Biomedical Engineering Reference
In-Depth Information
studies) or to introduce the gene into cells that are otherwise refrac-
tory to transfection. The three most common forms of viruses that are
used are adenovirus, vaccinia virus, and retroviruses, including
lentiviruses. These viral vectors are usually rendered “defective”,
so that infectious particles are not made in the transduced cells. To
render them infectious for use, these viral vectors must be “packaged”
in packaging cell lines in order to make infectious particles.
Baculovirus expression systems
A major drawback of expressing eukaryotic proteins in bacteria is the
lack of post-translational processing of the proteins that otherwise oc-
curs in eukaryotic cells. In addition, certain proteins do not fold properly
when expressed in prokaryotic (bacterial) cells. To help circumvent these
problems, the baculovirus expression system was developed. Baculovi-
urses are large double stranded DNA viruses that infect insect cells
and can express many gene products at high levels. When mammalian
genes are expressed using this system, they are usually processed nor-
mally and the expressed proteins often form higher level polymers in
the same way they are found in mammalian cells, even though the in-
sect cells are grown at 25°-27°C rather than 37°C used for growing
mammalian cell lines.
The most common baculovirus used for expressing proteins is
Autographa californica nuclear polyhedrosis virus, a virus than infects
2 members of the moth family. The insect cell lines commonly used are
the SF-9 or SF-21 cell lines, both ovarian cells derived from Spodoptera
frugiperda (the fall army worm), and High-Five cells from Trichoplusia
ni, (the cabbage looper). As you might imagine, these cell lines were not
developed in medical research settings but borrowed from agricultural
research laboratories studying insects!
C. Libraries
A library is a general term to identify the cloned DNA from an organ-
ism, a tissue, or a cell. Two types of libraries are commonly generated,
genomic libraries and cDNA libraries. Libraries can be cloned into either
plasmids or viruses. Bacteriophages are most commonly used because
of their higher cloning efficiency and the ability to package larger-sized
DNA inserts into the bacteriophage heads than can be accommodated
by plasmids.
Libraries are screened by colony or plaque hybridization or using an-
tibodies as described earlier.
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