Month: March 2012

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. 

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Combinatorial Activation and Repression by Seven Transcription Factors Specify Drosophila Odorant Receptor Expression

This paper (summarizing many years of work) describes the combinatorial code that controls the expression of olfactory receptor proteins in the fly sensory system.  It is unknown at present how much OR expression patterns  differ between species.  But if they do, this code would suggest a clear mechanism for their evolution.

Two papers in Nature Biotechnology describe massively parallel functional dissection of mammalian enhancers:

Massively parallel functional dissection of mammalian enhancers in vivo

Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay

These papers translate the good old idea of systematic structure-function analysis from proteins to regulatory sequences.  The ultimate goal is to characterize the functional significance of every nucleotide position (and, ideally, their interactions) within a cis-regulatory element.  Two make this happen, you need two things: the ability to generate hundreds of thousands of synthetic sequences, and the ability to analyze the output of all these sequences – and you need to accomplish both of these things in an automated, high-throughput way.  That’s exactly what these folks did, in somewhat different ways.  These tricks would not work for complex developmentally regulated enhancers, but it’s a good start.

Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks