Quantification of transgene copy number

Real-time quantitative PCR (qPCR) was applied to quantify transgene copy numbers in ESC lines. For this purpose, we extracted genomic DNAs from each cell line as described above. Tenfold serial dilutions of each genomic DNA sample were prepared in nuclease-free water and applied as the template in qPCRs. Three sets of qPCR reactions were performed using primer pairs (Table 1) specific for GFP (representing the transgene), Sry (single copy endogenous target) and Fgf10 (double copy endogenous target). qPCR was conducted with 2 μL of the diluted DNA in duplicate on a Rotor-Gene 6000 Real-time Thermal Cycler (Corbett Research Pty. Ltd., Australia). Acquired quantification cycles (Cqs) were applied for calculation of efficiency and copy numbers as described previously (18,20). Briefly, we calculated amplification efficiencies to ensure that the method’s requirements (amplification efficiency >90%) were met. Acquired Cqs in each dilution were normalized by the respective Cqs of Sry. GFP copy numbers were determined relative to respective internal controls, Sry and Fgf10, using the comparative Cq method (∆∆Cq). Transgene copy number were estimated with seven qPCR replicates for each transgenic cell line.