Konstitütif promotörlerin mühendisliği ve çeşitli çin hamsteri yumurtalik hücre hatlarinda sistematik karşilaştirmasi

Following the developments in molecular biotechnology over the past two decades, the production of recombinant biological drugs has increased significantly. Chinese Hamster Ovary (CHO) cells are essentially the most preferred mammalian cell expression system for industrial manufacturing of biotherapeutics because of easy adaptation to grow in suspension, human-like post translational modifications and well-characterized transfection and gene amplification systems. In the present study, using recombinant DNA technology, we engineered a novel all-in-one dual-promoter reporter system to systematically compare the strength of natural viral, mammalian and endogenous promoters for high level of protein expression in various lines of CHO cells. We firstly studied a large panel of candidate promoters (CMV, SV40, HSV TK, PGK, EFS, EF1a, UBC, CAG and CHEF1a) in CHO-WT, CHO-DG44 and CHO-DG44 suspension cell lines for transient gene expression. Of nine promoters, luciferase assay revealed that CMV achieved the highest reporter activity. We supported our luciferase assay results via repeating the comparison of three strong promoters and one weak promoter with flow cytometry. Then, five strongest promoters were selected and placed on the backbone having mouse DHFR coding sequence to test the promoter strength in stably transfected cells. The recovery of stable cells is still in progress. So, after recovery, we will be able to compare these five strong promoters with our dual promoter system in long term culture. Conclusively, the dual-promoter reporter system eliminated the problems in co-transfection assays and proved to be a useful system to identify strong regulatory elements ensuring high levels of expression in CHO cells. Key Words: CHO, recombinant DNA technology, promoter strength, dual promoter system