Prokaryotes recombinant gene expression

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. 

Whether A-DNA occurs in cells is uncertain, but there is evidence for some short stretches (tracts) of Z-DNA in both prokaryotes and eukaryotes. These Z-DNA tracts may play a role (as yet undefined) in regulating the expression of some genes or in genetic recombination. 

To understand how recombinant pharmaceutical products are manufactured we first need to review some of the essential DNA manipulation techniques used to generate these recombinant products. We will start by looking at ways to cut and join fragments of DNA and then examine step by step how these techniques can be exploited to clone and express any genes from eukaryotic and prokaryotic cells. 

DNA (section 2.4.1) than eukaryotic RNA (section 2.4.2). The result will be the construction of a collection of cloned DNA fragments propagated in bacteria that is called the genomic library. This library should contain representatives of every sequence in the chromosome of a prokaryotic cell and every expressed gene in the case of a eukaryotic cell. The final step consists of the screening of every recombinant clone to identify the required gene(s). 

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