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Medium used after transient transfection?


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#1 Ije

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Posted 01 April 2011 - 11:15 AM

Hello everybody,

After performing a transient transfection, would it make a difference if I change the transfection medium with reduced serum medium (0.1% FBS) rather than medium with 10% FBS ? Would this affect the expression of my transgene? I would like to incubate my transfected cells in reduced serum, stimulate with my growth factor and then harvest for WB analysis. I'm considering the 24-72h window period, but I don't know if my transgene can still be expressed in reduced serum/serum free conditions.

Any advice/comments would be greatly appreciated.

Thanks.

#2 Rsm

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Posted 02 April 2011 - 11:25 AM

0.1% serum induces severe changes in cellular activity, for example reduction of most mRNA transcription. See the excellent paper by Coller et al (http://www.plosbiolo...al.pbio.0040083). So your gene may not be expressed at all, depending on the promoter.
I would rather suggest to express your gene normally, and then 1) use a defined serum without your growth factor, 2) incubate your cells 2 days at normal condition, then make a serum starvation for 4hrs before feeding your growth factor or 3) don't give a damn about the levels of growth factor in the serum, but feed some extra growth factor anyway. That would be what most people do. I routinely do option 2), but I'm into immediate-early response genes...

Edited by Rsm, 02 April 2011 - 11:25 AM.

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