Neuronal Promoter Analysis by in Vitro Transcription Using Nuclear Extracts from Rat Brain
We present a reliable method for making nuclear extracts from rat brain and for using these extracts in an in vitro transcription assay. Our nuclear extract technique allows for the isolation of nuclear proteins from post mitotic neurons that presumably contain all of the factors required for the transcriptional regulation of neuron-specific genes. The use of brain as a source of nuclear extract is in contrast to the more commonly used cultured cell lines in which expression of neuron-specific genes may be poor or lacking in tissue specificity. In vitro transcription using nuclear extracts from expressing (brain) and nonexpressing (liver) tissues provides a direct assessment of promoter strength and level of tissue specificity apart from the effects of higher order DNA structure (i.e., chromatin). In vitro transcription is also amenable to DNA competitor studies and to the addition of purified or partially purified protein factors and antibodies. In this way, true neuron-specific transcriptional activator or repressor sequences or factors can be characterized. The approach is rapid, reliable, and easily quantifiable. Finally, advancements in the field of nucleosome reconstitution have allowed the coupling of in vitro transcription to nucleosome assembly systems and provide a novel and promising approach to the understanding of neuronal-specific gene expression.
Schwartz
ML
Division of Neuropathology, University of Pennsylvania Medical School, Philadelphia, Pennsylvania, 19104
Schlaepfer
WW