Eukaryotic culture systems

Use of Prokaryotic and Eukaryotic Culture Systems for Examining Biological Activity of Food Constituents... 

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 vitro genotoxicity studies are summarized in Table 2-17. Eukaryotic cell systems were used for detecting the effects of 2,3,7,8-TCDD exposure on DNA. Exposure to 2,3,7,8-TCDD did not stimulate the unscheduled DNA synthesis in cultural human cells (Loprieno et al. 1982), but inhibited DNA, ribonucleic acid (RNA), and protein synthesis in mouse lymphocytes (Luster et al. 1979) caused gene mutations in mouse lymphoma cells (Rogers et al. 1982) and induced sister chromatid exchanges in Chinese hamster cells (Toth et al. 1984). 

Although particularly problematic in the Porifera, the problem of contamination, mainly by unicellular eukaryotic organisms, is an area of focus. This obstacle necessitates the validation of cell type in the culture system. Other areas in which new approaches are being sought include the selection of cell sources for the initiation of cultures and cryopreservation protocols for the maintenance of source cells for long-term studies. 

Insect cell systems represent multiple advantages compared with mammalian cell cultures (1) they are easier to handle (Table 2.1) (2) cultivation media are usually cheaper (3) they need only minimum safety precautions, as baculovirus is harmless for humans (4) they provide most higher eukaryotic posttranslational modifications and heterologous eukaryotic proteins are usually obtained in their native conformation (5) the baculovirus system is easily scalable to the bioreactor scale. However, because of the viral nature of the system, continuous fermentation for transient expression is not possible - the cells finally die. 

Expression of Potential Vaccine Antigens. In general, in the future, eukaryotic cell culture is likely to be the method of choice for the production of subunit vaccine antigens where the organism to be vaccinated against replicates in eukaryotic cells. E. coli are unable to posttranslationally modify some vaccine candidates for example, bacterial systems cannot add carbohydrate which is important in the antigenicity and structure of many protective antigens. 

Today, there are a wide variety of laboratory protein expression systems available, ranging from cell-free systems over bacterial and yeast cultures to eukaryotic models including the Xenopus oocytes or insect and mammalian cell cultures, some of which even form polarised epithelial-like cells layers. In Table 24.1, an overview of the most important systems, as well as their particular strength and weaknesses in the expression of transmembrane transport proteins is provided. 

Vectors that include replication systems derived from more than one host species are known as shuttle vectors. Such vectors commonly include a replication system able to function in E. coli and one that works in a second host, which may be bacterial or eukaryotic. Initial cloning and amplification of the DNA segment to be studied is often carried out in E. coli because it is easier to make large quantities when culturing in E. coli. The recombinant DNA molecule, consisting of the bifunctional vector plus the cloned segment of DNA, is then introduced into the second host, where the purpose is usually to measure the expression of the genes carried by the vector. Shuttle vectors that can replicate in both E. coli and yeast are the most common. 

---