We have a gene with two isoforms, one with a longer 5' UTR and one with a shorter 5' UTR. It's been demonstrated that which isoform is predominant changes over development.
We want to study how the UTR might influence the translational efficiency of the transcripts. In the past, we tried to create a luciferase fused construct that had our UTR, our gene, and luciferase. However, the results were somewhat messy and hard to interpret and we questioned whether the luciferase construct itself was affecting translation.
Does anyone have any experience with alternative methods of studying 5' UTR's effect on translation, or have better experience with luciferase constructs?
I've worked with 5'UTR constructs (151 and 171bases long) for an invertase gene in plant systems. However, the one UTR which gave me a lot of trouble was 22b long. I had fused them upstream to GUS in a modified pCAMBIA1391z. I noticed a significant increase in reporter expression, which was traced back to the UTRs using functional assays later. The shorter the lengths of UTRs, the messier it is.
Hope it helps!
Don't use luciferase because too many variables affect the assay. Transfection efficiency, reaction times, substrate availability, etc. Just do western blots to examine protein levels directly. Control for transfection efficiency by using a second control plasmid, such as GFP.