Mammalian expression vectors cell culture

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. 

It is worth emphasizing that all biopharmaceuticals mentioned here are produced from mammalian cell culture. The protein production system based on insect cells known as BEVS (baculovirus expression vector system) is widely employed for the expression of a wide range of proteins, but, due to regulatory issues, biopharmaceuticals produced by insect cells are not yet in the market. However, some of them are being evaluated,... 

Most of the expression vectors currently in use are constructed and propagated in E. coli bacterium. Large amounts of the vector DNAs are extracted from E. coli cultures and introduced into the chosen host cell. (A notable exception to this generalization are vectors derived from mammalian and insect viruses, but even in these cases at least some of the components of the expression vector are assembled in E. coli.) Thus, vectors assembled by the manipulation of DNA by restriction nucleases and ligase are introduced in the naked-DNA form into E. coli rendered competent to accept extraneous DNA. This process is termed Transformation Once the vector enters the E. coli cell, it has to be replicated and maintained in order to avoid its loss due to the dilution that accompanies rounds of cell division. The presence of a DNA sequence on the vector which functions as the origin of replication permits the vector to replicate in the host cell. 

Insect and Mammalian Cell Cultures. Animal cell cultures are the systems with highest similarity to human cells with respect to the pattern and capacity of posttranslational modifications. However, their cultivation is more complicated and costly and usually yields lower product titers. Among the known systems, insect cells transformed by baculovirus vectors have reached a comparable popularity as Pichia among yeasts because they are considered to be more stress resistant, easier to handle, and more productive compared with mammalian systems and are thus frequently employed for high-throughput protein expression. The highest reported... 

Their rather short in vivo half-life can be overcome by conjugation to polyethyleneglycol molecules. The phage display format also allows facile cloning into IgG vectors, so that the human IgG from mammalian cell cultures can be expressed. 

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. 

Two other viral vectors are extensively used. Vaccinia virus, a large DNA-containing virus, replicates in the cytoplasm of mammalian cells, where it shuts dovm host-cell protein synthesis. Baculovirus infects insect cells, which can be conveniently cultured. Insect larvae infected with this virus can serve as efficient protein factories. Vectors based on these large-genome viruses have been engineered to express DNA inserts efficiently. 

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