Much of what we know about gene interactions in development has been learned using nematodes, yeast, flies, and bacteria. This is due, in part, to the relative ease of genetic manipulation of these well-characterized genomes. However, of great interest are gene interactions involving complex diseases in humans. Wang and White [(2011). Nature Methods 8(4):341–346] describe work using RNAi to examine the interactive proteome in mammalian cells. They mention that knockdown inefficiencies and off-target effects of introduced RNAi species are areas that need particular improvement if the methodology is to be fruitful.
How might one use RNAi to study developmental pathways?

Using the components in the accompanying diagram, design regulatory modules (i.e., enhancer/silencer modules) required for 'your' gene to be expressed only in differentiating (early) and differentiated (late) liver cells. Answer the three questions presented below by describing the roles that activators, enhancers, repressors, silencers, pioneer factors, insulators, chromatin remodeling complexes, and chromatin readers, writers, and erasers will play in the regulation of expression of your gene, that is, what factors will bind and be active in each case? Specify which transcription factors need to be pioneer factors. How will the gene be activated in the proper cell type?

The majority of this chapter focused on gene regulation at the transcriptional level, but the quantity of functional protein product in a cell can be regulated in many other ways as well. Discuss possible reasons why transcriptional regulation or posttranscriptional regulation may have evolved for different types of genes.

What role do ubiquitin ligases play in the regulation of gene expression?

Much of what we know about gene interactions in development has been learned using nematodes, yeast, flies, and bacteria. This is due, in part, to the relative ease of genetic manipulation of these well-characterized genomes. However, of great interest are gene interactions involving complex diseases in humans. Wang and White [(2011). Nature Methods 8(4):341–346] describe work using RNAi to examine the interactive proteome in mammalian cells. They mention that knockdown inefficiencies and off-target effects of introduced RNAi species are areas that need particular improvement if the methodology is to be fruitful.

Comment on how 'knockdown inefficiencies' and 'off-target effects' would influence the interpretation of results.

We discussed several specific cis-elements in mRNAs that regulate splicing, stability, decay, localization, and translation. However, it is likely that many other uncharacterized cis-elements exist. One way in which they may be characterized is through the use of a reporter gene such as the gene encoding the green fluorescent protein (GFP) from jellyfish. GFP emits green fluorescence when excited by blue light. Explain how one might be able to devise an assay to test for the effect of various cis-elements on posttranscriptional gene regulation using cells that transcribe a GFP mRNA with genetically inserted cis-elements.

Incorrectly spliced RNAs often lead to human pathologies. Scientists have examined cancer cells for splice-specific changes and found that many of the changes disrupt tumor-suppressor gene function [Xu and Lee (2003). Nucl. Acids Res. 31:5635–5643]. In general, what would be the effects of splicing changes on these RNAs and the function of tumor-suppressor gene function? How might loss of splicing specificity be associated with cancer?

Mutations in the low-density lipoprotein receptor (LDLR) gene are a primary cause of familial hypercholesterolemia. One such mutation is a SNP in exon 12 of the LDLR. In premenopausal women, but not in men or postmenopausal women, this SNP leads to skipping of exon 12 and production of a truncated nonfunctional protein. It is hypothesized that this SNP compromises a splice enhancer [Zhu et al. (2007). Hum Mol Genet. 16:1765–1772]. What are some possible ways in which this SNP can lead to this defect, but only in premenopausal women?