Department of Metabolic Disorders, Diabetes Research Center, National Center for Global Health and Medicine, Tokyo 162-8655, Japan.
Endocrinology. 2013 Nov;154(11):4388-95. doi: 10.1210/en.2012-2248. Epub 2013 Sep 12.
The luciferase reporter system is useful for the assessment of various biological processes in vivo. The transcription factor pancreatic and duodenal homeobox 1 (Pdx1) is critical for the formation and the function of pancreatic β-cells. A novel reporter system using secreted Gaussia princeps luciferase (GLuc) under the control of a Pdx1 promoter was generated and activated in rat and mouse β-cell lines. This Pdx1-GLuc construct was used as a transgene for the generation of reporter mice to monitor Pdx1 promoter activity in vivo via the measurement of secreted GLuc activity in a small aliquot of blood. Significantly increased plasma GLuc activity was observed in Pdx1-GLuc mice. Analysis of Pdx1-GLuc mice by bioluminescence imaging, GLuc reporter assays using homogenates of various organs, and immunohistochemistry revealed that GLuc expression and activity were exponentially higher in pancreatic β-cells than in pancreatic non-β-cells, the duodenum, and other organs. In addition, GLuc activity secreted into the culture medium from islets isolated from Pdx1-GLuc mice correlated with the number of islets. The transplantation of Pdx1-GLuc islets into severe combined immunodeficiency mice elevated their plasma GLuc activity. Conversely, a partial pancreatectomy in Pdx1-GLuc mice reduced plasma GLuc activity. These results suggest that a secreted luciferase reporter system in vivo enables not only the monitoring of promoter activity but also a quantitative and minimally invasive assessment of physiological and pathological changes in small cell masses, such as pancreatic β-cells.
荧光素酶报告系统可用于评估体内的各种生物过程。胰岛转录因子 1（Pdx1）对于胰腺β细胞的形成和功能至关重要。我们构建了一种新型的报告系统，该系统利用受 Pdx1 启动子控制的分泌型海肾荧光素酶（GLuc），在大鼠和小鼠β细胞系中激活。该 Pdx1-GLuc 构建体被用作转基因，用于通过测量小部分血液中的分泌型 GLuc 活性，在体内监测 Pdx1 启动子活性。在 Pdx1-GLuc 小鼠中观察到显著增加的血浆 GLuc 活性。通过生物发光成像、使用各种器官匀浆进行的 GLuc 报告测定和免疫组织化学分析表明，GLuc 在胰腺β细胞中的表达和活性比胰腺非β细胞、十二指肠和其他器官高得多。此外，从 Pdx1-GLuc 小鼠分离的胰岛分泌到培养基中的 GLuc 活性与胰岛的数量相关。将 Pdx1-GLuc 胰岛移植到严重联合免疫缺陷小鼠中会提高其血浆 GLuc 活性。相反，Pdx1-GLuc 小鼠的部分胰腺切除术会降低血浆 GLuc 活性。这些结果表明，体内分泌型荧光素酶报告系统不仅能够监测启动子活性，还能够对小细胞群（如胰腺β细胞）的生理和病理变化进行定量和微创评估。
