RNA Interference For Gene Knockdown

RNA is generally single-stranded but it
can also form double-stranded structures it can form hairpin loops and bulges
that allows RNA to have functions beyond being a mere messenger of information
contained in DNA RNA is with hairpin structures can be used to control the
output of the genome by inhibiting or repressing transcribed DNA – so the
messenger RNA. So in this way double-stranded RNA can be used to
control protein expression and play regulatory roles. This system is
actually endogenous and is used by the cell to control it’s protein expression. An
understanding of this system led to the production of synthetic small
interfering RNAs – or siRNAs for short – and short hairpin RNA – or shRNA – to
investigate gene function. The RNA interference pathway was actually
characterized in 1998. It was by Andrew Fire and Craig Mello and by 2006 the tech,
the technology had been used by so many people that actually won a Nobel Prize
in Medicine and Physiology in 2006. How siRNA or shRNA knockdown genes is quite
an intricate and complex process and it’s absolutely fascinating and you
would definitely find videos on here – on YouTube- about how the process works but
briefly and actually in summary siRNA results in a transient knockdown of
genes where knockdown occurs for up to 48 hours – all the knockdown you will get happens within 48hrs and you
don’t get further knockdown after the 48 hours whereas shRNA allows stable
knockdown of genes. so briefly how it works is sequence specific shRNA can be
processed by an an RNAase III endonuclease known as ‘Drosha’ in the nucleus and
when it comes out into the cytoplasm it is processed by another
enzyme called ‘Dicer’ before it enters what is known as an RNA-Induced-
Silencing-Complex (RISC) the RNA Induced Silencing Complex functions to degrade
the corresponding messenger RNA RISC in my head is analogous to the dead Cas9
if you’ve studied how the catalytically dead Cas9 operates, in
that it is guided by RNA to repress transcription in this case by degrading
messenger RNA that is complementary to it. Short interfering RNAs or siRNA for
short are processed by ‘Dicer’ in the cytoplasm they are not processed in the
nucleus and once they’re processed in the cytoplasm by Dicer
they are loaded onto RISC and processed just like shRNA. I mentioned that the
technique actually got a Nobel Prize in medicine and physiology in 2006 and in
that same year the Broad Institute set up an RNAi consortium that identified-
well it set up the goal of identifying and cloning shRNA sequences for every
gene in humans and mouse and these are distributed commercially via Sigma.
once you identify construct sequences that are targeting your gene you can
introduce it into the cell via various methods using lentivirus is a common
method so that you can have long term expression that are reproducible
when you repeat experiments with the lentivirus vector. Just keep in mind that
you may want one that is inducible, so that you can study the effects of the
knockdown at precise time point. This is because constitutive… so constant
knockdown of a gene may lead to long term adaptive responses so you… it’s… it’s..
you may want to create a setup where you knock down the gene precisely and
study its effects rather than having it being constitutively knocked down. All the best with your experiments.

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