Generation of LexA enhancer-trap lines in Drosophila by an international scholastic network

Abstract

Conditional gene regulation in Drosophila through binary expression systems like the LexA-LexAop system provides a superb tool for investigating gene and tissue function. To increase the availability of defined LexA enhancer trap insertions, we present molecular, genetic and tissue expression studies of 301 novel Stan-X LexA enhancer traps derived from mobilization of the index SX4 line. This includes insertions into distinct loci on the X, II and III chromosomes that were not previously associated with enhancer traps or targeted LexA constructs, an insertion into ptc, and seventeen insertions into natural transposons. A subset of enhancer traps was expressed in CNS neurons known to produce and secrete insulin, an essential regulator of growth, development and metabolism. Fly lines described here were generated and characterized through studies by students and teachers in an international network of genetics classes at public, independent high schools, and universities serving a diversity of students, including those underrepresented in science. Thus, a unique partnership between secondary schools and university-based programs has produced and characterized novel resources in Drosophila, establishing instructional paradigms devoted to unscripted experimental science.

CRISPR/Cas9 gene editing inDrosophilavia visual selection in a summer classroom

“…Our course uses Drosophila , a safe, fast and economic genetic model organism that supplements basic theoretical training in mendelian and non-mendelian genetics widespread in most secondary school and university biology curricula. Our programs enable student-scientists to execute experiments and to present data to peers and the community of science, including through oral presentations, and manuscripts or publications (Kockel et al 2016, 2019; Chang et al 2022; Kim et al 2023; Rankin et al 2023). This short course has been ideal for adaptation by Stan-X partner schools that have recently adopted it for instruction in the regular academic year.…”

“…To address a global desire for LexA lines with cell type-specific expression patterns, we and others conducted enhancer trap screens or a directed cloning approach (Pfeiffer et al 2010; Kockel et al 2016, Kockel et al 2019, Kim et al 2023, Wendler et al 2022). However, the enhancer trapping approach requires extensive molecular characterization, including insertion site and expression mapping.…”

“…The Drosophila Gal4 genome editing class took place in summer 2023 as a seven week ‘Animal Transgenesis” course in the Harvard University Department of Continuing Education (DCE). The class was held at the Biolabs of Harvard University, with instruction by personnel from Stanford University School of Medicine, The Lawrenceville School (NJ), and by two teaching assistants from Phillips Exeter Academy and the Lawrenceville School, both previously educated through the Stan-X program (Kim et al, 2023). The classroom was staffed with one-to-two instructors and two TAs every day.…”

“…The generation of multiple binary expression systems has been limited by the relative paucity of specific driver lines. While several thousand stocks of Gal4 lines are available, the number of publicly-available LexA driver lines remains comparatively low, though efforts have begun to address this resource gap (Pfeiffer et al 2010, Kockel et al 2016; Kockel et al 2019; Kim et al 2023). Recently, homology-assisted CRISPR knock-in (HACK) and similar CRISPR/Cas9-based methods were built to convert the large number of existing Gal4 lines into driver lines expressing QF or LexA (Lin and Potter, 2016, Chang et al 2022; Karuparti et al 2023).…”

Expanding the Drosophila toolkit for dual control of gene expression

“…Furthermore, the FlyLight lines use a p65 transcriptional activation domain and therefore are not compatible with the GAL80 temperature-sensitive GAL4 repression system. A second large collection of ~180 LexA-based enhancer trap fly stocks has been generated (the StanEx collection) ( Kockel et al, 2016 ; Kockel et al, 2019 ; Kim et al, 2023 ). On average, each StanEx line expresses LexA activity in five distinct cell types, with only one line showing expression in just one tissue, unfortunately limiting usefulness of these reagents ( Kockel et al, 2016 ).…”