A gene-specific T2A-GAL4 library for Drosophila
- Baylor College of Medicine, United States
- Harvard Medical School, United States
- Howard Hughes Medical Institute, Carnegie Institution for Science, United States
Abstract
We generated a library of ~1,000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3' of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36(70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function.
Article and author information
Author details
Benjamin E Housden
Funding
National Institutes of Health (R01GM067858)
- Pei-Tseng Lee
Dana-Farber/Harvard Cancer Center (5 P30 CA06516)
- Stephanie E Mohr
Howard Hughes Medical Institute
- Karen L Schulze
National Institute of General Medical Sciences (GM084947)
- Norbert Perrimon
Howard Hughes Medical Institute
- Yuchun He
Howard Hughes Medical Institute
- Hongling Pan
Howard Hughes Medical Institute
- Stephanie E Mohr
Howard Hughes Medical Institute
- Robert W Levis
Howard Hughes Medical Institute
- Allan C Spradling
Howard Hughes Medical Institute
- Norbert Perrimon
Howard Hughes Medical Institute
- Hugo J Bellen
Eunice Kennedy Shriver National Institute of Child Health and Human Development (U54HD083092)
- Hugo J Bellen
National Institutes of Health (U54NS093793)
- Shinya Yamamoto
National Institute of General Medical Sciences (GM067761)
- Jonathan Zirin
National Institute of General Medical Sciences (GM067761)
- Yanhui Hu
Robert A. and Renee E. Belfer Family Foundation
- Hugo J Bellen
Huffington Foundation
- Shinya Yamamoto
Alzheimer's Association (NIRH-15-364099)
- Shinya Yamamoto
Simons Foundation (368479)
- Shinya Yamamoto
Naman Family Fund for Basic Research
- Shinya Yamamoto
Caroline Wiess Law Fund
- Shinya Yamamoto
National Institute of General Medical Sciences (GM067761)
- Stephanie E Mohr
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India
Version history
- Received: January 31, 2018
- Accepted: March 16, 2018
- Accepted Manuscript published: March 22, 2018 (version 1)
- Version of Record published: April 13, 2018 (version 2)
Copyright
© 2018, Lee et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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A gene-specific T2A-GAL4 library for Drosophila
eLife 7:e35574.
https://doi.org/10.7554/eLife.35574
Further reading
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- Genetics and Genomics
We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.
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- Chromosomes and Gene Expression
- Developmental Biology
Developmental programming involves the accurate conversion of signalling levels and dynamics to transcriptional outputs. The transcriptional relay in the Notch pathway relies on nuclear complexes containing the co-activator Mastermind (Mam). By tracking these complexes in real time, we reveal that they promote the formation of a dynamic transcription hub in Notch ON nuclei which concentrates key factors including the Mediator CDK module. The composition of the hub is labile and persists after Notch withdrawal conferring a memory that enables rapid reformation. Surprisingly, only a third of Notch ON hubs progress to a state with nascent transcription, which correlates with polymerase II and core Mediator recruitment. This probability is increased by a second signal. The discovery that target-gene transcription is probabilistic has far-reaching implications because it implies that stochastic differences in Notch pathway output can arise downstream of receptor activation.
