Mammalian expression vectors

The selection of appropriate introns is important for efficient RNA processing and to eliminate the utilization of the cryptic splice site in the coding region. Introns with splice sites and branch points that closely match the established consensus sequences are spliced more efficiently and accurately than ones that do not. Several introns have been used widely in mammalian expression vectors, including the second intron of the rabbit, 0-globin gene (O Hare et al., 1981), and intron A of CMV (Hartikka et al., 1996). Some expression vectors include a hybrid intron, e.g., the 5 portion of an adenovirus sequence and the 3 portion of an immunoglobulin sequence (Choi et al., 1991). 

Foecking, M.K. and Hofstetter, H. (1986) Powerful and versatile enhancer-promoter unit for mammalian expression vectors. Gene, 45, 101-105. 

Mizushima, S. and Nagata, S. (1990) pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res., 18, 5322. 

Gene therapy refers to the delivery of genetic material to cells for the correction or replacement of defective or absent endogenous genes. In vitro this is readily achievable using any number of mammalian expression vectors that can be found on the market. However, when the same delivery strategies are used in vivo the outcomes are not so promising. To date only limited success has been achieved in rare cases of... 

The field of DNA vaccines centers on the ability to deliver genetic material, coding, for desired protein antigens in the context of a mammalian expression vector (Donnelly et al., 1997). It was realized that the immunostimulatory properties observed with DNA vaccines, namely the ability to induce a Thl biased or cell mediated immune response, was attributable in part to the presence of CpG motifs within the plasmid DNA (Klinman et al., 1997). In fact, it was demonstrated that the quality of immune activation by DNA vaccines could be influenced by the deliber-ate alteration of CpG content (Krieg et al., 1998). Further, with the identification of neutralizing CpG motifs it became possible to remove any neutralizing influence from the DNA vaccine plasmids. The end result was the optimization of DNA vaccine plasmids, with respect to immune activation (Krieg et al., 1998). 

The fourth approach, which eventually proved successful, was expression cloning. In this strategy cDNA from a tissue or cell line expressing the receptor was cloned into a mammalian expression vector and transfected in to a cell line devoid of opioid receptors. Transfected cells could then be screened... 

Direct cloning of ORFs has also been used for Hving cell arrays where the cDNAs, cloned in mammalian expression vectors, were spotted onto microscope slides. Mammalian cells growing onto these printed areas took up the cDNA and expressed the corresponding gene product (Ziauddin and Sabatini, 2001). 

Baneijea, A. C., Brechling, K. A., Ray, C. A., Erikson, H., Pickup, D. J., andjoklik, W. K. (1988). High-level synthesis of biologically active reovirus protein crl in a mammalian expression vector system. Virology 167, 601-612. 

More recently, inducible promoters have been developed for mammalian expression vectors because of their potential use in gene therapy where constitutive expression would frequently not be desired. One of the beneficial aspects of induction would be the separation of culture phases for... 

The complete 7 kb protein-coding sequence of human factor VIII was assembled from portions of overlapping cDNA and genomic clones and Introduced into appropriate mammalian expression vectors (9,10). Following transformation into either hamster kidney cells (9) or COS-1 monkey cells (11) factor VIII expression in transfected cell lines was characterized. The recombinant protein was shown to be biologically active, demonstrating the ability to activate factor IX and to reduce clotting time in plasma derived from patients affected with hemophilia A. Additionally, factor VIII activity of transfected cells was inhibited by a factor Vlll-specific antibody. 

Cytomegalovirus (CMV) A strong viral promoter used in mammalian expression vectors. 

---