Figure 6

PAM gene copy numbers, mRNA expression, and C-terminal amidation for the clones transfected using ZFN mRNA. The PAM gene copy numbers (a), PAM mRNA expression levels (b; calculated per ACTB housekeeping gene), and prolinamide contents of the recombinant mAb determined by CEX (c), for the three best ZFN knock-out clones derived from the CHO Der2 parental cell line, where ZFN mRNA was used for the transfection. The two clones identified (K62 and K68) were knocked-out for all PAM gene copies at the genomic DNA level, with mRNA expression and a prolinamide content of 6% still observed. The copy numbers and the mRNA expression levels were determined using qPCR, and the data are means ± standard deviations of two technical replicates. Prolinamide content was determined by CEX, results are presented as an average of two biological replicates.