Kishore Shivendra, Khanna Amit, Stamm Stefan
Department of Molecular and Cellular Biochemistry, B283 Biomedical Biological Sciences Research Building, 741 South Limestone, University of Kentucky, College of Medicine, Lexington, KY 40536-0509, USA.
Gene. 2008 Dec 31;427(1-2):104-10. doi: 10.1016/j.gene.2008.09.021. Epub 2008 Oct 1.
Almost all human protein-coding transcripts undergo pre-mRNA splicing and a majority of them is alternatively spliced. The most common technique used to analyze the regulation of an alternative exon is through reporter minigene constructs. However, their construction is time-consuming and is often complicated by the limited availability of appropriate restriction sites. Here, we report a fast and simple recombination-based method to generate splicing reporter genes, using a new vector, pSpliceExpress. The system allows generation of minigenes within one week. Minigenes generated with pSpliceExpress show the same regulation as displayed by conventionally cloned reporter constructs and provide an alternate avenue to study splice site selection in vivo.
几乎所有人类蛋白质编码转录本都经历前体mRNA剪接，其中大多数还会发生可变剪接。用于分析可变外显子调控的最常用技术是通过报告基因小基因构建体。然而，它们的构建耗时，并且常常因合适的限制性酶切位点有限而变得复杂。在这里，我们报告了一种基于重组的快速简便方法，使用新载体pSpliceExpress来生成剪接报告基因。该系统可在一周内生成小基因。用pSpliceExpress生成的小基因显示出与传统克隆的报告构建体相同的调控，并为体内研究剪接位点选择提供了另一条途径。
