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Applicatons of Antisense Technology

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26 views3 pages

Applicatons of Antisense Technology

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mohijitmy19
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© © All Rights Reserved
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APPLICATION

The possibilities that aRNA has to offer the scientific and medical fields seem endless. Projects
are in the works already on how to use antisense technology for the deactivation of
oncogenes25, for the suppression of viral RNA expression24, and to understand naturally
occurring aRNA that can be used for genomic imprinting 25.
Antisense technology has already been successful in suppressing the gene for the
protein that makes

tomatoes – Poil Flavor saur tomatoes were transgenic tomatoes constructed to have
artificial DNA that coded for aRNA that was complementary to the RNA that coded for the
protein that caused spoiling. The aRNA suppressed the expression of this spoilage gene by
10% which was enough to save the tomatoes from rotting while being shipped to grocery
stores. The tomatoes are no longer on the market due to complications in the
harvesting process25.

In vitro application
Antisense technology has been applied successfully in two general areas. The first is in
fundamental research where the introduction of antisense oligonucleotides can help
determine the role of a specific gene in a specific physiological process10-12. For
example, in the laboratory of Alton ochsmir medical foundation, New Orleans. L.A
(U.S.A) was interested for same time in the idea that local components of the rennin
angiotensin system can be produced by specific cells. They hypothesized that the
production of angiotensin II by cell feeds back on those cells resulting in cell growth and
other changes.

In their view this tissue (cellular rennin angiotensin system) could therefore potentially
play a role in a wide variety of cardiovascular disorders, including
atheroeclerosis and vascular hypertrophy. It is difficult to demonstrate that a cellular
system is operative in any given process. However, because a circulatory rennin
antiotensin system exists that produces angiotensin II in tissue culture medium as well as
in tissues. To approach this problem they developed oligonucleotides to inhibit the
synthesis of angiotensinogen, the substrate from which cells make angiotensin II18.

Thus, through the application of antisense technology, they are able to demonstrate the
biological principle that cells can make their own angiotensin II with growth promoting
effects. Now they extended this antisense work to certain cancers and demonstrated, for
the first time, that neuroblastoma cells18.
Therapeutic Application
A second application of this technology, and one that is potentially of more immediate
relevance to the practicing physician, in the use of this technology in therapy2-3,14-19. In principle,
antisense oligonucleotides complementary to viral RNAs can suppress a wide variety of viral
infections, a tremendous amount of research is ongoing in this area, similarly, antisense
oligonucleotides directed towards the products of oncogenes can play a role in reducing the
growth of cancer cells, and this leads in being hotly pursued20.

Perhaps the most widely discussed application of antisense technology lies in its
applications to gene therapy. In this case, a variety of vectors is used to introduce antisense
encoding genes into a large number of cells in a patient or animal to produced long term
inhibition of a protein. For example, in animal models the introduction of vectors encoding
antisense angiotensin II

receptor sequences results in long term normotension in spontaneously hypertensive animals20.

These are but a few of the possible application of antisense technology. As familiarity
with the relevant chemistry increases, it is likely that more effective oligonucleotides and
gene vector will be developed thereby providing the ability to interfere at will with the
translation of specific mRNA.
Obstacles of Antisense
The successful result of any of those methods is reduced protein production as long as
the surrounding conditions are favorable (Driver). However, there are more problems
that need to be overcome then just inserting the antisense molecule into the cell.
If antisense is being used as a treatment in the human body it might be degraded
before stopping any protein production if it is unmethylated because the
body would recognize it as an invader26. When antisense is used is living organisms,
there are also many complications that can arise such as high blood pressure and a low
white blood cell count26.
Antisense and how it applies to Flavr Savr
The first step is to create a cDNA of the PG gene. This involves generating an
mRNA, then re-transcribing it back into a cDNA. This cDNA contains the entire
PG gene. Next a 730 base pair (bp) region, including a 50bp non- coding region is
excised called the Hinf1 fragment. This is cloned into a plasmid. Next the cloned
fragment was excised and legated into a second plasmid. The insertion occurred just
after the CaMV35s (Cauliflower Mosaic Virus Promoter) finally, this plasmid was
inserted into an agrobacteria, where the complete fragment was inserted yet again, into
another plasmid, this one containing the KanR gene19.

Figure 5

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