There is an enormous variety of expression vectors and systems for use with mammalian cells, and a full treatment of this field is way beyond the scope of this book. Although the details are more complex than the systems described so far, the general principles remain familiar.
Most vectors use the enhancer/promoters from the human cytomegalovirus (CMV), the SV40 virus, or the herpes simplex virus thymidine kinase (HS-TK) to drive transcription. These give high level, constitutive, expression. As in prokaryotic systems, it is sometimes desirable to control the onset of expression. One way of achieving such regulation is by interposing the operator sequence (tetO) from the bacterial tetracycline resistance operon between the promoter and the cloned gene. If the mammalian cells are cotransfected with a second plasmid containing the tetracycline repressor gene (tetR), also expressed using the CMV promoter, the TetR protein will bind to the tetO site, thus preventing transcription. When tetracycline is added to the culture medium it will bind to the TetR protein, altering its conformation and releasing it from the DNA, thus derepressing transcription of the cloned gene. The advantage of this is that, because this system is of prokaryotic origin, its activation does not affect the induction of native mammalian genes. Similarly, an insect ecdysone-responsive element is sometimes used to induce gene expression in mammalian cells. Like the tetracycline system, ecdysone does not have any effect on native mammalian genes, and the induction is therefore specific to the genes placed under its control.
Additional sequences can be added to enable targeting of the product to specific cellular locations such as the nucleus, mitochondria, endoplasmic reticulum or cytoplasm, or secretion into the culture medium.
In contrast to bacterial cells, the introduction of DNA into mammalian cells does not depend on the independent replication of the vector; the introduced DNA can be stably integrated into the nuclear DNA. However, some expression vectors can be stably maintained at high copy extrachromosomally, such as those containing the origin of replication from the Epstein Barr Virus (EBV); with a suitable promoter system these are capable of allowing high levels of protein expression. The use of retroviral vectors for integrating foreign genes in mammalian genomes, and for obtaining gene expression, was con sidered in Chapter 6; for the application of these techniques in whole animals, see Chapter 17.
The advantage of using mammalian cells for expression of eukaryotic genes, especially those from mammalian sources, rather than the other systems described, lies in the greater likelihood of a functional product being obtained. This is especially relevant for studies of structure-function relationships and the physiological effect of the protein on cell function. However, the relative difficulty, and cost, of scaling up production, compared with either Pichia or baculovirus systems, makes mammalian cells less attractive if the objective is the large-scale production of recombinant proteins for other uses.
Was this article helpful?