 The Art Of Gene Expression
May 24 '07
Mammalian cells provide an excellent expression system for the production of proteins for a number of reasons. Mammalian cells manage to properly fold many protein molecules that fail to do so in other systems. In addition, mammalian cells generally preserve the appropriate post-translational modifications, which are crucial for the function of many proteins. For example, mammalian cells allow the formation of proper N-linked glycosylation and of authentic O-glycosylation patterns on protein molecules. However, robust production in mammalian cells can be very demanding. Potential problems might occur at the transcriptional-translational level, with RNA processing, mRNA stability, etc. Many of these problems can be overcome with careful design of the whole process, starting with the selection of an appropriate vector. A useful vector must have a promoter that is recognized by the cell type you will be using in your experiments. In addition, you’ll want to consider whether the promoter operates constitutively or after induction with an appropriate agent, thus allowing the control of the gene expression. An inducible promoter is important if you will be expressing a toxic protein. Other elements that must be present are termination codons, mRNA cleavage and polyadenylation sites, signals that ensure successful RNA splicing, selection markers (e.g. resistance to an antibiotic), a prokaryotic origin of replication for maintenance of the vector in bacterial cells, etc. While most vectors include all of these features, you may want to look for those that include termination codons in all 3 reading frames for flexibility in cloning. The vectors listed below can help you design an appropriate strategy for the production of your molecules in a variety of mammalian expression systems, thus allowing you to take advantage of their unique properties as protein factories.
Marios-Frantzeskos Sardis
Graduate Student
University of Crete
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| Company |
Promega |
Invitrogen |
Novagen |
| Item |
pCI Mammalian Expression Vector |
Gateway™ pCMV•SPORT6 Not I/Sal I Cut |
pTK-neo DNA |
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| Description |
The pCI Mammalian Expression Vector is designed to promote constitutive expression of cloned DNA inserts in mammalian cells. The major difference between the pCI and pSI Mammalian Expression Vectors is the enhancer/promoter region controlling the expression of the inserted gene...
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Offers a simplified primer-adapter strategy for directional cloning and includes an oligo(dT) primer adapter containing a Not I site for first-strand synthesis and ligation of Sal I adapters after second-strand synthesis |
The pTK-neo vector can select stably transformed mammalian cell lines using G418. A minimal thymidine kinase (TK) promoter controls expression of the neomycin resistance gene. This promoter facilitates selection for stable integration of both the selection plasmid and a cotransfected expression plasmid into transcriptionally active sites in the genome...
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| Catalog Number |
E1731 |
12209011 |
71284-3 |
| Size |
4006 bp |
4396bp |
2872 bp |
| Selectable Marker |
Amp |
Ampicillin |
G 418 |
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