Stirland J A, Seymour Z C, Windeatt S, Norris A J, Stanley P, Castro M G, Loudon A S I, White M R H, Davis J R E
School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK.
J Endocrinol. 2003 Jul;178(1):61-9. doi: 10.1677/joe.0.1780061.
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.
尽管利用发光成像分析荧光素酶活性为深入了解活组织中基因表达的动态调控提供了新的视角，但体外研究依赖于稳定转染的克隆细胞系，这限制了细胞类型和物种的选择，或者依赖于DNA显微注射，而这既费力又具有高度选择性。我们在此报告首次使用一种重组腺病毒，其中萤火虫荧光素酶报告基因受催乳素基因启动子调控，以研究启动子活性的时间动态。该载体用于感染垂体GH3细胞系以及叙利亚仓鼠垂体细胞的原代培养物。我们表明，腺病毒转导的细胞通过适当信号保留了启动子-报告转基因的正常调控。此外，显微成像研究表明，克隆垂体细胞和原代垂体细胞均被有效转导，长时间实时产生易于检测的发光信号。最后，单细胞表达模式分析表明，催乳素启动子活性具有高度动态性，基因表达存在脉冲，这表明在克隆细胞中观察到的转录不稳定性是正常垂体细胞的一个特征。腺病毒载体为在无法使用传统转基因和克隆细胞系的基因调控研究中提供了一种有价值的工具。
