transfection

Peter Cherepanov peter.cherepanov at uz.kuleuven.ac.be
Wed Apr 24 05:32:57 EST 2002


Hi,

as far as I know, IF the purpose of your transfection is to obtain a stable
cell line (through integration of the transfected DNA into chromosome) then
your chances are much higher if you use linearized plasmid (chose an enzyme
that cuts your vector somewhere in the non-essential part, like in the ApR
gene - often ScaI site is good for it). That is explained by the fact that
dsDNA breaks are much more efficient in recombination then intact circular
DNA (they activate NHEJ pathway and immediately are joined to anything else
that has free ds end, often to another molecule of your plasmid and to the
chromosomal DNA). Another good thing is that you control the way of
integration (site were you plasmid is opened and joined to the chromosome).

I heard some people linearize plasmid even for transient transfections - if
you want to reduce transient expression levels (can be usefull if you see
your protein overexpressed and agregated). I did not try it for that purpose
yet.

As for the promoter - you do not like SV40 promoter because it also contains
SV40 ori? (the only reason I know of why this promoter not good sometimes,
and only for the host cell lines expressing SV40 large T antigen).

CMV is a very strong promoter, I would not use it to express the neoR gene
from it, it will take ages before transient expression will die out and real
selection start. There are nice IRES-containing vectors with neoR, I used
pLX-IN (Clontech). Advantage is that you can use it as such or as a
retroviral vecotor if you want. Disadvantage is that its MCS is quite
primitive (but there must be other vectors available).

good luck,
Peter




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