Sheets Michael D
Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, WI, USA.
Methods Mol Biol. 2019;1920:33-39. doi: 10.1007/978-1-4939-9009-2_3.
The earliest steps of animal development depend upon posttranscriptional events that drive the embryonic cell cycle and guide cell fate decisions. The analysis of post-transcriptional regulatory events has relied upon the use of chimeric reporter mRNAs that encode firefly luciferase fused to potential regulatory sequences. A new and more sensitive luciferase developed recently called NanoLuc has the potential to improve reporter studies and provide new insights into the regulation of embryonic processes. Here I describe how to create and analyze reporter mRNAs encoding NanoLuc luciferase using extracts from microinjected Xenopus embryos.
动物发育的最早阶段依赖于驱动胚胎细胞周期并指导细胞命运决定的转录后事件。转录后调控事件的分析依赖于使用嵌合报告mRNA，这些mRNA编码与潜在调控序列融合的萤火虫荧光素酶。最近开发的一种新的、更灵敏的荧光素酶NanoLuc有潜力改进报告基因研究，并为胚胎发育过程的调控提供新的见解。在这里，我描述了如何使用显微注射的非洲爪蟾胚胎提取物来创建和分析编码NanoLuc荧光素酶的报告mRNA。
