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 StanEx 1 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 StanEx 1 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 a model that contributes to experiential 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 , 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, 2 US secondary schools.…”

“…For example, simultaneous use of 2 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 , 2019 ; Wendler 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 , 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 , 2019 ; Wendler 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 , 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 well-characterized, 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 , 2019 ). Although these resources are slowly growing, there are many well-characterized Gal4 enhancer lines that lack cognate LexA lines.…”

Identification, characterization and use of novel thermogenetic tools in drosophilia melanogaster

“…Cas9, that was isolated from a bacteria, Staphylococcus, has proven extremely useful in gene editing with the CRISPR-Cas9 system [176,177]. Similarly, two other proteins Cre recombinase [178][179][180], and lex a repressor [84,181,182] that were isolated from bacteria and viruses respectively, are used extensively in making transgenic mammals. Yet another example is that of the bioluminescent protein, luciferase obtained from a firefly that is used as a reporter for gene expression [183][184][185][186].…”