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Transfection Expression Level Issues


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#1 glenk

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Posted 23 June 2009 - 12:26 PM

Hi All,
This is my first post here, but I've checked through the archives and this seems like the place to ask about such an obscure problem as this.

We have an experiment in which the expression level of a cDNA construct is critical, too much or too little not being good. Our construct is in a highly modified pGEM3Z, which usually gives us a whopping expression, even in the MEFs that we're stuck using. However, in this case that's not what we want.

The problem is that we need a more moderate, maybe even low level of expression in mammalian cells. Does anyone know of any plasmid that would be of use for this? Everything I find seems to use the CMV or actin promoter, both of which I feel are going to give too high of expression.

I must admit that I'm a bit of a novice at all this cell-culture expression stuff, so pardon my ignorance if the answer is obvious to all but me. :)

Any help would be greatly appreciated.


Thanks,
Guy

#2 cellcounter

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Posted 26 June 2009 - 08:29 AM

Hi All,
This is my first post here, but I've checked through the archives and this seems like the place to ask about such an obscure problem as this.

We have an experiment in which the expression level of a cDNA construct is critical, too much or too little not being good. Our construct is in a highly modified pGEM3Z, which usually gives us a whopping expression, even in the MEFs that we're stuck using. However, in this case that's not what we want.

The problem is that we need a more moderate, maybe even low level of expression in mammalian cells. Does anyone know of any plasmid that would be of use for this? Everything I find seems to use the CMV or actin promoter, both of which I feel are going to give too high of expression.

I must admit that I'm a bit of a novice at all this cell-culture expression stuff, so pardon my ignorance if the answer is obvious to all but me. :huh:

Any help would be greatly appreciated.


Thanks,
Guy


1. Stable cell lines, in general have a lower expression than transients, so if it helps, you may want to make stable clones/pool.

2. Different cell lines give different expression levels with different promoters, so rather than assuming, you may want to try out several promoters.

3. And the easy one, just transfect your cells with limiting amount of plasmid DNA, that would result in less number of plasmids transfected per cell, and you will get lower expression levels. This however may result in spotty transfection (some cells getting huge amount of plasmids, some none).

#3 KevinK

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Posted 26 June 2009 - 10:06 AM

Guy,
I'm surprised this doesn't get asked more frequently actually, as expression of popular proteins like GFP with a strong CMV promter can cause all sorts of biological issues. At Promega, we actually just released a series of CMV deletions to help moderate the expresion level of a reporter protein called HaloTag. You may be able to look at our sequences and use one of the deletions to get an appropriate expression level in your system. Here is a link with an article describing the properties of these promoters: http://www.promega.c...16/16620_16.pdf.

If you have any questions, feel free to post here or you can always contact Promega Technical Services.

Regards,
Kevin

Promega Corporation
Promega Corporation
Madison, WI

#4 genehunter

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Posted 29 June 2009 - 09:17 AM

RSV promoter is 2-3 fold and SV40 promoter is several fold to 10-fold less active than CMV.

#5 KevinK

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Posted 30 June 2009 - 06:28 AM

Genehunter,
While that may generally be true, this is also VERY cell line specific. We have tested cell ines where SV40 is equal to or greater than CMV. It all depends on which transcription factors are in the promoter and expressed by the cell.

Another idea: If you truly need to moderate your expression level an inducible system may be the best option.


Kevin

Promega Corporation
Promega Corporation
Madison, WI

#6 genehunter

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Posted 30 June 2009 - 07:31 AM

Forgot about beta-actin promoter which is much weaker.

Kevin, you are right. I have seen that in host cells that are transformed with SV40 T and in particular when transfection is done with with less DNA.

#7 glenk

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Posted 02 July 2009 - 12:01 PM

Thanks all! I Will try to lower the expression level with DNA amount transfected, and after I think I'll try the halo-taged CMV deletion line of plasmids.

Thanks for all the ideas!

-Guy




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