In vitro transcription to study WT1 function

Abstract

In vitro transcription methods using mammalian nuclear extracts have been available for over 30 years and have allowed sophisticated biochemical analyses of the transcription process. This method has been extensively used to study the basic mechanisms of transcription, allowing the identifi cation of the general transcription factors and elucidation of their mechanisms of action. Gene-specifi c transcriptional regulators have also been studied using in vitro transcription. This has facilitated the identifi cation of their cofactors and provided information on their function that is invaluable to facilitate their study in a more physiological setting. Here we describe the application of in vitro transcription methods to study the mechanism of action of WT1. Coupling transcription assays with methods to purify transcription complexes, and protein affi nity chromatography, has provided insights into how WT1 can both positively and negatively regulate transcription.