Overexpression of mouse constructs in human cell lines (View forum version)



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Posted 23 March 2011 - 09:04 PM

Dear colleagues,

I have a question regarding the overexpression studies. We have identified a new de novo mutation in a human and would like to perform some functional analysis using cell lines. We think that it's better to use human cell lines since the mutation observed in humans, however, we would like to overexpress mouse construct tagged with GFP or whatever else ... (one of the reasons is that we don't have suitable human cDNA to clone the construct). So the question is would it be the case for reviewers to pay attention on this fact? How reasonable to overexpress proteins from other species?

I saw quite a lot of papers where people did it in this way and since they got published they apparently had no so many difficulties...

The homology between human and mouse proteins more than 90%, the protein indirectly regulates calcium signalling.

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Posted 24 March 2011 - 05:59 PM

Hey cloning Guru where are you :)

Rsm

Posted 25 March 2011 - 12:00 AM

You don't have a roblem with cloning, but with review, it seems... If I were the reviewer, I would want to ask if this particular residue is highly conserved (otherwise it makes no sense) and if there is really no cDNA available (for example from IMAGE or RIKEN). But I think it should be fine to use mouse cDNA on human cells (if this residue is conserved). Does your cell line express the endogeneous protein?

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Posted 27 March 2011 - 05:55 PM

thanks for reply Post Dog.
You are right, we can buy human cDNA basicaly it's just a matter of money...
Human cell line which we use express its own endogeneous protein. I thought of transfecting the cell line with siRNA to reduce the competition with our overexpressed protein that would also an explanation for reveiwers why we use protein from another species (to supress only endogenous protein but not the one we overexpress). What do you say is a right direction to follow?
P.S. protein is highly conserved.

Rsm

Posted 27 March 2011 - 11:18 PM

thanks for reply Post Dog.
You are right, we can buy human cDNA basicaly it's just a matter of money...
Human cell line which we use express its own endogeneous protein. I thought of transfecting the cell line with siRNA to reduce the competition with our overexpressed protein that would also an explanation for reveiwers why we use protein from another species (to supress only endogenous protein but not the one we overexpress). What do you say is a right direction to follow?
P.S. protein is highly conserved.


Yes, the old money issue <_<
Maybe you can find someone who has the full length ORF in some plasmid, and is willing to provide you with? Most people are, if you acknowledge them (and cite).
If your cell line expresses this protein, then you should knock it down first, to have a clearer effect of the mutation. So your approach is quite good.
How about conservation of this particular residue (of your point mutation)?

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Posted 28 March 2011 - 10:10 PM

Maybe you can find someone who has the full length ORF in some plasmid, and is willing to provide you with? Most people are, if you acknowledge them (and cite).


I am from Australia. It costs about 1500 dollars to get a shipment on dry ice from US, so that's a bit complicated.

If your cell line expresses this protein, then you should knock it down first, to have a clearer effect of the mutation. So your approach is quite good.

In a case if I want to check collocalisation of my overexpressed protein tagged by GFP should I knockdown endogenous protein? or it is not neccecary since the amount of overexpressed protein is usually 5-10 times higher? does it make sence to reduce amount of overexpressed protein in order to make it more comparable with endogenous one?

How about conservation of this particular residue (of your point mutation)?

good point, I have to check :)

Rsm

Posted 28 March 2011 - 10:44 PM

A plasmid on Whatman paper in a standard letter costs $1.50 from US to Australia, so it's not very complicated.
If I were you, I would start with simple overexpression of the mutant protein. If you have some phenotype, good for you, if not you can still knock down the endogenous protein. How do you want to control levels of overexpression?