Eukaryotes recombinant gene expression

Volume 306. Expression of Recombinant Genes in Eukaryotic Systems Edited by Joseph C. Glorioso and Martin C. Schmidt... 

DNA plasmid-based treatment ( gene therapy ) is considered an alternative to the one based on classical chemical drugs or proteins recovered from recombinant cells. Treatment of acquired and inherent genetic diseases as well as the use of DNA for the purpose of vaccination are potential applications of plasmid DNA (pDNA). The plasmid carries information that allows protein expression in the targeted human cells as well as eukaryotic regulatory elements and specific prokaryotic sequences that control replication in the host cell, see Fig. 10. Formulation is required for ex- or in-vivo administration. Selected systems for gene expression can be viral or non-viral. 

If the end goal of cloning is to have a cloned gene expressed in a cell, the entire coding sequence must be doned intact. Furthermore, if a cloned eukaryotic gene is to be expressed in bacteria (to make recombinant proteins), the gene must not contain introns, which could not be processed in a prokaryotic cell. In these cases it is more convenient to done cDNA rather than DNA restriction fragments. 

There exist a variety of vectors for cloning into eukaryotic systems, ranging from yeast (Saccharomyces as well as Pichia) through insect cells (Baculovims) and plants (Ti plasmid from Agrobacterium tumefaciens) to mammalian cells (transfected by viral or mammalian vectors). As expression in eukaryotic hosts is less efficient than bacterial expression in terms of yield and time and more complicated in terms of vector structure and culture conditions, such eukaryotic expression systems are only used for genes whose proteins require posttranslational modification which is not possible in bacteria. Yeast is the preferred option as a relatively easily culturable single-cell system but posttranslational modification capabilities is limited. The additional complexity can be circumvented in part by exploiting the ability of eukaryotic vectors to act as shuttle vectors, which can be shuttled between two evolutionarily different hosts. Thus, eukaryotic vectors can be replicated and analyzed in bacteria and transfected into eukaryotic cells for expression of the recombinant product. 

Volume 306. Expression of Recombinant Genes in Eukaryotic Systems... 

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