transfection

delios97 at hotmail.com delios97 at hotmail.com
Wed Apr 24 20:40:32 EST 2002


Wrong, there is no statistical significance between generation of
stable cells  using Linearized vs. Plasmid...OLD WIVES TALE


On 24 Apr 2002 11:32:57 +0100, peter.cherepanov at uz.kuleuven.ac.be
("Peter Cherepanov") wrote:

>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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