Abstract
Chinese hamster ovary (CHO) cells and dihydrofolate reductase (dhfr)/methotrexate gene amplification system are routinely used to generate stable producer CHO cell clones in biopharmaceutical industries. The present study proposes a novel method by the co-amplification of the dhfr-targeting RNA silencing vector targeted to dhfr gene for improvements of selecting high-producing clones in CHO cells. Initially, three target sequences (sd1, sd2, sd3) located in the conserved sequences of the mouse and Chinese hamster dhfr genes were selected to silence dhfr genes in CHO cells using DHFR-EGFP fusion protein as reporter. The silencing vector psd2 demonstrated to be most effective to silence dhfr RNA transcripts was approved to enhance EGFP expression in dhfr-deficient CHO/dhFr- cells through dhfr/MTX gene amplification. The utilization of the silencing vector also enhanced the stability of EGFP expression in the stable CHO/dhFr- cells grown in MTX-free medium. CHO-K1 cell clones co-transfected with the silencing vector reveal the similar productivity, similar efficiency of transfection and similar production stability through dhfr/MTX gene amplification. This method was further applied to the antibody expression in CHO and NS0 cells. Higher level of IgG expression and more stable productivity in MTX-free medium was achieved in amplified stable clone from dhfr-deficient and wild-type CHO cells. Unfortunately, this method was unsuccessful to obtain the amplified producer clones for IgG expression in NS0 cells. The new strategy proposed here can be applied to obtain high producer cell clones for recombinant antibody or other biologics expression in both dhfr deficient and wild-type CHO cells with equally efficient stable transfection, which can be benefit the production of recombinant protein therapeutics in bioindustry.