antisense technology?

Mart Speek mspeek at tamm.eenet.ee
Tue Nov 26 03:16:34 EST 1996


Elizabeth Rosenberg (erosenbe at gpu2.srv.ualberta.ca) wrote:
: Hi!

: 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.

Good luck!

Mart (mspeek at ebc.ee in Estonia)




More information about the Methods mailing list