Biomedical Engineering Reference
In-Depth Information
enhancer sequences. Eukaryotic expression of a gene requires the pres-
ence of an ATG codon (encoding the amino acid methionine) placed in
an efficient context for optimal expression of the cloned gene (a Kozak
sequence), and a polyadenylation signal sequence downstream of the
gene to be expressed. While these can be provided by the cloned gene,
most expression vectors contain these sequences.
Transient or stable expression of eukaryotic
expression vectors
Eukaryotic expression plasmids can be introduced into cells so that
they are expressed in a transient or stable fashion. For transient expres-
sion studies, the plasmid is introduced into the cell in its circular form by
transfection. A number of transfection methods have been developed, in-
cluding electroporation of the DNA, adding DNA in the presence of salts
(such as calcium chloride) or together with weak base carriers (such
as DEAE dextran), or by first incorporating the DNA into lipid vesicles,
to name but a few. The plasmid need not replicate, and the eukaryotic
gene is expressed for a limited period of time (usually 1 to 3 days). In
transient expression studies, only a small fraction of the cells take up
and express the transfected DNA.
For stable expression of a DNA in eukaryotic cells, the plasmid is
linearized prior to transfection so that it can be incorporated into the
host genome. In addition, these vectors contain a selectable marker. A
selectable marker is a gene that produces a product that protects cells
that have taken up the DNA from an antibiotic that would normally kill the
cells. In this way, the cells that have incorporated the transfected DNA
can be selected from the bulk of the cells, since the non-transfected
cells will die in the presence of the antibiotic. The most frequently used
selectable markers are genes encoding the neomycin resistance gene
(neo) and the hygromycin resistance gene.
A commonly used eukaryotic expression vector is the pcDNA3 vec-
tor used for expressing genes from cDNA. However, many commercial
companies have generated expression cloning vectors that contain se-
quence tags (such as the HIS, FLAG, or GST tags mentioned earlier)
or other features to facilitate cloning, detection, or purification of the
expressed protein.
Viral vectors
In addition to plasmids, viral vectors can be used to introduce genes
into eukaryotic cells. These vectors are often used to increase the fre-
quency of cells expressing the transduced gene (in transient transfection
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