Mammalian transfectants vector transfection

Also, special vectors allowing expression in both insect cells and mammalian cell cultures from the same vector (pMamaBac [11] andpBacMam [12]) were described, though the amount required for mammalian transfection with one of these vectors is twofold higher than for insect cells, which makes it applicable only for assessment of suitability for a certain cell culture. 

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... 

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. 

It should be emphasized at this point that the use of physicochemical methods is so far the only way to demonstrate the import of transgene DNA into the mitochondrial matrix in living mammalian cells. The unavailability of a mitochondria-specific reporter plasmid designed for mitochondrial expression severely hampers current efforts toward the development of effective mitochondrial expression vectors. Although any new nonviral transfection system (i.e., cationic lipids, polymers, and others) aimed at the nuclear-cytosolic expression of proteins can be systematically tested and subsequently improved by utilizing anyone of many commercially available reporter gene systems, such a methodical approach to develop mitochondrial transfection systems is currently impossible. 

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. 

Teshigawara K, Katsura Y (1992), A simple and efficient mammalian gene expression system using an EBV-based vector transfected by electroporation in G2/M phase, Nucleic Acids Res. 20 2607-2611. 

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