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Plasmid Vector - CMV enhancer / promoter fluorescent reporter, a positive or neg

vector fluorescent positive control CMV promoter enhancer transfection

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#1 ImposterSyndrome

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Posted 03 September 2013 - 04:12 AM

We have a technique where we can transfect primary human cells using a plasmid vector construct that contains a fluorescent reporter driven by our promoter sequence insert. The sequence is inserted between the CMV reporter/promoter and MCS. In parallel I aim to transfect primary cells using the original unadulterated vector as a control. Now the question is - will the fluorescent reporter signal in this control be ubiquitous whereas signal from our construct will be specific, or will it be instead that this control will have no signal at all? As these are primary cells, I have no idea whether many of us are casually carrying endogenous (viral?) proteins that will activate the fluorescent reporter via the CMV promoter. Or whether or not I have completely missed the point altogether (i'm new to this).    

 

 

 

 

 

 

 

 



#2 bob1

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Posted 03 September 2013 - 12:34 PM

Viral promoters like CMV tend to work well in mammalian cells as the viruses use these to run the genes that allow them to replicate themselves.  There is no need for transformed or altered cells for this to work (think about viral infection of yourself - it still works).

 

If you have both promoters in the plasmid, how do you know which one is working?



#3 ImposterSyndrome

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Posted 04 September 2013 - 01:37 AM

Thanks for your reply,

 

In the construct, when I say 'between' I meant the use of cloning sites within the MCS and CMV promoter, I assume that the insert at these cloning sites would render the CMV promoter inert (?).  



#4 bob1

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Posted 04 September 2013 - 03:13 PM

Only if that insert disrupts the promoter sequence.







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