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 promoterreporter 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.

Dynamic organisation of prolactin gene expression in living pituitary tissue

“…Activity of both the prolactin and GH gene promoters, as measured by low light luminescence imaging of luciferase reporter gene expression, has been shown to exhibit dramatic dynamic heterogeneity in both cell lines (Friedrichsen et al, 2006;McFerran et al, 2001;Norris et al, 2003;Stirland et al, 2003;Takasuka et al, 1998) and primary pituitary cells (Shorte et al, 2002). In each case studied so far, large-amplitude, non-co-ordinated and non-circadian pulses have been observed in promoter activity, resulting in marked heterogeneity of gene expression at any single time-point, with the pulsing lasting for many hours in vitro.…”

An adenoviral vector for probing promoter activity in primary immune cells

“…rAdV containing DNA regulatory regions upstream of a luciferase reporter have been used to monitor gene expression in cardiomyocytes, primary pituitary cells, and salivary gland epithelial cells (Zheng et al, 2001;Stirland et al, 2003;Wilkins et al, 2004). To determine whether such rAdV would be useful for analyzing gene expression in immune cells, we developed a rAdV that is based on the pAdEasy system (Stratagene) (He et al, 1998).…”

“…Recently, several groups have also used rAdV as a tool to probe gene promoter activity in different types of non-immune cells (Zheng et al, 2001;Stirland et al, 2003;Wilkins et al, 2004), suggesting that rAdV could also readily be used to probe promoter activity in primary immune cells. We thus developed a novel adenoviral vector, pShuttle-luciferase-GFP (pSLUG), that allows for the generation of rAdV that are highly efficient transducers of multiple primary immune cell types.…”

Pulsatile patterns of pituitary hormone gene expression change during development

“…Quantification of PRL gene expression in real-time in pituitary lactotroph cells previously detected pulsatile transcription activity in cell lines (Takasuka et al, 1998;McFerran et al, 2001) and isolated primary cells (Stirland et al, 2003). To assess the transcription dynamics of the PRL gene within individual cells in intact tissue, and the level of cell-to-cell coordination, transgenic F344 rats with firefly luciferase or destabilised EGFP reporter gene expression controlled by more than 155 kbp of the human PRL gene locus were generated (Semprini et al, 2009).…”