期刊
出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2304730120
关键词
Drosophila; split-Gal4; intersectional genetics; single-cell transcriptomics
The split-Gal4 system based on a self-excising split-intein enables genetic labeling of specific cell types and tissues in Drosophila with temporal control. It can be repressed by Gal80 and can be combined with the drug-inducible GeneSwitch system for inducible control. In addition, the system can generate highly specific genetic drivers based on scRNAseq datasets and provides a plasmid toolkit for efficient driver creation.
The split-Gal4 system allows for intersectional genetic labeling of highly specific cell types and tissues in Drosophila. However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is repressible by Gal80. We demonstrate the potent inducibility of split-intein Gal4 in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell type-specific genetic drivers based on in silico predictions generated by single-cell RNAseq (scRNAseq) datasets, and we describe an algorithm (Two Against Background or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible.
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