当前位置: X-MOL 学术J. Neurogenet. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
A quick and versatile protocol for the 3D visualization of transgene expression across the whole body of larval Drosophila
Journal of Neurogenetics ( IF 1.9 ) Pub Date : 2021-03-10 , DOI: 10.1080/01677063.2021.1892096
Oliver Kobler 1 , Aliće Weiglein 2 , Kathrin Hartung 3 , Yi-Chun Chen 2 , Bertram Gerber 2, 4, 5 , Ulrich Thomas 3, 6
Affiliation  

Abstract

Larval Drosophila are used as a genetically accessible study case in many areas of biological research. Here we report a fast, robust and user-friendly procedure for the whole-body multi-fluorescence imaging of Drosophila larvae; the protocol has been optimized specifically for larvae by systematically tackling the pitfalls associated with clearing this small but cuticularized organism. Tests on various fluorescent proteins reveal that the recently introduced monomeric infrared fluorescent protein (mIFP) is particularly suitable for our approach. This approach comprises an effective, low-cost clearing protocol with minimal handling time and reduced toxicity in the reagents employed. It combines a success rate high enough to allow for small-scale screening approaches and a resolution sufficient for cellular-level analyses with light sheet and confocal microscopy. Given that publications and database documentations typically specify expression patterns of transgenic driver lines only within a given organ system of interest, the present procedure should be versatile enough to extend such documentation systematically to the whole body. As examples, the expression patterns of transgenic driver lines covering the majority of neurons, or subsets of chemosensory, central brain or motor neurons, are documented in the context of whole larval body volumes (using nsyb-Gal4, IR76b-Gal4, APL-Gal4 and mushroom body Kenyon cells, or OK371-Gal4, respectively). Notably, the presented protocol allows for triple-color fluorescence imaging with near-infrared, red and yellow fluorescent proteins.



中文翻译:

一种快速而通用的协议,用于对整个果蝇幼虫体内的转基因表达进行 3D 可视化

摘要

幼虫果蝇在许多生物学研究领域被用作基因可及的研究案例。在这里,我们报告了一种快速、稳健且用户友好的果蝇全身多荧光成像程序幼虫;该协议专门针对幼虫进行了优化,系统地解决了与清除这种小型但角质化有机体相关的陷阱。对各种荧光蛋白的测试表明,最近引入的单体红外荧光蛋白 (mIFP) 特别适合我们的方法。这种方法包括一种有效、低成本的清除方案,处理时间最短,所用试剂的毒性降低。它结合了足够高的成功率以允许小规模筛选方法和足以通过光片和共聚焦显微镜进行细胞水平分析的分辨率。鉴于出版物和数据库文件通常仅在给定的感兴趣器官系统内指定转基因驱动系的表达模式,目前的程序应该具有足够的通用性,可以系统地将此类文件扩展到整个机构。例如,覆盖大部分神经元或化学感觉、中央脑或运动神经元子集的转基因驱动系的表达模式在整个幼虫体体积的背景下记录(使用 nsyb-Gal4、IR76b-Gal4、APL-Gal4和蘑菇体 Kenyon 细胞,或 OK371-Gal4,分别)。值得注意的是,所提出的协议允许使用近红外、红色和黄色荧光蛋白进行三色荧光成像。在整个幼虫体体积的背景下记录(分别使用 nsyb-Gal4、IR76b-Gal4、APL-Gal4 和蘑菇体 Kenyon 细胞或 OK371-Gal4)。值得注意的是,所提出的协议允许使用近红外、红色和黄色荧光蛋白进行三色荧光成像。在整个幼虫体体积的背景下记录(分别使用 nsyb-Gal4、IR76b-Gal4、APL-Gal4 和蘑菇体 Kenyon 细胞或 OK371-Gal4)。值得注意的是,所提出的协议允许使用近红外、红色和黄色荧光蛋白进行三色荧光成像。

更新日期:2021-03-10
down
wechat
bug