Transgenic Drosophila lines for LexA-dependent gene and growth regulation

Abstract

Conditional expression of short hairpin RNAs (shRNAs) 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 two 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-shRNA 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 shRNA expression vector to achieve shRNA-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.

A genetic strategy to measure insulin signaling regulation and physiology in Drosophila

“…Compared to kinase regulators of insulin signaling [1, 6] phosphatases remain relatively understudied. Drosophila genetic screens have revealed phosphatases governing wing disc circadian systems, and embryonic development [12, 24-26]. In a genetic screen of Ser/Thr phosphatases, we found that Pp1-87B suppression potently inhibited Akt phosphorylation.…”

“…Our findings suggested the pAktHF ELISA could be used to identify novel genetic regulators of insulin resistance, an established risk for type 2 diabetes [5]. To investigate this possibility, we first assessed pAktHF changes in flies after genetic suppression of known insulin signaling regulators ( Figure 4A ; [12]). After shRNA knockdown in fat body of the insulin receptor ( InR ) or chico , the Drosophila ortholog of IRS1/2, we observed a significant reduction of pAktHF in ad libitum fed flies ( Figure 4B ).…”

“…For LexA phospho-Akt experiments flies were generated on a yellow white (yw) background, with the following genotype: yw ; r5-LexA, Tubp-Gal80TS ; Ilp2-Gal4, LexAop-Akt-HF. These flies were crossed to LexAop-RNAi lines [12]. For Gal4 phospho-Akt experiments, flies were generated on a yellow white (yw) background, with the following genotype: yw ; Ilp2-LexA, Tubp-Gal80TS ; Lpp-Gal4, UAS-Akt-HF.…”

An international scholastic network to generate LexA enhancer-trap lines forDrosophila

“…In our prior studies, we produced and characterized novel fly enhancer trap lines through an interscholastic partnership of secondary school and university-based researchers in the U.S. (Kockel et al 2016, 2019; Chang et al 2022). This involved development of curricula permitting flexible scheduling of laboratory-based ‘modules’: in some schools, this occurred in year-long courses ( Suppl.…”

“…There were also multiple positive outcomes for students and teachers at partnering schools that were unanticipated. These included (1) emergence of student course alumni as instructors at their home institution or another Stan-X partner site (n = 6), (2) interscholastic collaboration and data development through sharing of Stan-X fly strains and other resources, (3) regular video conferencing and in-person multi-institutional student symposia organized independently by Stan-X instructors, (4) additional professional development opportunities for adult teachers, including presentation of pedagogy at professional meetings, promotion, and travel to other Stan-X partner schools, (5) development of new courses founded on CRISPR and other approaches to generate novel LexA or LexAop strains (Chang et al 2022; Wendler et al 2021) or fly genomics (see Kockel et al 2019), (6) development of Stan-X summer school courses at Harvard, Oxford, Lawrenceville and Exeter ( Suppl. Figure 1B , https://stan-x.org), and (7) philanthropic funding for science curricular innovation and infrastructure modification to Stan-X partners.…”