Stanford University, Stanford, CA 94305, USA.
Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, USA.
G3 (Bethesda). 2022 Mar 4;12(3). doi: 10.1093/g3journal/jkac018.
Conditional expression of short hairpin RNAs with binary genetic systems is an indispensable tool for studying gene function. Addressing mechanisms underlying cell-cell communication in vivo benefits from simultaneous use of 2 independent gene expression systems. To complement the abundance of existing Gal4/UAS-based resources in Drosophila, we and others have developed LexA/LexAop-based genetic tools. Here, we describe experimental and pedagogical advances that promote the efficient conversion of Drosophila Gal4 lines to LexA lines, and the generation of LexAop-short hairpin RNA lines to suppress gene function. We developed a CRISPR/Cas9-based knock-in system to replace Gal4 coding sequences with LexA, and a LexAop-based short hairpin RNA expression vector to achieve short hairpin RNA-mediated gene silencing. We demonstrate the use of these approaches to achieve targeted genetic loss-of-function in multiple tissues. We also detail our development of secondary school curricula that enable students to create transgenic flies, thereby magnifying the production of well-characterized LexA/LexAop lines for the scientific community. The genetic tools and teaching methods presented here provide LexA/LexAop resources that complement existing resources to study intercellular communication coordinating metazoan physiology and development.
带有二元遗传系统的短发夹 RNA 的条件表达是研究基因功能不可或缺的工具。解决体内细胞间通讯的机制得益于同时使用 2 个独立的基因表达系统。为了补充果蝇中现有的大量 Gal4/UAS 资源，我们和其他人开发了基于 LexA/LexAop 的遗传工具。在这里，我们描述了促进将 Drosophila Gal4 系高效转化为 LexA 系，以及生成 LexAop-短发夹 RNA 系以抑制基因功能的实验和教学进展。我们开发了一种基于 CRISPR/Cas9 的敲入系统，用 LexA 替换 Gal4 编码序列，以及一种基于 LexAop 的短发夹 RNA 表达载体，以实现短发夹 RNA 介导的基因沉默。我们展示了这些方法在多种组织中实现靶向基因功能丧失的应用。我们还详细介绍了我们开发的中学课程，使学生能够创建转基因苍蝇，从而为科学界放大产生经过良好表征的 LexA/LexAop 系。这里介绍的遗传工具和教学方法提供了 LexA/LexAop 资源，补充了现有的资源，以研究协调后生动物生理学和发育的细胞间通讯。
