Shimomura Kenju, Galvanovskis Juris, Goldsworthy Michelle, Hugill Alison, Kaizak Stephan, Lee Angela, Meadows Nicholas, Quwailid Mohamed Mohideen, Rydström Jan, Teboul Lydia, Ashcroft Fran, Cox Roger D
Henry Wellcome Center for Gene Function, Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, United Kingdom.
Methods Enzymol. 2009;457:451-80. doi: 10.1016/S0076-6879(09)05025-3.
Nicotinamide nucleotide transhydrogenase (NNT) is an inner mitochondrial membrane transmembrane protein involved in regenerating NADPH, coupled with proton translocation across the inner membrane. We have shown that a defect in Nnt function in the mouse, and specifically within the beta-cell, leads to a reduction in insulin secretion. This chapter describes methods for examining Nnt function in the mouse. This includes generating in vivo models with point mutations and expression of Nnt by transgenesis, and making in vitro models, by silencing of gene expression. In addition, techniques are described to measure insulin secretion, calcium and hydrogen peroxide concentrations, membrane potential, and NNT activity. These approaches and techniques can also be applied to other genes of interest.
烟酰胺核苷酸转氢酶（NNT）是一种线粒体内膜跨膜蛋白，参与NADPH的再生，并伴有质子跨内膜转运。我们已经表明，小鼠中Nnt功能的缺陷，特别是在β细胞内，会导致胰岛素分泌减少。本章描述了检测小鼠中Nnt功能的方法。这包括通过点突变构建体内模型以及通过转基因表达Nnt，以及通过基因表达沉默构建体外模型。此外，还描述了测量胰岛素分泌、钙和过氧化氢浓度、膜电位以及NNT活性的技术。这些方法和技术也可应用于其他感兴趣的基因。
