Antisense oligo experiments - help

Jack M. Bernstein, M.D. Bernstein at WSU-ID.Dayton.OH.US
Sat Jul 15 09:11:04 EST 1995


grggta at picr.cr.man.ac.uk (Graham Atherton) wrote:


>Has anyone experience of using 22mer antisense oligo's to give 
>biological effects when added to adherent mammalian cells eg interfere
>with cell cycle. All my experiments so far show as much effect when
>sense (control) oligo's are used as when antisense are used.
>I just add phosphorothioate oligo's to the growth medium at 5-15uM.
>Would it be better to use lipofectin (or similar) to aid uptake of the
>oligo and hence increase specific effect - does anyone have a good
>protocol for this? 

>Cheers
>Graham

Many of us have played with this issue. I do not have access to the
data but if you do a search of the literature, you will find that
there are several papers out there speaking to the distribution of
oligos after several different methods of uptake.

Since you are trying to take them up in the nucleus, lipofectin/
lipofectamine should work fine. Oligos introduced in this manner tend
to transit to the nucleus quite quickly. Treat the oligos as if they
were plasmid DNA and they should go in quite well. Be careful with
your lipofectin concentration as that itself can be somewhat toxic.

If you need a specific protocol, e-mail me and I will have my grad
student send it out next week.

Hope the weather in England is better than here. We hit 39C yesterday,
in OHIO!

Cheers.

Jack





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