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Generating double stable cell line


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#1 kulla

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Posted 23 April 2009 - 07:37 AM

Hey,

I have a problem concerning my cell line. All DNA constructs, prior used with another cell line, use the neomycine resistance gene for generation of stable cell lines. My cell line was immortalized with pE1A-neo plasmid which means that this cell line is already neomycin resistant. Alternatives are e.g. puromycin or hygromycin etc. Who has experiences with selection alternatives to neomycine? Which plasmids are recommended for this attempt?

Thanks

#2 Bomber

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Posted 23 April 2009 - 08:35 AM

A plasmid we recently used is the pIRESpuro3 from Clontech.
It contains CMV promoter and allows the expression of your gene of interest in fusion with the selection marker (it is a mRNA fusion not a protein fusion).
Thereby you can reduce the screening effort a bit since resistent clones are likely to express your gene of interest as well.
I find puromycin selection also faster and more reliable.

Good luck

#3 bob1

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Posted 26 April 2009 - 05:57 PM

I have used G418, hygromycin, blasticidin and zeocin effectively. They all work well, but it is worth the extra time to do a cell death assay to choose the appropriate dose.

#4 kulla

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Posted 28 April 2009 - 07:12 AM

Thanks for answering so fast.




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