Murphy Daniel, Kolandaivelu Saravanan, Ramamurthy Visvanathan, Stoilov Peter
Department of Biochemistry, School of Medicine, West Virginia University, 1 Medical Center Drive, Morgantown, WV, 26505, USA.
Methods Mol Biol. 2016;1421:269-86. doi: 10.1007/978-1-4939-3591-8_20.
In vivo alternative splicing is controlled in a tissue and cell type specific manner. Often individual cellular components of complex tissues will express different splicing programs. Thus, when studying splicing in multicellular organisms it is critical to determine the exon inclusion levels in individual cells positioned in the context of their native tissue or organ. Here we describe how a fluorescent splicing reporter in combination with in vivo electroporation can be used to visualize alternative splicing in individual cells within mature tissues. In a test case we show how the splicing of a photoreceptor specific exon can be visualized within the mouse retina. The retina was chosen as an example of a complex tissue that is fragile and whose cells cannot be studied in culture. With minor modifications to the injection and electroporation procedure, the protocol we outline can be applied to other tissues and organs.
体内可变剪接受组织和细胞类型特异性方式的控制。复杂组织的单个细胞成分通常会表达不同的剪接程序。因此，在多细胞生物中研究剪接时，确定位于其天然组织或器官背景下的单个细胞中的外显子包含水平至关重要。在这里，我们描述了荧光剪接报告基因与体内电穿孔相结合如何用于可视化成熟组织中单个细胞内的可变剪接。在一个测试案例中，我们展示了如何在小鼠视网膜内可视化光感受器特异性外显子的剪接。视网膜被选为一个复杂组织的例子，该组织很脆弱，其细胞无法在培养中进行研究。对注射和电穿孔程序进行微小修改后，我们概述的方案可应用于其他组织和器官。
