1. Chromosomes and Gene Expression
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A gene-specific T2A-GAL4 library for Drosophila

  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  Is a corresponding author
  1. Baylor College of Medicine, United States
  2. Harvard Medical School, United States
  3. Howard Hughes Medical Institute, Carnegie Institution for Science, United States
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Cite this article as: eLife 2018;7:e35574 doi: 10.7554/eLife.35574

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.

Reviewing Editor

  1. K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

Publication history

  1. Received: January 31, 2018
  2. Accepted: March 16, 2018
  3. Accepted Manuscript published: March 22, 2018 (version 1)
  4. 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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Further reading

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    Foldback inversions, also called inverted duplications, have been observed in human genetic diseases and cancers. Here we used a Saccharomyces cerevisiae genetic system that generates gross chromosomal rearrangements (GCRs) mediated by foldback inversions combined with whole-genome sequencing to study their formation. Foldback inversions were mediated by formation of single-stranded DNA hairpins. Two types of hairpins were identified: small-loop hairpins that were suppressed by MRE11, SAE2, SLX1, and YKU80 and large-loop hairpins that were suppressed by YEN1, TEL1, SWR1, and MRC1. Analysis of CRISPR/Cas9-induced double strand breaks (DSBs) revealed that long-stem hairpin-forming sequences could form foldback inversions when proximal or distal to the DSB, whereas short-stem hairpin-forming sequences formed foldback inversions when proximal to the DSB. Finally, we found that foldback inversion GCRs were stabilized by secondary rearrangements, mostly mediated by different homologous recombination mechanisms including single-strand annealing; however, POL32-dependent break-induced replication did not appear to be involved forming secondary rearrangements.

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