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.
| Original language | English (US) |
|---|---|
| Article number | e22279 |
| Journal | eLife |
| Volume | 6 |
| DOIs | |
| State | Published - Feb 17 2017 |
Bibliographical note
Funding Information:We thank T Mosca and members of the Clandinin Laboratory for helpful discussion and comments on this manuscript. We thank D Bieli (University of Basel), M Müller (University of Basel), L Luo (Stanford University), T Mosca (Stanford University), A DeAntonio (Washington University in St. Louis), B Ganetzky (University of Wisconsin), R Ordway (Pennsylvania State University), the Drosophila Genome Resource Center (DGRC), and the Bloomington Drosophila Stock Center (BDSC) for reagents. We thank R Volkan for an unpublished qRT-PCR primer sequence; M Bennett, C Bennett, T Li, M Fu, and S Liddelow (Stanford University) for advice about qRT-PCR experiments; and B Barres (Stanford University) for use of a qRT-PCR machine. We thank the Harvard Image and Data Analysis Core (IDAC) for access to Imaris software for analysis of immunostaining. YEF was supported by a National Science Foundation Fellowship. HHY was supported by a Stanford Graduate Fellowship and a Stanford Interdisciplinary Graduate Fellowship. DMG was supported by a Ruth L Kirschstein NRSA Postdoctoral Fellowship (F32 EY020040) from the National Eye Institute. This work was funded by R01 EY022638 and U01 MH109119 (to TRC). Funding Funder Grant reference number Author National Eye Institute R01 EY022638 Thomas R Clandinin National Institute of Mental Health U01 MH109119 Thomas R Clandinin National Science Foundation Yvette E Fisher Stanford University School of Medicine Helen H Yang National Eye Institute F32 EY020040 Daryl M Gohl The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Publisher Copyright:
© Fisher et al.