Targeting of a porcine EGFP line mediated by ZFNs to establish cloned red fluorescent primary cell lines suitable for Cre-mediated RMCE

Recently site specific nucleases (ZFN, Tal Effector and CRISPR) emerged as powerful tools for gene modification of different cells types 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, Clon. Stem. Cells) we generated an EGFP transgenic porcine line (Verro2GFP) 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 modify a transcriptionally active GFP-locus into one suitable for Cre-mediated RMCE, using EGFP-specific ZFNs. Homology arms for promoter-less targeting vector were derived from pCAGGS-EGFP vector (promoter fragment = Left-Homology-Arm = LHA; polyA sequence = Right-Homology-Arm = RHA). Cloning floxed (lox2272/lox5171) DsRedT4 reporter gene between LHA and RHA sequences, we generated the targeting/RMCE vector (pB50 30 DsRed4-PL) and its positive control (C?) for PCR set-up (100\u20131,000 plasmid copies). Verro2GFP fibroblasts cultured in DMEM ? M199(1:1) ? 10 %FCS, bFGF in 5 %CO2, 5 %O2, were transfected using Nucleofector (V-024 program). In ZFNs-mediated gene targeting, 2 lg of each ZFNs coding vectors (Sigma-CompoZr) and 2 lg of pB50 30 RedT4-PL/NotI vector were used to 00nucleofect00 1x106 Verro2GFP fibroblasts. Part of transfected cells were plated (3,000 cells/plate) in 10 Petri dishes ([ = 100 mm) and cultured without selection till well growing colonies appeared. Four RFP?/GFPcolonies were identified using epifluorescence, re-plated in two 150 mm dishes/colony and PCR verified. Finally 3 colonies were used in zona-free SCNT to produce reconstructed embryos, and 159 D6 blastocysts/compacted morulae were transferred into two synchronized sows that both became pregnant. One pregnancy is ongoing, another was interrupted at D58 and 7 fetuses (61.14 g \ub1 17.98 g) were used to generate RFP primary cell lines, finally verified by PCR. This experiments demonstrated that ZFN-mediated gene targeting can be successfully done without any selection cassette addition creating a feasible SCNT-tested platform for further Cre-mediated site-specific gene modifications