FlpStop, a tool for conditional gene control in Drosophila

  1. Yvette Erica Fisher
  2. Helen H Yang
  3. Jesse Isaacman-Beck
  4. Marjorie Xie
  5. Daryl M Gohl
  6. Thomas R Clandinin  Is a corresponding author
  1. Harvard Medical School, United States
  2. Stanford University, United States
  3. University of Minnesota Genomics Center, United States

Abstract

Manipulating gene function cell type-specifically is a common experimental goal in Drosophila research and has been central to studies of neural development, circuit computation, and behavior. However, current cell type-specific gene disruption techniques in flies often reduce gene activity incompletely or rely on cell division. Here we describe FlpStop, a generalizable tool for conditional gene disruption and rescue in post-mitotic cells. In proof-of-principle experiments, we manipulated apterous, a regulator of wing development. Next, we produced conditional null alleles of Glutamic acid decarboxylase 1(Gad1) and Resistant to dieldrin (Rdl), genes vital for GABAergic neurotransmission, as well as cacophony (cac) and paralytic (para), voltage-gated ion channels central to neuronal excitability. To demonstrate the utility of this approach, we manipulated cac in a specific visual interneuron type and discovered differential regulation of calcium signals across subcellular compartments. Thus, FlpStop will facilitate investigations into the interactions between genes, circuits, and computation.

Article and author information

Author details

  1. Yvette Erica Fisher

    Department of Neurobiology, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Helen H Yang

    Department of Neurobiology, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5140-9664
  3. Jesse Isaacman-Beck

    Department of Neurobiology, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Marjorie Xie

    Department of Neurobiology, Stanford University, Stanford, United States
    Competing interests
    The authors declare that no competing interests exist.
  5. Daryl M Gohl

    University of Minnesota Genomics Center, Minneapolis, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Thomas R Clandinin

    Department of Neurobiology, Stanford University, Stanford, United States
    For correspondence
    trc@stanford.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6277-6849

Funding

National Eye Institute (R01 EY022638)

  • Thomas R Clandinin

National Institute of Mental Health (U01 MH109119)

  • Thomas R Clandinin

National Science Foundation

  • Yvette Erica Fisher

Stanford University School of Medicine

  • Helen H Yang

National Eye Institute (F32EY020040)

  • Daryl M Gohl

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2017, Fisher 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.

Metrics

  • 10,277
    views
  • 2,135
    downloads
  • 57
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Yvette Erica Fisher
  2. Helen H Yang
  3. Jesse Isaacman-Beck
  4. Marjorie Xie
  5. Daryl M Gohl
  6. Thomas R Clandinin
(2017)
FlpStop, a tool for conditional gene control in Drosophila
eLife 6:e22279.
https://doi.org/10.7554/eLife.22279

Share this article

https://doi.org/10.7554/eLife.22279

Further reading

    1. Chromosomes and Gene Expression
    Carmina Lichauco, Eric J Foss ... Antonio Bedalov
    Research Article

    The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA (rDNA) arrays. We have previously reported that in the absence of SIR2, a de-repressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.

    1. Chromosomes and Gene Expression
    Carlos Moreno-Yruela, Beat Fierz
    Insight

    Specialized magnetic beads that bind target proteins to a cryogenic electron microscopy grid make it possible to study the structure of protein complexes from dilute samples.