mspeek at tamm.eenet.ee
Tue Nov 26 03:16:34 EST 1996
Elizabeth Rosenberg (erosenbe at gpu2.srv.ualberta.ca) wrote:
: Our group is about to embark on the glorious journey of downregulation of
: a gene by use of antisense constructs. We are very new to this, and would
: appreciate any and all feedback anyone might have.
: Now we would like to insert an antisense fragment of the same gene into
: the pREP10 vector and tranfect this construct into "normal" CHO cells that
: are the "parent" of our mutant strain.
: Does this strategy seem reasonable?
I would not recommend antisense experiments such as yours. This is because
it is remarkably inefficient (for review see NAR 21:4383-91 (1993))
I spent 1.5 yrs to study RNA-RNA hybridization/annealing between
a pair of naturally occurring sense-antisense mRNAs, but found
very little of them in duplex (Mol. Endo.9:1655-65 (1995))
What you should know here is that all mRNAs/RNAs are in the form
of heterogeneous ribonucleoprotein particles (hnRNPs)and it is very
hard to compete with proteins for RNA hybridization/binding.
However, as you are set for your antisense experiment, the best strategy
to use antisense RNA to inhibit translation (if this is what you want)
is blocking 5'UTR/Met by antisense RNA prepared against this region.
Also try to make sure that antisense RNA is produced in excess (<100)and
you dont kill cells simply by producing too much of your antisense RNA.
This is all empirical, but luckily can be tested.
Mart (mspeek at ebc.ee in Estonia)
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