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.
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
- Evolutionary Biology
Repression of retrotransposition is crucial for the successful fitness of a mammalian organism. The domesticated transposon protein L1TD1, derived from LINE-1 (L1) ORF1p, is an RNA-binding protein that is expressed only in some cancers and early embryogenesis. In human embryonic stem cells, it is found to be essential for maintaining pluripotency. In cancer, L1TD1 expression is highly correlative with malignancy progression and as such considered a potential prognostic factor for tumors. However, its molecular role in cancer remains largely unknown. Our findings reveal that DNA hypomethylation induces the expression of L1TD1 in HAP1 human tumor cells. L1TD1 depletion significantly modulates both the proteome and transcriptome and thereby reduces cell viability. Notably, L1TD1 associates with L1 transcripts and interacts with L1 ORF1p protein, thereby facilitating L1 retrotransposition. Our data suggest that L1TD1 collaborates with its ancestral L1 ORF1p as an RNA chaperone, ensuring the efficient retrotransposition of L1 retrotransposons, rather than directly impacting the abundance of L1TD1 targets. In this way, L1TD1 might have an important role not only during early development but also in tumorigenesis.
