Cell Culture Technology for Pharmaceutical and Cell-Based by Sadettin Ozturk, Wei-Shou Hu

By Sadettin Ozturk, Wei-Shou Hu

Edited by means of of the main unique pioneers in genetic manipulation and bioprocess expertise, this bestselling reference offers a complete evaluate of present mobilephone tradition know-how utilized in the pharmaceutical undefined. Contributions from a number of prime researchers show off the significance of gene discovery and genomic know-how improvement within the construction of biotechnology items, tissue engineering, and cell-based cures. delivering distinct suggestions, they hide the stairs best as much as the construction of manageable remedies together with host telephone choice, cloning and gene amplification, bioreactor layout and operation, protein purification, optimization, scale-up, and facility layout.

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The ‘‘dominant’’ feature of these markers refers to the fact that there is no background activity of this sort expressed by the Recombinant DNA Technology and Cell Line Development 23 Table 1 Selective Agents Commonly Used in Constructing Mammalian Expression Vectors Selectable marker Dominant Aminoglycoside phosphotransferase (aph) Hygromycin-B phosphotransferase (hph) Selective agent Enzyme inhibitor used for gene amplification Geneticin (G418)=Neomycin Hygromycin B Puromycin N-acetyltransferase (pac) Puromycin Zeocin resistance gene (sh ble) Recessive Zeocin Glutamine synthase (GS) Glutamine-free media Methionine sulfoximine (MSX) Dihydrofolate reductase (DHFR) Media lacking glycine, hypoxanthine and thymidine Methotrexate (MTX) cell, and as a result, selection using these markers does not rely on availability of a host cell that contains a mutant or impaired endogenous gene.

The average production life of the transient expression system is usually limited by toxicity of the rapidly multiplying DNA, or the loss of DNA from the cell population during division. While not suitable for long-term production or commercial manufacture, expression levels in transient systems may range from >1 to 100 mg=L, making these systems extremely useful for rapid production 15 16 Shen et al. Figure 1 Cell line development flow chart. of small quantities of research materials for drug candidate identification and in vivo evaluation.

Modifications of this system as well as other similarly convertible on=off systems based on gene regulation by other antibiotics have also recently been described. The attractiveness of these expression systems is their ability to be used modularly to regulate both the product gene and one or more functional genes involved in complex regulatory pathways in a concerted fashion (42,43). 22 Shen et al. Figure 3 Diagram of expression system used to provide tetracycline-regulatable gene expression in mammalian cells.

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