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Good stable mammalian promoter


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#1 Doda

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Posted 11 May 2010 - 12:08 AM

Hi, everyone!
I'm quite new to this field of cloning, and from what I've been reading, like any other field, there is a bunch of information of whatever one's looking for.
My question is, has anyone know, or at least has some references that I can look for, about good stable promoter(s) for mammalian cells? It is, I am to transfect a reporter gene (let's say, GFP) into mouse ES cells, generate viable germline chimaeras which have this reporter gene activated, and from what I know, transgenes are generally silenced after a while.
Any help is more than welcome, please!

#2 Rsm

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Posted 11 May 2010 - 06:53 AM

Do you want to do a knock-in or a transgene?

Expression from transgenic insertions depends on integration site effects, but is not generally silenced after a while. I have worked with CAG-CAT-eGFP mouse, which are GFP positive since more than 10 years... The CAG promoter is a very strong artificial promoter, by the way (Kawamoto, FEBS Lett, 2000).

A promoter for knock-in, especially if you're looking for constitutive ubiquitous expression, is the Rosa26 promoter (Zambrowicz, PNAS, 1997). This is an endogenous but rather weak promoter with a very good recombination efficiency (normally around 10-30%).

There are also vectors around where you can combine the strong CAG promoter with the nice targeting efficiency of Rosa26.

BTW, if you'll get germline chimeras does not depend on your promoter, but rather on your skills and lots of luck...


Cheers,

Minna
I got soul, but I'm not a soldier




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