Overexpressing miR - rescue by co-overexpressing its target - (Mar/31/2013 )

Hi,

I've written a hypothetical research proposal for an exam and will be giving an oral presentation of my proposal in a week. I am preparing my presentation now and having trouble with planning my transfection.

I am looking at a miRNA in glioblastoma cells. I want to overexpress the miRNA (and a scramble ctrl) to investigate the effect on the cells' invasive, tumorigenic and angiogenic abilities in vitro and in vivo.

I would also like to overexpress the putative target mRNA of the miRNA to see to what extent this will rescue the effect of the miRNA.

So I am debating how to best design the DNA constructs and stably transfect the cells so I can investigate this in vivo.

The miRNA is 22 bp, the full-length protein is ca. 1800 bp. I was looking at OriGene plasmids to generate the following lines:

scr
miRNA
miRNA-target gene

What would be the best way to design the constructs and what would be a good way to stably transfect it into glioblastoma cells for xenografting. I was thinking to put miRNA-target gene under a CMV promoter and insert and IRES or a splice site between the two transcripts.

As a final complication, it would be very nice to have also a Luciferase expressing system so I can do non-invasive imaging of the growth of the tumors inside the mice. So I was thinking to have a separate plasmid with Luc and a plasmid with scr, miRNA or miRNA-target gene.

I am working in the lab, but have never performed transfection of mammalian cells myself, so I don't have the hands-on experience to say what till work. I would be very grateful for any feedback.

For the rescue experiment, you don't need to put the miR and the protein coding gene in the same vector. they should be in separate vectors and the protein coding gene to be cloned should not have its 3'UTR.

To do the rescue experiment, you need to use a control vector (such as empty vector) to control for the vector carrying the protein coding gene.

If you want to track cells in vivo, you can use an existing GFP or luc stable line to do all the miRNA and gene overexpressing vector transfection.