Analysis of the PuroR genomic integration site in 293-Flp-mEGFP cells. (A) The integrity of the mEGFP gene in non-green puromycin resistant colonies of 293-Flp-mEGFP cells was analyzed after transfection with pDonor together with the ZFNs. Genomic DNA isolated from 25 individual colonies was subjected to PCR using primer pairs as indicated above the panels. The genomic mEGFP specific primer (P1) was used together with the puromycin specific primers in opposite orientations (Pu-1 and Pu-2). Control PCR's (rightmost gel, ctr) using pEGFP-BABE-puro as template was performed with the primer pairs: P-amp + Pu-1 (lane 1), P1 + Pu-1 (lane 2), P1 + Pu-1 without template (lane 3) and PCR amplification produced the anticipated amplification products (2364 bp, 1429 bp and no product) verifying that primers worked under the given conditions. (B) PCR analysis of genomic DNA from 26 individual colonies displaying both green fluorescence (HR) and puromycin resistance (illegitimate recombination) after transfection with the pDonor and ZFNs. The genomic mEGFP specific primer (P1) was used together with the puromycin specific primers (Pu-1 and Pu-2) to investigate if the ΔEGFP and PuroR cassette had integrated in the genome of the 293-Flp-mEGFP cells as a single fragment or not. M represents the size marker and sizes of relevant bands are specified. Position of the PCR reactions from the individual colonies is indicated below the lanes.