morulae/blastocysts were transferred into two synchronized sows, one became pregnant and went to term delivering seven live and four stillborn piglets. Cre-mediated hygromycin cassette insertion into the V2G locus was PCRdetected in all the animals that resulted also negative for insertions of puromycin and EGFP cassettes. Combining ZFNmediated targeting with Cre-mediated cassette exchange, we demonstrated that two sequential site-specific modifications could be easily done with high efficiency producing live cloned animals. Moreover, we have validated the V2G locus as a feasible SCNT-tested platform for further site-specific gene modifications.