Methods for Assessing the Molecular Mechanisms Controlling Gene Regulation
Regulation of gene expression is a complex process that can be controlled at several steps, including transcription, pre-mRNA splicing and export, mRNA stability, translation, protein modification, and protein half-life. Because transcriptional regulation often involves DNA-protein interactions, several techniques are used, including nuclear run-off assays, DNase I footprinting analysis, and mobility shift assays. Together these assays can determine transcriptional rates, as well as locate, identify, and characterize DNA-protein interactions. Functional analyses to assess the role of specific regulatory regions in gene regulation often requires the introduction of reporter genes under the control of regulatory elements being investigated into mammalian cells. This is often accomplished using transient transfections or, more recently, adenovirally mediated gene delivery. Adenovirus-mediated gene delivery is useful for cells that are difficult to transfect with conventional methods, such as hepatic stellate cells, and when close to 100% of transfection efficiency is needed. Posttranscriptional regulation is often involved in regulating gene expression and may involve mRNA stabilization or translational regulation. Together, these techniques can provide information about which step a particular gene is predominantly regulated. This chapter will detail common methodology used to assess molecular mechanisms involved in controlling gene regulation.
Key WordsGene expression gene regulation mRNA mRNA stability transcription DNase I footprinting nuclear run-off mobility shift assay adenovirus construction gene delivery transient transfection nuclear extract RNase protection assay hybridization actinomycin D reporter genes
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