A LexAop > UAS > QUAS trimeric plasmid to generate inducible and interconvertible Drosophila overexpression transgenes

Abstract

The existence of three independent binary systems for conditional gene expression (Gal4/UAS; LexA/LexAop; QF/QUAS) has greatly expanded versatile genetic analyses in the Drosophila melanogaster; however, the experimental application of these tools is limited by the need to generate multiple collections of noninterchangeable transgenic fly strains for each inducible gene expression system. To address this practical limitation, we developed a 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 QUAS modules with spatial and temporal control, thus offering unprecedented possibilities and logistical advantages for in vivo genetic modulation and efficient interconversion of overexpression transgenic fly lines.

Simplified homology-assisted CRISPR for gene editing in Drosophila

“…To expand the collection of LexA drivers, we and others have generated novel LexA lines using enhancer trap screens ( Kockel et al 2016 , 2019 ; Kim et al 2023 ) or by cloning enhancers to direct the LexA expression ( Pfeiffer et al 2010 ; Wendler et al 2022 ). While these approaches are sound, the novel lines generated by random transposon insertion or putative genomic enhancer fragments require extensive characterization, including insertion site mapping or expression specificity.…”

“…To train instructors with little to no experience with Drosophila or CRISPR, we developed a week-long, intensive teacher training academy, called Discover Now . This approach of “teaching the teachers” has fostered the autonomy of Stan-X instructors and their schools ( Chang et al 2022 ; Wendler et al 2022 ; Kim et al 2023 ). Currently, partnering teachers from 4 additional schools are training to adopt HACKy-based experiments and instruction (S.P.…”

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

“…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 Clustered Regularly Interspaced Short Palindromic Repeats and other approaches to generate novel LexA or LexAop strains ( Chang et al . 2022 ; Wendler et al . 2022 ) or fly genomics (see Kockel et al .…”

Expanding the Drosophila toolkit for dual control of gene expression

“…There remains an unmet need for a single vector that would allow for UAS/LexAop/QUAS control of different shRNAs. However, recent innovations in multi-module vectors and multiplexed drug-based genetics allow researchers to more efficiently generate UAS/QUAS/lexAop transgenic fly strains ( Matinyan et al, 2021 ; Wendler et al, 2022 ). In summary, we have generated a set of stocks, vectors, and protocols that when combined with the wide array of GAL4 lines will greatly expand the ability of Drosophila researchers to modulate gene expression in multiple tissues simultaneously.…”