ausblenden:
Schlagwörter:
cortex; cerebellum; microarrays; transcriptome; microdissection; GFP GENE-EXPRESSION ANALYSIS; CENTRAL-NERVOUS-SYSTEM; TRANSGENIC MICE; REGULATED GENES; BRAIN-FUNCTION; IN-SITU; MOUSE; MICROARRAYS; HYBRIDIZATION; GLYCOLYSIS
Zusammenfassung:
The enormous cellular complexity of the brain is a major obstacle for gene expression profiling of neurological disease models, because physiologically relevant changes of transcription in a specific neuronal subset are likely to be lost in the presence of other neurons and glia. We solved this problem in transgenic mice by labeling genetically defined cells with a nuclear variant of GFP. When combined with laser-directed microdissection, intact RNA from unfixed, freeze-dried sections can be isolated, which is a prerequisite for high-quality global transcriptome analysis. Here, we compared gene expression profiles between pyramidal motor neurons and pyramidal somatosensory neurons captured from layer V of the adult neocortex. One striking feature of motor neurons is the elevated expression of ribosomal genes and genes involved in ATP synthesis. This suggests a molecular adaptation of the upper motor neurons to longer axonal projections and higher electrical activity. These molecular signatures were not detected when cortical layers and microareas were analyzed in toto. Additionally, we used microarrays to determine the global mRNA expression profiles of microdissected Purkinje cells and cellularly complex cerebellar cortex microregions. In summary, our analysis shows that cellularly complex targets lead to averaged gene expression profiles that lack substantial amounts of cell type-specific information. Thus, cell type-restricted sampling strategies are mandatory in the CNS. The combined use of a genetic label with laser-microdissection offers an unbiased approach to map patterns of gene expression onto practically any cell type of the brain.
