綜觀現今生技製藥產業， 中國倉鼠卵巢(Chinese hamster ovary, CHO)細胞與二氫葉酸還原脢(dhfr)/Methotrexate(MTX)基因增幅選擇法所建立之穩定細胞株，已廣泛應用於高品質重組藥用蛋白質之產製。本研究提出一全新方法，在基因增幅選擇法中加入以dhfr為標的之RNA干擾載體，藉此改善篩選高產量穩定純系細胞株的製程。首先，以來自小鼠與中國倉鼠dhfr基因內的序列保留區為依據，構築出三種(sd1, sd2, sd3) 干擾載體，並以含有EGFP的報導基因篩選出最有效干擾dhfr基因表現的載體。所選出的干擾載體sd2，進一步被證實可以在dhfr/MTX基因增幅選擇法的製程中，提高CHO/dhFr-（dhfr基因表現缺失的細胞株）穩定純系細胞株所表現的外源EGFP蛋白。利用RNA干擾法所獲得的穩定增幅細胞株，長期生長於無MTX培養基中，同時顯現出較高的重組蛋白表現穩定度。加入RNA干擾載體的dhfr/MTX基因增幅選擇法，亦可成功應用於dhfr基因表現正常的CHO-K1細胞株， 所獲得的穩定增幅細胞株，除了擁有較高的EGFP產量，同時製程中篩選穩定細胞株的效率與重組蛋白表現的穩定度，也都有較突出的表現。本研究亦嘗試利用RNA干擾載體於CHO與NS0細胞，來提升穩定增幅細胞株所生產的嵌合性抗體。結果顯示，此一方法成功地篩選出高穩定度與高產量的穩定增幅細胞株，而製程中用以產製抗體的CHO細胞，不但可來自於CHO/dhFr-細胞株，亦可選用CHO-K1細胞株。但利用RNA干擾載體的基因增幅選擇法，並不能成功地應用於NS0細胞生產嵌合性抗體。利用本研究所提出之RNA干擾載體於dhfr/MTX基因增幅選擇法，可篩選出高穩定性與高產量的穩定增幅細胞株，用以生產抗體或其他生技藥品，對生技製藥產業而言，極富應用性。

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.

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