Stringent Analysis of Gene Function and Protein–Protein Interactions Using Fluorescently Tagged Genes
Another invention based on the MiMic gene trap system. From the abstract:
GFP traps have mainly been used to study the endogenous expression patterns of trapped genes or the subcellular localization of their protein products. Here, we show that the GFP tag can also be used to interfere with gene function by RNAi-mediated knockdown of the tagged transcripts. This method, which we refer to as “tag-mediated loss-of-function,” addresses major shortcomings of the classical RNAi approach in which gene-specific sequences are targeted.
The basic idea is that you modify your favorite gene in vivo so it becomes tagged with GFP, and then direct your RNAi targeting constructs against the GFP tag rather than the protein itself. The advantage is that you can test several different but “universal” shRNA constructs, then find out which ones are the strongest and use those to target different genes. It is also easier to detect and circumvent off-target effects, and it also becomes possible to perform sort-of direct comparisons between the phenotypes of different genes.
If your favorite genes have MiMic alleles, why not compare this new technique with traditional RNAi? Does anyone want to give this a try?