Scottish Fish Immunology Research Centre, Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, AB24 2TZ Scotland, UK.
Fish Shellfish Immunol. 2010 Feb;28(2):312-9. doi: 10.1016/j.fsi.2009.11.010. Epub 2009 Nov 14.
An interferon (IFN)-gamma responsive stable cell line RTG-3F7 has been developed for rainbow trout by modifying the RTG-2 cell line through transfection with a plasmid construct (pGL4.14[luc2/hygro]-PrTAP2) containing a promoter element from the IFN-gamma responsive gene TAP2 linked to a luciferase reporter gene and a hygromycin resistance gene. Following transfection single clones were selected in 96 well plates using hygromycin B, and those showing specific activation after rIFN-gamma stimulation were maintained. Five clones that showed the highest reporter activity to rIFN-gamma were incubated with different stimuli to examine specificity. No significant induction of luciferase was observed following exposure to recombinant type I IFN, LPS, PHA or poly I:C. The cell line was responsive to rIFN-gamma at concentrations between 150 pg and 20 ng ml(-1). Supernatants of primary cultures of head kidney leucocytes stimulated with PHA, known to induce IFN-gamma gene expression, were also used to assess the reporter activity of the stable cell line. A dose-dependent induction of the promoter activity was observed with these supernatants indicating the presence of IFN-gamma. These results indicate that the stable cell line RTG-3F7 is an excellent tool for monitoring the presence of trout IFN-gamma in biological samples, and in addition, enables the study of intracellular signalling pathways of IFNs, their receptor interactions, and other closely related signalling networks.
已通过转染包含干扰素 (IFN)-γ 反应基因 TAP2 启动子元件的质粒构建体 (pGL4.14[luc2/hygro]-PrTAP2)，将虹鳟 RTG-2 细胞系修饰为干扰素 (IFN)-γ 反应稳定细胞系 RTG-3F7。该基因与荧光素酶报告基因和潮霉素抗性基因相连。转染后，使用潮霉素 B 在 96 孔板中选择单克隆，然后维持对 rIFN-γ 刺激有特异性激活的单克隆。用不同的刺激物孵育显示出对 rIFN-γ 具有最高报告基因活性的 5 个克隆，以检查其特异性。用重组 I 型 IFN、LPS、PHA 或聚肌苷酸刺激后，未观察到荧光素酶的显著诱导。该细胞系对浓度在 150 pg 和 20 ng ml(-1) 之间的 rIFN-γ 有反应。还使用 PHA 刺激的头肾白细胞原代培养物的上清液来评估稳定细胞系的报告基因活性，PHA 已知可诱导 IFN-γ 基因表达。用这些上清液观察到启动子活性的剂量依赖性诱导，表明存在 IFN-γ。这些结果表明，稳定细胞系 RTG-3F7 是监测生物样品中虹鳟 IFN-γ 存在的极好工具，此外，还能够研究 IFN 的细胞内信号通路、它们的受体相互作用以及其他密切相关的信号网络。
