title: Real-time imaging of gene promoter activity using an adenoviral reporter construct demonstrates transcriptional dynamics in normal anterior pituitary cells.
abstract: Although analysis of luciferase activity using luminescence imaging has provided new insights into the dynamic regulation of gene expression in living tIssues, studies in vitro have relied on stably transfected clonal cell lines, limiting the choice of cell type and species, or DNA microinjection, which is arduous and highly selective. We report here the first use of a recombinant adenovirus in which the firefly luciferase reporter gene was regulated by the prolactin gene promoter, to study temporal dynamics of promoter activity. This vector was used to infect the pituitary GH3 cell line, and also primary cultures of Syrian hamster pituitary cells. We show that adenovirally transduced cells retained normal regulation of the promoter-reporter transgene by appropriate signals. Furthermore, microscopic imaging studies indicated that both clonal and primary pituitary cells were transduced efficiently, giving readily detectable luminescence signals in real-time over long periods. Finally, analysis of single-cell expression patterns indicated that prolactin promoter activity was highly dynamic with pulses in gene expression, revealing that the transcriptional instability seen in clonal cells is a feature of normal pituitary cells. Adenoviral vectors offer a valuable tool for studies of gene regulation where conventional transgenesis and clonal cell lines are not available.
authors.affiliation: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.
authors.forename: J A
authors.initials: JA
authors.lastname: Stirland
authors.forename: Z C
authors.initials: ZC
authors.lastname: Seymour
authors.forename: S
authors.initials: S
authors.lastname: Windeatt
authors.forename: A J
authors.initials: AJ
authors.lastname: Norris
authors.forename: P
authors.initials: P
authors.lastname: Stanley
authors.forename: M G
authors.initials: MG
authors.lastname: Castro
authors.forename: A S I
authors.initials: AS
authors.lastname: Loudon
authors.forename: M R H
authors.initials: MR
authors.lastname: White
authors.forename: J R E
authors.initials: JR
authors.lastname: Davis
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000293:Adolescent; D000704:Analysis of Variance; D002478:Cells, Cultured; D002999:Clone Cells; D005576:Colforsin / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors / Q000008:administration & dosage; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008163:Luminescent Measurements; D008854:Microscopy, Electron; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011388:Prolactin / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D019076:Transgenes
chemical_list: D005576:Colforsin; D011388:Prolactin; D008156:Luciferases