Eukaryotic vectors

Niwa, H., Yamamura, K. and Miyazaki, J. (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene, 108, 193-199. 

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. 

In most countries, about one third of the mutations that are identified wfll not have been reported previously in AIP and may represent rare polymorphisms rather than disease-specific mutations. Criteria that suggest that such novel mutations cause disease include production of a frameshift or stop codon, the absence of any other sequence abnormality in the gene, segregation with disease, and nonconservative change of an amino acid residue that is conserved between species and/or known to have a functional role in catalysis. Mutations of consensus bases in splice sites are also likely to be disease specific, but ideally all putative splicing defects should be confirmed by analysis of mRNA. Proof that a missense mutation causes disease may require expression and characterization of the mutant enzyme in a prokaryotic or eukaryotic vector. 

The production of selectable vectors for use in animals cells often used Szybalski s system or further developments based on selective protocols directed to the purine synthesis pathway, for example, Bacchetti and Graham [198], who transferred the Herpes simplex thymidine kinase into a human cell line, albeit with very low efficiency in 1977 and Mulligan and Berg [199] who developed a selection protocol for the expression of an E. coli gpt gene in a eukaryotic vector in 1981. 

Eig. 5. Restriction map of the yeast artificial chromosome (YAC) vector used for cloning very large fragments of eukaryotic DNA. Terms defined in text... 

The field of DNA vaccination started when eukaryotic expression vectors were injected into the muscle of laboratory animals [2]. The authors observed protein expression for more than 2 months after injection and noted that no special delivery system was required to obtain this expression. Subsequently, it was demonstrated that antibodies can be induced simply by injecting plasmid DNA into the muscle of mice [3]. Subsequent studies found that the injection of expression plasmids also leads to the induction of a cytotoxic T-cell response. After injection, the DNA enters cells of the vaccinated host and the encoded gene becomes expressed. This eventually leads to the induction of a cellular cytotoxic T-cell, T-helper, and/or humoral (antibody) immune response. 

There are various protocols of administering eukaryotic expression vectors aiming to deliver (i.e. transfect) the DNA into the cytoplasm of the host cells (see Figure 2). The DNA is subsequently imported into the nucleus of the transfected cells allowing expression of the... 

The eukaryotic expression cassette is the part of an expression vector that enables production of a protein in a eukaryotic cell. The cassette consists of a eukaryotic promoter for mRNA transcription, the gene and an mRNA termination and processing signal (Poly-A signal). 

PCA Passive cutaneous anaphylaxis pCDM8 Eukaryotic expression vector PCNA Proliferating cell nuclear antigen... 

Transfection is the process of introducing DNA or RNA into eukaryotic ceils. The use of transfection is to study the role and regulation of proteins or to understand the mechanisms of a pathway. Transfection can be transient for rapid analysis or stable , mostly for induction of expression. There are various methods of transfection which include electroporation, viral vectors, DEAE-Dextran, calcium phosphate or Lipofectamine. The choice of transfection depends on the cell type used. The most desirable technique is the one which gives high efficiency of nucleic acid transfection with less interference to the cells physiology and high reproducibility. 

E. coli Economical, fast, easy, high yield, well characterized genetics, large number of cloning vectors Insolubility and misfolding of proteins, no glycosylation possible, difference in codon usage between prokaryotes and eukaryotes... 

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