| 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 particles 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 costs are 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; if not cultured in suspension, the cells should also be able to grow in adherence. The abilities of cultured cells mentioned above cannot 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 challenging environments. Considering Chinese hamster ovary (CHO) cells are the most frequently used host cells for biopharmaceutical production, we are intended to over-express or suppress some critical genes in CHO cells to make the cells more desirable for large-scale culture in bioreactors.1. Construction of high efficiency expression vectorsExpression vectors are critical to high efficiency expression of foreign proteins. In this study, we first constructed a vector containing human elongation factor la subunit promoter (PEF-1 ) for transcription of genes of interests, and mouse dihydrofolate reductase (dhfr) gene under control of SV40 promoter (Psv4o) for clonal selection and amplification. The vector was named pED5. The expression efficiency difference between pED5 and pCdhfrl, a vector utilizing CMV enhancer/promoter (PCMV-IE) for foreign protein production, was analyzed using human interferon-p (IFN- ) gene and human secreted alkaline phosphatase (SEAP) gene as reporters. When analyzed in transient expression, pED5 showed a little more protein produciton than pCdhfrl.However, in continuous expression, when serum concentration was lessened to slow down cell proliferation, pED5 expressed 3.1 times more reporter proteins than pCdhfrl, which implied that PEF-IO was less affected by cell cycle status in contrast to PCMV-IE, making pED5 a good expression vector for foreign protein production. To further enhance protein productivity of expression vectors, two artificial transcription activating domains, AH and VP2, were linked to cl represser protein of phage through a soft linker, respectively, and thus two artificial transcription activators were created. The OR2-OR1 sequence of phage K was inserted into PEF-IO about 200bp before TATA box. The artificial transcription activators can bind to OR2-OR1 sequence, and are thus conscripted near to the TATA box of PCMV-IE, activating gene transcription with artificial activating domain AH or VP2. All the components mentioned above were integrated into pED5, producing two vectors: pER-AH and pER-VP2. The two vectors were tested for their efficiency of expressing IFN- and SEAP reporter genes in comparison with pERFRT, a vector similar to pER-AH and pER-VP2 except for lacking of artificial transcription activators. To abolish transcription efficiency affected by copy number or integration site in chromosome, all the vectors were integrated into FRT site by co-transfecting with an Flp recombinase producing plasmid. The FRT site was pre-inserted into CHO cell chromosome by Invitrogen Corporation. It was showed that pER-AH was only a little more efficient tha...
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