Institute of Metabolic Disease, Baylor University Medical Center, Dallas, TX, USA.
Gene Ther. 2009 Oct;16(10):1202-9. doi: 10.1038/gt.2009.114. Epub 2009 Sep 3.
This study was done to improve efficiency and islet specificity of the rat insulin promoter (RIP). Various RIP lengths were prepared and tested in vitro to drive luciferase reporter gene expression in INS1-cells, alpha-cells, acinar cells, ductal cells and fibroblasts. The CMV promoter was used as a positive control. In addition, the DsRed reporter gene was administered in vivo to rat pancreas by ultrasound-targeted microbubble destruction (UTMD). Confocal microscopy was used to detect the presence and distribution of DsRed within the pancreas after UTMD. A modified RIP3.1 promoter, which includes portions of the insulin gene after its transcription start site is fivefold more active in INS-1 cells than the full-length RIP promoter or the CMV promoter. RIP3.1 is regulated by glucose level and various islet transcription factors in vitro, and exhibits activity in alpha-cells, but not in exocrine cells. In vivo delivery of RIP3.1-DsRed resulted in expression of DsRed protein in beta-cells, and to a lesser extent in alpha-cells under normal glucose conditions. No DsRed signal was present in exocrine pancreas under RIP3.1. A modified RIP, RIP3.1, efficiently and specifically directs gene expression to endocrine pancreas.
这项研究旨在提高大鼠胰岛素启动子（RIP）的效率和胰岛特异性。制备了不同长度的 RIP，并在 INS1 细胞、α 细胞、胰岛细胞、导管细胞和成纤维细胞中进行了体外测试，以驱动荧光素酶报告基因的表达。CMV 启动子被用作阳性对照。此外，通过超声靶向微泡破坏（UTMD）将 DsRed 报告基因体内给药到大鼠胰腺。使用共聚焦显微镜检测 UTMD 后 DsRed 在胰腺中的存在和分布。一种改良的 RIP3.1 启动子，其包括转录起始位点后胰岛素基因的部分，在 INS-1 细胞中的活性比全长 RIP 启动子或 CMV 启动子高五倍。RIP3.1 在体外受葡萄糖水平和各种胰岛转录因子调节，在 α 细胞中具有活性，但在外分泌细胞中没有活性。在正常葡萄糖条件下，RIP3.1 体内递送 RIP3.1-DsRed 导致 DsRed 蛋白在 β 细胞中表达，在 α 细胞中表达程度较低。在 RIP3.1 下，外分泌胰腺中没有 DsRed 信号。一种改良的 RIP，RIP3.1，可高效且特异性地将基因表达导向内分泌胰腺。
