Warzecha Claude C, Hovhannisyan Ruben, Carstens Russ P
Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
Methods Mol Biol. 2012;867:273-87. doi: 10.1007/978-1-61779-767-5_18.
Alternative splicing of pre-mRNA transcripts is a critical and extensively utilized mechanism of gene regulation. In this chapter, we describe a series of fluorescent and luminescent minigene reporters our lab has used to facilitate the study of alternative splicing regulation in cultured cells. Through the use of different versions of these minigenes, the inclusion level of a cassette exon can be directly ascertained by fluorescence or luciferase activity, thereby making it possible to establish cell-based assays for induced exon splicing or skipping. A successful application of these minigenes in a high-throughput cDNA screen led to the identification of a cell type-specific regulator of FGFR2 splicing, illustrating the power of these reporters to yield novel insights into alternative splicing. The methods and minigenes described are adaptable for genetic screens to uncover novel regulators of a broader set of alternative splicing events in other gene transcripts. These reporters also have a dynamic range that is suitable for small molecule screening for compounds that can regulate splicing.
前体mRNA转录本的可变剪接是一种关键且被广泛利用的基因调控机制。在本章中，我们描述了一系列荧光和发光微型基因报告基因，我们实验室利用它们来促进对培养细胞中可变剪接调控的研究。通过使用这些微型基因的不同版本，盒式外显子的包含水平可以通过荧光或荧光素酶活性直接确定，从而有可能建立基于细胞的检测方法来检测诱导的外显子剪接或跳跃。这些微型基因在高通量cDNA筛选中的成功应用导致鉴定出一种FGFR2剪接的细胞类型特异性调节因子，说明了这些报告基因在深入了解可变剪接方面的强大作用。所描述的方法和微型基因适用于基因筛选，以发现其他基因转录本中更广泛的可变剪接事件的新型调节因子。这些报告基因还具有适合小分子筛选的动态范围，以筛选可调节剪接的化合物。
