transfection

Peter Cherepanov peter.cherepanov at uz.kuleuven.ac.be
Fri Apr 26 17:23:28 EST 2002


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

enlightening indeed ...
at hotmail.com may be,
in real life things are more complicated

there are different things to consider, one for example is it worth at all
to respond to an anonimous letter and the rest of them are listed here:

1. linerized DNA is more recombinative then circular - fact (dont they do
gene nock-outs with linear DNA?).

2. over-expression of any gene during transfection leads to cell death,
linear DNA gives lower expression levels - fact
that gives you two effects - lower transient expression of the selected gene
and lower expression of your gene at the start.

3. linear DNA will probably polymerize (NHEJ) leading to possibly higher
copy number - I think

4. method of transfection and the cell line - how many DNA molecules you
introduce into your cell (level of initial expression and therefore cell
killing).

5. kind of plasmid - I heard people could poduce stable cell lines based on
COS7 (expresses SV40 large T antigen) transfecting them with linear DNA that
contained SV40 promoter/ori  - can someone confirm that?? I would very much
like to know if it is true.

6. luck or lack of luck - unfortunate fact

generally, I think the consensus is that it is better to linearize your
expression construct, I am sure with good spirits one can do it anyway he or
she wants and produce a great clone.

BTW, the subject has been discussed here before (check those links and
follow the responses).

http://bionet.hgmp.mrc.ac.uk/hypermail/methods/methods.200111/0180.html
http://bionet.hgmp.mrc.ac.uk/hypermail/methods/methods.199602/1043.html
http://bionet.hgmp.mrc.ac.uk/hypermail/methods/methods.199411/0973.html

okay, that is getting really late, my cells are ready and I must be off

good luck with your stuff,

Peter

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