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Real‐Time Analysis of Preprotachykinin Promoter Activity in Single Cortical Neurons
Author(s) -
Walker Paul D,
Andrade Rodrigo,
Quinn John P,
Ban Michael J
Publication year - 2000
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2000.0750882.x
Subject(s) - green fluorescent protein , gene expression , biology , microbiology and biotechnology , confocal microscopy , fluorescence microscope , transfection , fluorescence , promoter , gene , endogeny , confocal , biochemistry , physics , quantum mechanics , geometry , mathematics
Technological limitations have hindered the study of gene elements regulating transcription within CNS neurons. In the present stuides, rat cortical brain slices endogenously expressing the preprotachykinin (PPT) gene were transfected with gene constructs encompassing green fluorescent protein (GFP) under the control of the PPT promoter. These slices were maintained in organotypic culture so that the fluorescence intensity within individual living cells could be quantified using laser scanning confocal microscopy before and after application of stimulatory agents. Combined treatment with forskolin and elevated potassium significantly increased expression of both endogenous PPT mRNA and the PPT promoter‐GFP construct. The ability to follow fluorescence changes within single neurons in real time offers a powerful “within‐subject” experimental approach for analysis of neural gene promoters.