Bonano Vivian I, Oltean Sebastian, Garcia-Blanco Mariano A
Department of Molecular Genetics and Microbiology, Center for RNA Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Nat Protoc. 2007;2(9):2166-81. doi: 10.1038/nprot.2007.292.
Imaging technologies are influencing the way we study regulatory processes in vivo. Several recent reports use fluorescence minigenes to image alternative splicing events in living cells and animals. This type of reporter is being used to generate transgenic mice to visualize splicing regulation in diverse tissues and cell types. In this protocol, we describe how to develop animals that report on alternative splicing and how to assess reporter expression in excised organs and tissue sections. The entire procedure, from making the reporters to imaging organs and tissues in adult transgenic mice, should take approximately 1.5 years. Fluorescence reporters can be used to image many splicing decisions in normal tissues and organs and can be extended to the study of disease states.
成像技术正在影响我们在体内研究调控过程的方式。最近的几份报告使用荧光微型基因对活细胞和动物中的可变剪接事件进行成像。这种类型的报告基因正被用于生成转基因小鼠，以可视化不同组织和细胞类型中的剪接调控。在本方案中，我们描述了如何培育能够报告可变剪接的动物，以及如何评估切除器官和组织切片中的报告基因表达。从制备报告基因到对成年转基因小鼠的器官和组织进行成像，整个过程大约需要1.5年。荧光报告基因可用于对正常组织和器官中的许多剪接决定进行成像，并可扩展到疾病状态的研究。
