Real-time analysis of preprotachykinin promoter activity in single cortical neurons.
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.
Walker
P D
PD
Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, USA.
Andrade
R
R
Quinn
J P
JP
Bannon
M J
MJ
D000818:Animals; D000831:Animals, Newborn; D002540:Cerebral Cortex / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009924:Organ Culture Techniques; D011188:Potassium / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015320:Tachykinins / Q000235:genetics*; D013997:Time Factors; D014158:Transcription, Genetic
D008164:Luminescent Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D015320:Tachykinins; C044206:preprotachykinin; D049452:Green Fluorescent Proteins; D005576:Colforsin; D011188:Potassium