antisense oligonucleotides

[Gavin Fischer]

Jan Eggermont (Jan.Eggermont at med.kuleuven.ac.be) wrote:
: Hello all,

: I am planning to use antisense oligonucleotides to silence the expression
: of a gene in permanent adherent cell line. In view of this I would greatly
: appreciate your input on:
: 1) the chemistry of oligo: ordinary or phosphoro-thioates

In heat treated Serum you would be OK with normal oligos, but it is
better to use Phosphorothioates anyway.
: 2) the target of the oligo in the mRNAe: can you have antisense oligo's
: against any part of the mRNA or are preferential target sites for
: anti-sense oligo's.

Traditionally they are desighned against the translational start site. In our
hands there was a bit of variablity as you moved around. 

: 3) effective concentration range of oligo's and duration of treatment to
: get maximal inhibition

Varies by gene. You have to make sure that the treatment is longer than the
1/2 life of the protein. YOU HAVE to do westerns to check that it is 
working, as we showed (Fischer et al. JEM Jan 94) that we got stabilization
with the addition of "antisense" molecules. With other sequences we got 
loss of gene product. This was weird, but I moved here before looking into 
it.

: 4) which control oligonucleotides to use

We always used the randomized version of the olgo, so as to keep the
base pair composition (percentages) correct. Other people use the sense
version. 

: Many thanks in advance.

: Jan

: --
: Jan Eggermont                       Jan.Eggermont at med.kuleuven.ac.be
: Laboratorium voor Fysiologie - KU Leuven
: Campus Gasthuisberg
: B-3000 Leuven                            tel 32-16-345938
: Belgium                                  fax 32-16-345991


Gavin Fischer 
Immunology Program Boston University