Modification Of Chinese Hamster Ovary Cells

Mammalian cells are among the best systems for biopharmaceutical production due to their excellence in post-translational modifications. The products they produced are much more similar to their natural forms than those produced by prokaryotic, yeast or insect cells. Some complicated molecules such as genetic engineered antibodies and virus proteins for gene therapy can only be produced in mammalian cells. However, the drawbacks of mammalian cell expression systems are apparent: they are difficult to culture in large scale, especially in serum-free medium; the productivity is much lower than other expression systems and the cost is higher. A successful cell strain for biopharmaceutical production must conform to the following characteristics: the cells could produce high level of recombinant proteins; the cell should be adapted to serum-free or protein-free medium; the cells should be resistant to adverse culture conditions, which means the cells should have some anti-apoptosis property. The abilities of cultured cells mentioned above can not be obtained just by optimization of medium formulations and the implementation of high-performance bioreactor configurations. Modifying the cells themselves by gene technology to change their physiological exhibitions may provide a better way to make the cells more robust to challenge environments. Considering Chinese hamster ovary (CHO) cells are the most frequently used host cells for biopharmaceutical production, we are intended to over-express some critical genes in CHO cells to make the cells more desirable for large-scale culture in bioreactors.1. Construction of robust CHO cell lines by overexpression of CyclinD1/Survivin genesSerum-free or even protein-free medium are preferable for recombinant protein production. However, without serum to provide essential components such as hormones, growth factors and binding proteins, cells are easy to die. In this study, CHO DG44 cells were genetically engineered to overexpress Cyclin D1 and Survivin to adapt strict enviroment such as in serum-free MEM,and be resistant to apoptosis. Cyclin D1 is a cell cycle protein expressed continuously in G1 phase. When Cyclin D1 accumulates to certain amount, cell cycle was driven to S phase. So Cyclin D1 is a proliferation-promoting protein. Survivin, a 16. 3 kDa protein which has four exons and three introns, belongs to a family of proteins, known as inhibitor of apoptosis protein (IAP), which plays a key role in the regulation of apoptosis and cell division. It is characterized by the presence of single BIR repeat and lacks carboxyl terminal RING finger domain in its protein structure. Survivin plays a important rolein cell apoptosis and cell differentiation. It can interact with 3 and Caspase-7 and then prevent them from promoting apoptosis. By co-expressing Survivin and Cyclin D1 in CHO DG44 cells with the pIRES expression vector, we constructed the cell lines: CHO-SD. By RT-PCR we got three positive clones: 6, 13 and 31. And further more, the high expression of each protein was confirmed by Western blot. After synchronization the cell cycle was detected by flow cytometry, the result demonstrated that the reconstructed cell lines had more cells in S phase of the cell cycle. Apoptosis was analyzed by flow cytometry, and the three clones were found much more resistant to apoptosis induced by addition of Straurosporine than the CHO DG44 parent cell. Cell proliferation assay by MTT method showed that clone 6 proliferated much faster than CHO DG44 in MEM medium without serum. When compared to CHO DG44, the reconstructed cell lines exhibited much more viable cell numbers and faster growth rate.2.Construction of robust CHO cell lines resist to apoptosis by inducible expression of Survivin geneAfter the running out of nutrition, the product of metabolite cells will occur apoptosis in large-scale cell culture. To inhibite the apoptosis and made the cell adapt strict enviroment, We contrusted the cell lines overexpressing Survivin under the control of the tetracycline-inducible systems by two steps. First step, we got the clone which can produce high yield of tetracycline repressor by screening them with ransient transfection of pcDNA4/TO/EGFP, and it was named DG44-B2. The clones with high induction and low background of EGFP expression in response to Tet were freezed for the second transfection. Second step, We transfected the Tet-On cell line with the plasmid containing Survivin gene, and next, used 200μg/ml Zeocin and 8μg/ml Blasticidin to isolate the stable clones. We got three positive clones at last , which were named clone 9, clone 29 and clone 31. The ability of anti-apoptosis were analyzed by DNA ladder and flow cytometry. Both methods demonstrated that the cell lines which were regulated by Tet-On system could grow well in the strict enviroment.3. Construction of CHO-TD serum-independent cell lineTransferrin is an iron-binding protein that is responsible for transporting iron. And it is also a growth factor, which can promote the growth of cells by binding to specific receptor. By co-express Transferrin gene and Cyclin D1 gene in CHO cells with the pIRES expression vector, we developed the stably transfected cell line, which is named CHO-TD.