Cell Engineering : Transient Expression

The advantages of the baculovirus system are rooted in the properties of the virus and the host (insect, or cell lines derived from it). During the normal infection cycle, two forms of the virus are produced: an early budded virus (BY) form (Kost et al. , 2000), in which the viral DNA and structural proteins are surrounded by membrane derived from the infected cell; and a late occluded form (occlusion-derived virus, ODy), consisting of enveloped viral cores which are embedded in a crystal matrix of viral proteins. The principal component of the matrix is the abundantly expressed protein polyhedrin. The budded virus rapidly spreads the infection from cell to cell within the insect host, resulting ultimately in the complete liquefaction of the host, and release of occluded virus into the environment. The occluded form protects the released virus, allowing it to survive for long periods in the environment until ingested by another host. In the alkaline environment ofthe insect gut, the protective protein matrix is removed, and the life cycle is repeated. In insect cell cultures, only the BV form of baculovirus is required, and the polyhedrin gene may be replaced with the gene for the recombinant protein. An additional benefit of replacing or deleting polyhedrin is that it effectively makes the virus unable to survive outside the laboratory, an advantage in terms of environmental safety. The system is intrinsically safe to animals, being unable to replicate in species other than a limited range of insects. This volume focuses on the transient gene expression technologies for recombinant protein production and gene therapy. It provides coverage of an important issue in Cell Engineering by internationally authoritative experts. Topics covered range from the use of baculovirus, adenovirus, adeno-associated virus and alphavirus vectors in biopharmaceutical, genomics and gene delivery research, to large-scale transfection of plasmid DNA into cultured cells for bioreactor applications. This book illuminates all the scientific and technological issues involved in the research, development, production, and human use of viral and non-viral vectors for in vitro and in vivo expression of recombinant proteins, thus making it an essential reference for cell culture technologists, virologists, and scientists interested in gene therapy Front Matter....Pages i-vii Application of Recombinant Baculoviruses in Biopharmaceutical Research....Pages 1-28 Engineering Post-Translational Processing of Recombinant Proteins Produced in Insect Cell Culture....Pages 29-42 A Guide to Successful Scale-up of the Baculovirus Expression System....Pages 43-55 Transient Gene Expression in Mammalian Cells Based on the Calcium Phosphate Transfection Method....Pages 56-79 Adenovirus Vectors in Functional Genomics....Pages 80-154 Adeno-Associated Virus: A Promising Tool for Gene Delivery....Pages 155-181 Alphavirus Vectors: Highly Efficient Systems for Transient Gene Expression....Pages 182-210 Transient Expression in Mammalian Cells : Applications and Perspectives....Pages 211-218 Back Matter....Pages 219-223