2. RNA Interference
It’s a evolutionary conserved mechanism for gene regulation that is induced by small silencing RNA molecules
having sequence specificity.
Andrew Fire & Craig C. Mello found this mechanism in C.elegans in 1998 and were jointly awarded nobel prize for
their discovery in 2006.
Also known as post transcriptional gene silencing (PTGS), transgene silencing, quelling and now RNAi.
This mechanism is used by plants animals and mammels to defend against external viral infections, transposable
elements, and also for gene regulation at different stages of life cycle.
3.
4.
5. The findings proposed that externally introduced RNA molecule can produce interference with the endogenous RNA
thus affecting the expression levels of the gene.
RNA interference has been used widely in the nematode Caenorhabditis elegans to manipulate gene expression.
Despite the usefulness of RNA interference in C. elegans, two features of the process have been difficult to explain.
First, sense and antisense RNA preparations are each sufficient to cause interference. Second, interference effects can
persist well into the next generation, even though many endogenous RNA transcripts are rapidly degraded in the early
embryo.
Study reveled that double standard RNA molecules was more effective than single standard RNA.
To test whether double-stranded character might contribute to interference, they purified single-stranded RNAs and
compared interference activities of individual strands with the activity of a deliberately prepared double-stranded
hybrid.
6. EXPERIMENT
The unc-22 gene was chosen for initial comparisons of activity. unc22 encodes an abundant but nonessential myofilament
protein. Several thousand copies of unc-22 mRNA are present in each striated muscle cell.
PHENOTYPIC EFFECT: Decreases in unc-22 activity produce an increasingly severe twitching phenotype, whereas complete
loss of function results in the additional appearance of muscle structural defects and impaired motility.
Along with unc-22 , three different reference genes were
used for which expression pattern was known.
unc-54 encodes a body-wall-muscle heavy-chain isoform of
myosin that is required for full muscle contraction.
fem-1 encodes an ankyrin-repeat-containing protein that is
required in hermaphrodites for sperm production.
hlh-1 encodes a C.elegans homologue of myoD-family
proteins that is required for proper body shape and motility.
7. Interference with GFP and lacZ activity was assessed using C. elegans strain
PD4251. This strain is a stable transgenic strain containing an integrated array
(ccIs4251) made up of three plasmids: pSAK4 (myo-3 promoter driving
mitochondrially targeted GFP); pSAK2 (myo-3 promoter driving a nuclear-
targeted GFP–LacZ fusion); and a dpy-20 subclone26 as a selectable marker.
This strain produces GFP in all body muscles, with a combination ofmitochondrial
and nuclear localization. The two distinct compartments are easily distinguished
in these cells, allowing easy distinction between cells expressing both, either, or
neither of the original GFP constructs.
CONSTRUCT
9. Interferance produced by using unc-22 dsRNA was very specific and similar to the phenotype
produced by null mutant of unc-22. Similar results were found with other genes.
13. RNAi-Based Tumor Treatment
Compared with traditional gene therapy, RNAi has the benefits of higher
silencing efficiency and stability. Hence, it is widely used in tumor gene
therapy research. The application of RNAi in cancer is mainly manifested
in the following aspects:
1) inhibition of tumor anti-apoptosis genes,
2) study of tumor signal transduction pathway,
3) inhibition of tumor angiogenesis-related factors,
4) the effect on oncogenes,
5) tumor suppressor genes, and
6) reduction of tumor drug resistance.
14. Patisiran works by silencing the gene that underlies a rare disease called hereditary
transthyretin amyloidosis. In that illness, mutated forms of the protein transthyretin accumulate
in the body, sometimes impairing heart and nerve function.
