A toolkit to generate inducible and interconvertibleDrosophilatransgenes

Abstract

The existence of three independent binary systems for conditional gene expression (Gal4/UAS; LexA/LexAop; QF/QUAST) has greatly expanded versatile genetic analyses in the fruit fly Drosophila melanogaster; however, the experimental application of these tools is limited by the need to generate multiple collections of non-interchangeable transgenic fly strains for each inducible gene expression system. To address this practical limitation, we developed a multipurpose modular vector that contains the regulatory elements from all three binary systems, enabling Gal4-, LexA- or QF-dependent expression of transgenes. Our methods also incorporate DNA elements that facilitate independent site-specific recombination and elimination of regulatory UAS, LexAop or QUAST modules with spatial and temporal control, thus offering unprecedented possibilities and logistical advantages for in vivo genetic modulation and efficient interconversion of transgenic fly lines.

Transgenic Drosophila lines for LexA-dependent gene and growth regulation

“…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 ).…”

Transgenic Drosophila lines for LexA-dependent gene and growth regulation

“…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.…”

Discovering signaling mechanisms governing metabolism and metabolic diseases with Drosophila

“…Today, multiple binary systems (e.g., Gal4/ UAS, LexA/LexAop, Q systems) can be combined in a single fly to conduct genetic perturbations of multiple tissues simultaneously, and measure the effects of those perturbations (Figure 1). Together, these tools allow exquisite spatial and temporal control of gene expression and protein function, including the ability to genetically target different tissues or organs independently, manipulate the function of a gene in a given tissue or their activity, and determine the interactions among different tissues and organs (Kockel et al, 2019;Lin and Potter, 2016;Wendler et al, 2020) (and references therein). Use of genetic and physiological tools that can unequivocally establish the directionality and significance of signaling across organs is highlighted below.…”