choice of stable mammalian expression/knock-in

Ozan Aygun via methods%40net.bio.net (by metugenetics from yahoo.com)
Sat Aug 4 13:21:33 EST 2007


Hi,

I have been using one of the IRES systems of clontech
for some time an successfully generated a lot of
stable cell lines with these vectors.

I have to say the success depends very much on initial
transfection efficiency. If your protein is not very
large (i.e 100kDa <) you are unlikely to get any false
positives with a puromycin concentration of 1-3 ug/ml
(needs to be optimized for your cell line).

I strongly reccomend IRESpuro system if your protein
is not very large and expressed well in transient
transfections. You are very likely to get a lot of
stable clones after 3-4 weeks of transfection. Then
you can test the expression levels of individual
clones since this greatly varies among them.

 You can also generate your own gateway vector with
one of the IRES vectors. I strongly reccomend to use
puromycin marker as to our experience it is the best
drug to give zero background in very low doses.

I hope this may be any help, good luck !

Ozan.


       
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