Institute of Cellular and Integrative Neurosciences, INCI CNRS UPR3212, University of Strasbourg, Strasbourg, France.
Methods Mol Biol. 2022;2550:243-265. doi: 10.1007/978-1-0716-2593-4_28.
Genetic technology allows inserting transgenic reporters such as beta-galactosidase (LacZ) into the loci of the Mtnr1a (MT) and Mtnr1b (MT) receptor genes to track MT and MT melatonin receptor expression. Given the limited sensitivity of nonradioactive in situ hybridization and the problematic specificity of existing melatonin receptor antibodies for immunohistochemistry, this new technology is a key tool to study the localization and the phenotypes of cells expressing melatonin receptors. Here we describe two protocols to detect transgenic LacZ expression driven by the MT or MT promoters either by the enzymatic activity of the transgenic LacZ enzyme or by using specific antibodies against LacZ with immunohistochemistry. This approach has already yielded a detailed mapping of both MT and MT expression in the mouse brain and retina. Furthermore, we also phenotyped some of the most important types of cells expressing these two melatonin receptors.
遗传技术可将转基因报告基因如β-半乳糖苷酶（LacZ）插入 Mtnr1a（MT）和 Mtnr1b（MT）受体基因的基因座中，以跟踪 MT 和 MT 褪黑素受体的表达。鉴于非放射性原位杂交的灵敏度有限，以及现有的褪黑素受体抗体用于免疫组织化学的特异性问题，这项新技术是研究表达褪黑素受体的细胞的定位和表型的关键工具。在这里，我们描述了两种通过 MT 或 MT 启动子驱动的转基因 LacZ 表达的检测方案，要么通过转基因 LacZ 酶的酶活性，要么通过免疫组织化学中针对 LacZ 的特异性抗体。这种方法已经对小鼠大脑和视网膜中 MT 和 MT 的表达进行了详细的定位。此外，我们还对表达这两种褪黑素受体的一些最重要类型的细胞进行了表型分析。
