Diao Fengqiu, Ironfield Holly, Luan Haojiang, Diao Feici, Shropshire William C, Ewer John, Marr Elizabeth, Potter Christopher J, Landgraf Matthias, White Benjamin H
Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA.
Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK.
Cell Rep. 2015 Mar 3;10(8):1410-21. doi: 10.1016/j.celrep.2015.01.059. Epub 2015 Feb 26.
Genetically encoded effectors are important tools for probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here, we introduce a simple, versatile method for achieving cell-type-specific expression of transgenes that leverages the untapped potential of "coding introns" (i.e., introns between coding exons). Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted "plug-and-play" cassettes (called "Trojan exons") that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC (Minos-mediated integration cassette) transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system.
基因编码效应器是在活体动物中探究细胞功能的重要工具，但需要改进将其表达定向到特定细胞类型的方法。在此，我们介绍一种简单、通用的方法，用于实现转基因的细胞类型特异性表达，该方法利用了“编码内含子”（即编码外显子之间的内含子）尚未开发的潜力。我们的方法使用内含子插入的“即插即用”盒式结构（称为“特洛伊外显子”），将转基因的表达与在感兴趣细胞中表达的天然基因的表达相结合，该盒式结构带有一个剪接受体位点，其后是T2A肽的编码序列和一个效应器转基因。我们使用含有合适的MiMIC（米诺斯介导的整合盒）转座子的品系和一系列能够表达多种常用转录因子的特洛伊外显子，在果蝇中证明了这种方法的有效性。我们还引入了一种可交换的、类似MiMIC的特洛伊外显子构建体，它可以使用Crispr/Cas系统靶向到编码内含子。
