Does linearisation of DNA help stable trasfection

Tarja Kokkola tarja.kokkola at uku.fi
Wed Jan 14 08:15:58 EST 2004


"Morten Lindow" <morten at binf.ku.dk> kirjoitti
viestissä:<bu0l2f$qkv$1 at news.net.uni-c.dk>...
>
> sriadi at hotmail.com wrote:
> > Does some one know if linearization of Plasmid DNA will increase the
chances of getting the stable clones. I am using exgen500 from fermentas as
the vector which is a linear Polyethylenimine.
> > Thank you very much in advance.
> >
>
> I've seen several protocols for generation of stable cell lines that
> includes linearization. So I guess some people thinks it helps. Not sure
> why though - perhaps because you get to choose where the vector is cut,
> instead of a more random cut that might break your ORF?
>
> Morten
>

I have used both circular and linear plasmids for stable transfection with
success.

Circular vector enters the cell more easily in the transfection but is
randomly cut in the integration, which can break the ORF as Morten has
written above. The random integration can also break the promotor or the
polyA signal of your construct.

Linear vector has more trouble entering the cell, but the integration of the
linear DNA occurs from the ends, so you can trust that your ORF is not
disturbed.

In my hands, transfection with linear plasmid produced less colonies (less
cells surviving the transfection) but more cell lines that actually
expressed my gene. You could try transfection with linear plasmid first (be
sure you know where to cut the plasmid) and if unsuccesful, shift to using
circular plasmids.

-Tarja





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