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

  1. Pei-Tseng Lee

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7501-7881
  2. Jonathan Zirin

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  3. Oguz Kanca

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  4. Wen-Wen Lin

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  5. Karen L Schulze

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1368-729X
  6. David Li-Kroeger

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  7. Rong Tao

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  8. Colby Devereaux

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  9. Yanhui Hu

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  10. Verena Chung

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  11. Ying Fang

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  12. Yuchun He

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  13. Hongling Pan

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  14. Ming Ge

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  15. Zhongyuan Zuo

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
  16. Benjamin E Housden

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9134-4279
  17. Stephanie E Mohr

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9639-7708
  18. Shinya Yamamoto

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2172-8036
  19. Robert W Levis

    Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, United States
    Competing interests
    No competing interests declared.
  20. Allan C Spradling

    Department of Embryology, Howard Hughes Medical Institute, Carnegie Institution for Science, Baltimore, United States
    Competing interests
    Allan C Spradling, Reviewing editor, eLife.
  21. Norbert Perrimon

    Department of Genetics, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7542-472X
  22. Hugo J Bellen

    Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States
    For correspondence
    hbellen@bcm.edu
    Competing interests
    Hugo J Bellen, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5992-5989

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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  1. Pei-Tseng Lee
  2. Jonathan Zirin
  3. Oguz Kanca
  4. Wen-Wen Lin
  5. Karen L Schulze
  6. David Li-Kroeger
  7. Rong Tao
  8. Colby Devereaux
  9. Yanhui Hu
  10. Verena Chung
  11. Ying Fang
  12. Yuchun He
  13. Hongling Pan
  14. Ming Ge
  15. Zhongyuan Zuo
  16. Benjamin E Housden
  17. Stephanie E Mohr
  18. Shinya Yamamoto
  19. Robert W Levis
  20. Allan C Spradling
  21. Norbert Perrimon
  22. Hugo J Bellen
(2018)
A gene-specific T2A-GAL4 library for Drosophila
eLife 7:e35574.
https://doi.org/10.7554/eLife.35574

Share this article

https://doi.org/10.7554/eLife.35574

Further reading

    1. Genetics and Genomics
    Oguz Kanca, Jonathan Zirin ... Hugo J Bellen
    Research Advance Updated

    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.

    1. Chromosomes and Gene Expression
    2. Genetics and Genomics
    Hans Tobias Gustafsson, Lucas Ferguson ... Oliver J Rando
    Research Article

    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.