Successful double genetic modification of porcine EGFP primary cell line using ZFN and Cre-mediated cassette exchange

We previously generated EGFP transgenic porcine line (Verro2GFP) characterized by a single integration of pCAGGSEGFP cassette, high ubiquitous EGFP expression, mendelian transgene transmission and expression in F1 (Brunetti et al. 2008). Recently Zinc Finger Nucleases (ZFN) and TALENs emerged as powerful tools for gene modification of different cells types and Recombinase Mediated Cassette Exchange (RMCE) was widely tested in different species. Moreover EGFP-specific ZFNs were successfully used in rat (Geurtz et al. 2010) and in pig (Watanabe et al. 2010, Whyte et al. 2010). The purposes of our work were: a) to insert a vector suitable for RMCE into a transcriptionally active locus and b) to assess the possibility to obtain good quality cell colonies to be used in Somatic Cell Nuclear Transfer (SCNT) even after two sequential transgenic modifications (ZFN and RMCE), considering the finite life span of Verro2GFP primary fibroblasts. EGFP-specific ZFNs were purchased from Sigma (CompoZr®) and the promoter-less targeting vectors were created using a fragment of the promoter (Left-Homology-Arm = LHA) and the polyA sequence (Right-Homology-Arm = RHA) of pCAGGSEGFP expression vector. Cloning floxed (lox2272/lox5171) reporter genes (PuromycinR, HygromycinR ) between these homology sequences (LHA and RHA), we generated 2 targeting/exchanging vectors (pB53Puro-PL, pB53Hygro-PL) and their positive controls (C+). PCR on C+ was set up to obtain minimal sensitivity of 100–1000 plasmid copies. Verro2GFP fibroblasts (primary and colonies) cultured in DMEM + M199 (1:1) + FBS(10 %), 5 % CO2,5%O2, were transfected using Nucleofector (V-24 program) and selected starting at 48 h after transfection. Colonies were picked up at 8th selection day and expanded in 24-well plates for PCR screening, RMCE and/or SCNT. In ZFNs-mediated gene targeting, 2 μg of each ZFNs coding vectors (pZFN1 and pZFN2) and 2 μg of pB53Puro-PL/ KpnI vector were used to “nucleofect” primary Verro2GFP fibroblasts (3 105 ; Puromycin = 1 μg/ml). In 3 experiments, 15 PuroR colonies were PCR screened, and 13 (87 %) were positive. For subsequent RMCE, 4 colonies were pooled together and 4 105 cells were cotransfected with pB53Hygro-PL (2.5 μg) and pCAG-CRE:EGFP (2.5 μg-Cre) vectors, and selected with HygromycinB (175 μg/ml). One out of 3 HygroR colonies was positive. Finally 4 PuroR and 1 HygroR colonies were used in zona-free SCNT to produce 243 reconstructed embryos, and 51 (21 %, 25 PuroR 26 HygroR ) blastocysts/compacted morulae were transferred into two synchronized sows. These experiments demonstrated that ZFN-mediated gene targeting following by Cre-mediated cassette exchange can be successfully done during the life span of Verro2GFP fibroblasts thus creating a feasible platform for site-specific gene modification.