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
- Genetics and Genomics
Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA structure and nucleotide modifications can each interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We show that OTTR successfully captures both full-length tRNAs and tRNA fragments in budding yeast and in mouse reproductive tissues without any prior enzymatic treatment, and that tRNA cloning efficiency can be further enhanced via AlkB-mediated demethylation of modified nucleotides. As with other recent tRNA cloning protocols, we find that a subset of nucleotide modifications leave misincorporation signatures in OTTR datasets, enabling their detection without any additional protocol steps. Focusing on tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to "ground truth" Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRNA fragments - including both 5′ and 3′ tRNA halves derived from the majority of tRNAs – than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.
