Silencing of PAM using shRNA. The mRNA expression levels are shown after the transfections using the Ambion sh5 and sh6 shRNAs on the CHO Der2 and CHO Der3 parental cell lines and on the K25 and K62 clones derived from the CHO Der3 parental cell line. The data obtained for the parental cell lines are in grey. In comparison to the negative controls (CHO Der2 -K, and CHO Der3 -K), the highest silencing effects were achieved with shRNA sh6 and 5 μg/ml antibiotic selection (5 μg puromycin [PURO]). Up to 3.7-fold reduction in the mRNA expression levels was seen for the CHO Der2 cell line, and up to 2.6-fold reduction for the CHO Der3 cell line. The data for the mAb-expressing clones K25 and K62 are in black. In this case, the PAM expression level and the prolinamide content (%) are presented. In comparison to the negative controls (K25 -K, and K62 -K), the different antibiotic selections did not show any differences in mRNA expression levels and prolinamide content. Up to 3.5-fold reduction in mRNA expression levels was observed for K25, and up to 2.2-fold reduction in K62. Prolinamide was decreased from 3.5% to 3% for K25, and from 14% to 4.6% for K62, which represents a 3-fold decrease. The mRNA expression levels for parental cell lines and the K25 and K62 clones were determined using qPCR (calculated per ACTB housekeeping gene), and the data are means ± standard deviations of two biological replicates.