Reporter genes and eukaryotes? Why are they transcribed?

[Genecks]

I'm reading about embryonic development and tagging organisms with "reporter genes."

 

What I don't understand is why the eukaryotic organism would transcribe the reporter gene and the gene of interest at the same time. Supposedly, these can be hooked up somehow? Wouldn't that make the two genes like an operon? But eukaryotes don't have operons, right?

 

I don't understand.

[CharonY]

Several ways to do it. One is to insert the reporter gene in-frame, thus creating a hybrid protein.

[Genecks]

Yes, I was reading about this "hybrid" protein, during which a gene, such as GFP, does not cause the entire protein to lose functionality when the GFP becomes part of the protein. I'm guessing it could, but somehow scientists figured out how to do it. The mechanics behind it are unknown to me.

 

source: http://www.wormbook.org/chapters/www_reportergenefusions/reportergenefusions.html

 

 

 

But isn't another way to put the protein in before the gene of interest? Doesn't that make the system like an operon?

And what you're suggesting is a "translational reporter," right?

 

This is probably a better version of my first post's question: Why is GFP occurring as a transcriptional reporter?

Why is RNA polymerase II punching out both genes?

Edited January 31, 2010 by Genecks

[CharonY]

It isn't. There are no terminators therefore for transcriptional purposes it is a single gene. You may be confused with some of the nomenclature here. Each translational reporter is, in essence also a transcriptional reporter (but not vice versa).

 

Regarding fusion proteins, sometimes the GFP interferes with enzyme functions. One can play a couple of tricks but some fusions do not work well.

Edited January 31, 2010 by CharonY

[BrainWasher]

It's all about the vector. If it is a plasmid it wll depend on the instructions on the plasmid. As soon as it is in the cell it will start processing the plasmid.

[CharonY]

Uhm nope. Wrong application. The OP is talking about embryonic development.

[BrainWasher]

Char - So your saying the gene is now on a chromosome?

 

I'm just now looking at the page Geneck is referencing. My fault...

 

I see it's talking about it being a transcription reportor setup. I thought it was more about the novelty of making an organism fluoresce by introducing a plasmid.

 

It looks like the vector kit will answer the question.

http://www.addgene.org/docs/fire/andrew/datasheets.pdf

 

I think they (http://firelab.stanford.edu/) would not develop the vector kits if it would physiologically affect the target gene as this would change the expression and feedback mechanisms. I don't know... This is my opinion. But, the PDF file is really nice to reference to determine the genetic composition of the vectors. I'm guessing these would add a domain to the various proteins/genes.

 

http://www.addgene.org/pgvec1?f=c&cmd=showcol&colid=1

 

These Fire Vectors are realy nice! Worth looking into....

Edited February 2, 2010 by BrainWasher

[Mr Skeptic]

Wouldn't just putting the marker gene with a copy of the transcription factor of the gene of interest, somewhere in the genome, work?

[CharonY]

WB, there is always a underlying risk that the manipulation may affect physiology, regardless how you do it. It is just an accepted fact.

 

Mr. Skeptic, I cannot see how it would work and what it is supposed to accomplish. Maybe you may have misunderstood what a transcription factor is.

[Mr Skeptic]

Right, I meant the promoter sequence.

[CharonY]

That makes sense. Yes, you would have a transcriptional reporter system then. Two disadvantages: one is that you got two promoters in your system now, that may interfere with the regulation cascade, if it is tightly controlled.

Second that it is obviously only useful for transcriptional analyses. You lose information about other regulatory events as well as localization, etc.