An Interscholastic Network to Generate LexA Enhancer Trap Lines inDrosophila

Abstract

Binary expression systems like the LexA-LexAop system provide a powerful experimental tool kit to study gene and tissue function in developmental biology, neurobiology and physiology. However, the number of well-defined LexA enhancer trap insertions remains limited. In this study, we present the molecular characterization and initial tissue expression analysis of nearly 100 novel StanEx LexA enhancer traps, derived from the StonEx1 index line. This includes 76 insertions into novel, distinct gene loci not previously associated with enhancer traps or targeted LexA constructs. Additionally, our studies revealed evidence for selective transposase-dependent replacement of a previously-undetected KP element on chromosome III within the StanEx1 genetic background during hybrid dysgenesis, suggesting a molecular basis for the over-representation of LexA insertions at the NK7.1 locus in our screen. Production and characterization of novel fly lines were performed by students and teachers in experiment-based genetics classes within a geographically diverse network of public and independent high schools. Thus, unique partnerships between secondary schools and university-based programs have produced and characterized novel genetic and molecular resources in Drosophila for open-source distribution, and provide paradigms for development of science education through experience-based pedagogy.

Transgenic Drosophila lines for LexA-dependent gene and growth regulation

“…This illustrates the feasibility of building an interscholastic network to generate unfulfilled scientific resource needs, while providing paradigms for STEM education. Resources and outcomes described here significantly extend, develop and complement the interscholastic partnerships in experiment-based science pedagogy described in our prior studies, which focused on generating LexA enhancer trap lines (Kockel et al 2016(Kockel et al , 2019. The current study involved collaborations between Stanford and Oxford University investigators with students and teachers at the Lawrenceville School and the Phillips Exeter Academy, two U.S. secondary schools.…”

“…For example, simultaneous use of two independent binary expression systems allowed clonal analysis of multiple cell populations (Lai and Lee 2006;Bosch et al 2015), studies of tissue epistasis (Yagi et al 2010), and elucidation of otherwise undetected contacts between cells (Gordon and Scott 2009;Macpherson et al 2015). To address a growing need for unique fly strains permitting flexible intersectional approaches, we are expanding our efforts to create new genetic tools suitable for this purpose (Kockel et al 2016(Kockel et al , 2019Wendler et al 2020). Here we describe (1) a genetic tool to convert existing Gal4 lines to LexA.G4H by CRISPR/Cas9-mediated gene editing, (2) a universal cloning method to generate LexAop-based shRNA transgenic lines from functionally validated UAS-based shRNA transgenic lines, and (3) a research-based pedagogy to leverage effort by collaborating students in our secondary school network (Kockel et al 2016(Kockel et al , 2019.…”

“…To address a growing need for unique fly strains permitting flexible intersectional approaches, we are expanding our efforts to create new genetic tools suitable for this purpose (Kockel et al 2016(Kockel et al , 2019Wendler et al 2020). Here we describe (1) a genetic tool to convert existing Gal4 lines to LexA.G4H by CRISPR/Cas9-mediated gene editing, (2) a universal cloning method to generate LexAop-based shRNA transgenic lines from functionally validated UAS-based shRNA transgenic lines, and (3) a research-based pedagogy to leverage effort by collaborating students in our secondary school network (Kockel et al 2016(Kockel et al , 2019. Our results demonstrate that in vivo gene regulation by LexAop-based gene suppression succeeded in multiple tissue contexts.…”

“…To address this need, we and others have made systematic efforts to expand the number of wellcharacterized, publicly available LexA fly lines, either by linking defined enhancers to sequences encoding LexA (Pfeiffer et al 2010) or by mobilizing a LexA-containing transposable element to insert at genomic sites near enhancers, resulting in 'enhancer trap' LexA lines with unique expression patterns (Kockel et al 2016(Kockel et al , 2019. Although these resources are slowly growing, there are many wellcharacterized Gal4 enhancer lines that lack cognate LexA lines.…”

Spatial and temporal control of expression with light-gated LOV-LexA

“…There are thousands of enhancer-LexA or -GAL4 drivers targeting several cell types simultaneously [11,14,27,120,121]. The LOV-LexA can be placed downstream of broadly expressed enhancers, in order to elicit transgene expression in the neuron type of interest with delivery of light specifically to its somata.…”