Hello

Did you also find that the overexpression of the same gene in mouse (mouse gene not the human) have the same degenerative effect in the mice? if yes, then you report the function of that gene in the mouse, but tranfection of human gene to mouse may have many variable to check upon, thanks

Thanks! We haven't studied the mouse gene in vivo nor in vitro. There is no report of the same effect  of the mouse gene in mice. What's the problem in transfection of the human gene to mouse besides possible low expression and confounding factors to the disease model because it‘s xenogenic?

Hello, sorry but I'm not sure I get your question. Generally miRNAs function by down-regulating target genes expression. So, what gene would you like to over-express? A specific miRNA target gene (but why? the up-regulation of the miRNA wil down-regulate the target gene expression) or an indirectly up-regulated gene in consequence of the up-regulation of the miRNA in the disease?

Also, a cell infected with a lentiviral construct expressing the gene would not really be considered an "endogenous" expression... but maybe you meant something different.

Finally, if the human miRNA induces the same effect in the mouse as it is observed in human, it means the mechanism (besides the miRNA binding site, of course) is conserved between mouse and human, so the mouse gene is ok.

By the way, the 30% difference is at the mRNA or amino-acid level? Often 30% difference at the RNA level means very little difference at the amino-acid level due to the classical 3rd base variability in codon usage, which rarely alters the translation.

I am also confused by your question.   You mentioned that treatment with the miRNA led to injury/disease symptoms, which suggests that the disease is associated with DECREASED gene expression.  To model this, you would want to look into techniques for knocking out the target gene.  

Have you looked at protein expression for the target gene in your miRNA-treated mice?  If translation of the mouse target gene is regulated by the human miRNA, then you would expect to see decreased protein levels. 

Can you help me understand how your results led you to the conclusion that increased gene expression is associated with injury/symptoms of disease?  Hopefully this can help me or others to better answer your question.

Good luck!

Thank you for your questions! 'The gene' I mentioned above is referred to the DNA sequence which transcripts into the microRNA, not its target proteins. 

We found the miRNA increased in patients. Then we injected mimics of the human miRNA into mouse and mice showed the disease phenotype. By bioinformatic analysis we found several target genes of the miRNA.  The target genes are down regulated  on transcription  and translation levels in vitro. Now we want to see if a certain cell overexpress the miRNA  in mice can lead to the disease phenotype. But the miRNA gene sequence is different in mouse and human. So the problem is which miRNA to be overepressed in the cell, the human miRNA or the mouse homologous miRNA?

Ok, this is a lot clearer!

Now, one more point, the miRNA is only ca. 22 nt long, although it is transcribed in introns or cluster (mirtrons) as a pri-miRNA of a very variable length before all the Drosha and Dicer processing. So, when you mention the 30% difference between human and mouse, do you refer to the pri-miRNA or to the actual miRNA? If I get it right, you over-expressed the human miRNA in the mouse and it worked, in the sense that produced similar effects as in humans. So I imagine that the miRNA itself, at least in the seed region, is well conserved, but you are worried about the processing of the human pri-miRNA in the mouse. In any case if the miRNA is conserved, I would use the mouse gene to be sure the processing occurs. Or, why not just express the miRNA itself (or the pre-miRNA) in the viral construct? Then you could go for the human miRNA and show that over-production within the tissue is also having an effect (hopefully!).

Good luck!

 Thank you very much!  Your answer help us solve a key problem in our study.

Ohh!  That makes much more sense!  I'm sorry for any confusion!

I agree with Pietro that could express the human miRNA with the viral construct for this experiment, but it sounds like you may be at the beginning of a long series of experiments using this new model for the disease!  If this is true (which is very exciting!) then I think you should use the mouse gene and see if over-expression leads to disease symptoms.  If you see that function is conserved, it could make future experiments much more convenient (breeding mutant mice rather than treating them, and never worrying that cross-species processing issues are interfering with your results).  Also, if you see that function is not conserved when you use the mouse gene, then you could get valuable information about the mechanism by evaluating the specific differences between the species.

I do not know the details of your research, so your situation may be different--but these thoughts came to mind and I figured it couldn't hurt to post them for you to consider.  Good luck with everything!

the human miRNA 

Thanks a lot!  We are preparing to research in a new field to our institute which is really exciting. I'm lucky to have your illuminating answers and grateful for your kindly help.