Cell type-selective expression of green fluorescent protein and the calcium indicating protein, yellow cameleon, in rat cortical primary cultures.

摘要: A cell type-specific green fluorescent protein (GFP) expression system in rat cortical primary cultures has been developed for the fluorescence labeling of brain cells. Lipid-mediated transfection (lipofection) was employed, allowing establishment a convenient efficient analysis individual To achieve labeling, GFP vectors containing neuron-specific enolase (NSE) gene promoter, human glial fibril acidic (GFAP) elongation factor (EF-1alpha) or cytomegalovirus (CMV) immediate early promoter were constructed, and their specificities examined. Vectors CMV GFAP resulted primarily astrocytes, while those EF1-alpha NSE neurons. This applied to morphological living neurons type-selective calcium imaging. Confocal microscopy revealed that GFP-expressing had processes, which longer than 500 microm bore spine-like protrusions. calcium-indicating variant, yellow cameleon (YC2.1), expressed same system, imaging performed. On pharmacological stimulation, YC2.1-expressing responded depolarizing stimuli, but not metabotropic glutamate receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD), astrocytes only tACPD.