Reporter Gene

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Caty784
Caty784's picture
Reporter Gene

Hi.
I know how the reporter gene works in a transfected cell. My question for anyone who would know is, WHY does it work?
From the time you make the gene construct (with a gene of interest for a protein of interest, say, ErbB2) and until the cell culture starts to expresses that on the plasma membrane of the individual cell, there are a million things that can go wrong, but usually don't, at least in a large percentage of the cells. Why is that? Why is the cell, first of all, allowing that new stretch of DNA, which is exogenous, to be transcribed into mRNA and then translated into the respective protein (say beta-Gal)? Any papers in the literature about this that anyone might know about?
Thank you very much!!!

Edward Dougherty
Edward Dougherty's picture
Why is this so strange?

Why is this so strange? Viruses routinely infect cells with new DNA/RNA that is ultimately translated into functional protein products.

Omai
Omai's picture
Yes but viruses encode part

Yes but viruses encode part of their own machinery to facilitate this process. I've often wondered why transfected plasmids are expressed, as well.

Omai

Jason King
Jason King's picture
Omai wrote:Yes but viruses

Omai wrote:

Yes but viruses encode part of their own machinery to facilitate this process. I've often wondered why transfected plasmids are expressed, as well.

Omai

Maybe because they use viral expression control elements (CMV promoters, SV40 polyadenylation sites, retroviral splice donor / acceptor sequences, WPRE etc etc etc...

Shubhangi
Shubhangi's picture
It's all in the construction

It's all in the construction of gene insert.
The plasmids carry specific promoters that are very strong in the sense they are powerful in carrying out their work which is initiation of transcription.
Also the construct carries special sequences that when on mRNA show high affinity for ribosomal binding. This in turn facilitates translation.
Lots of protein …… :-)
 

Ivan Delgado
Ivan Delgado's picture
Hi Caty,

Hi Caty,
Your question could be rephrased to say: why does molecular biology work? It is no small thing, which is why the discovery of one of the most famous reporter genes, GFP, was awarded the Nobel prize last year.
The main concept to consider, as Shubjangi pointed out, is the way in which you put together your construct. Essentially you are tricking the cell into thinking that it is expressing something that is normal. For example a lot of constructs use a constitutive promoter that has all the necessary characteristics to be recognized by the cell's machinery so it is used. It is almost like driving a car in the middle of San Francisco that happens to have the steering wheel on the right side. It is "completely" different from all the other cars, but as long as you know the rules of the road you can drive around without any problems (of course, assuming that there are no laws against doing this :) ).
As for your question about a cell allowing the expression of genes within itself: it depends on which cell type we are talking about. If you are a prokaryotic cell, then you have a mechanism to recognize these foreign transcripts and degrade them using restriction endonucleases. If you've expressed proteins in bacteria you are likely aware that there are commercial competent cells out there that have been engineered for protein expression. This was achieved by removing the machinery within these bacteria that recognizes foreign transcripts (and proteins) so they are not degraded. In eukaryotic cells, like human or mouse cells, since they come from a multicellular organism with its own immune system, these individual cells do not have the machinery to recognize foreign genes. As a result, for the most part you do not need to engineer these cells since you can express whatever you want in them and they will not be aware something foreign is present. You could say that eukaryotic cells, unlike wild type prokaryotic cells, are blind to being modified genetically. In many ways, this is the price eukaryotic cells pay for being part of a complex multicellular organism (one of the many reasons why viruses are able to infect us early on before our immune system kicks in).
Cheers

RLS
RLS's picture
 Great response, Ivan, with

 Great response, Ivan, with fantastic metaphors!