Forbes Beadle Lauren, Sutcliffe Catherine, Ashe Hilary L
Faculty of Biology, Medicine and Health, The University of Manchester, Manchester M13 9PT, UK.
Development. 2024 Dec 15;151(24). doi: 10.1242/dev.204294. Epub 2024 Dec 16.
Live imaging of transcription in the Drosophila embryo using the MS2 or PP7 systems is transforming our understanding of transcriptional regulation. However, insertion of MS2/PP7 stem-loops into endogenous genes requires laborious CRISPR genome editing. Here, we exploit the previously described Minos-mediated integration cassette (MiMIC) transposon system in Drosophila to establish a method for simply and rapidly inserting MS2/PP7 cassettes into any of the thousands of genes carrying a MiMIC insertion. In addition to generating a variety of stem-loop donor fly stocks, we have made new stocks expressing the complementary coat proteins fused to different fluorescent proteins. We show the utility of this MiMIC-based approach by MS2/PP7 tagging of endogenous genes and the long non-coding RNA roX1, then imaging their transcription in living embryos. We also present live transcription data from larval brains, the wing disc and ovary, thereby extending the tissues that can be studied using the MS2/PP7 system. Overall, this first high-throughput method for tagging mRNAs in Drosophila will facilitate the study of transcription dynamics of thousands of endogenous genes in a range of Drosophila tissues.
利用MS2或PP7系统对果蝇胚胎中的转录进行实时成像，正在改变我们对转录调控的理解。然而，将MS2/PP7茎环插入内源基因需要繁琐的CRISPR基因组编辑。在这里，我们利用果蝇中先前描述的Minos介导的整合盒（MiMIC）转座子系统，建立了一种将MS2/PP7盒简单快速地插入数千个携带MiMIC插入的基因中的任何一个的方法。除了生成各种茎环供体果蝇品系外，我们还制作了表达与不同荧光蛋白融合的互补外壳蛋白的新品系。我们通过对内源基因和长链非编码RNA roX1进行MS2/PP7标记，然后在活胚胎中对其转录进行成像，展示了这种基于MiMIC方法的实用性。我们还展示了幼虫大脑、翅芽和卵巢的实时转录数据，从而扩展了可以使用MS2/PP7系统进行研究的组织范围。总体而言，这种在果蝇中标记mRNA的首个高通量方法将有助于研究果蝇一系列组织中数千个内源基因的转录动态。
