Plug-and-Play Genetic Access to Drosophila Cell Types using Exchangeable Exon Cassettes

Diao, Feiyang; Ironfield, Holly; Luan, Haojiang; Diao, Feici; Shropshire, William C; Ewer, John; Marr, Elizabeth; Potter, Christopher J.; Landgraf, Matthias; White, Benjamin H.

doi:10.1016/j.celrep.2015.01.059

Cited by 334 publications

(402 citation statements)

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“…All starting constructs were inserted into the SalI site of pBS-KS-attB1-2 and then modified by inserting effector transgenes into the BamHI sites in exon 4a or exon 4b. The effector transgenes were amplified by PCR from appropriate Trojan exons (Diao et al 2015) and included fusions of the T2A coding sequence to the sequences encoding: Gal4, Gal80, p65AD, or QF2. (The latter fragment was inserted into a BglII site placed in exon 4b rather than BamHI to allow the BamHI site placed in exon 4a to be used to insert a T2A-Gal4-encoding fragment.…”

Section: Molecular Biologymentioning

confidence: 99%

“…To more comprehensively investigate the function of both the ETHR gene and the neurons that express it in Drosophila, we here use the recently introduced Trojan exon method of genetic targeting (Diao et al 2015). Our results demonstrate that neurons that express the ETHRA isoform are required for ecdysis at all developmental stages and that functionally distinct subsets regulate not only behavior, but also fluid balance at the time of the pupal molt.…”

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The Splice Isoforms of the Drosophila Ecdysis Triggering Hormone Receptor Have Developmentally Distinct Roles

et al. 2015

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To grow, insects must periodically shed their exoskeletons. This process, called ecdysis, is initiated by the endocrine release of Ecdysis Trigger Hormone (ETH) and has been extensively studied as a model for understanding the hormonal control of behavior. Understanding how ETH regulates ecdysis behavior, however, has been impeded by limited knowledge of the hormone's neuronal targets. An alternatively spliced gene encoding a G-protein-coupled receptor (ETHR) that is activated by ETH has been identified, and several lines of evidence support a role in ecdysis for its A-isoform. The function of a second ETHR isoform (ETHRB) remains unknown. Here we use the recently introduced "Trojan exon" technique to simultaneously mutate the ETHR gene and gain genetic access to the neurons that express its two isoforms. We show that ETHRA and ETHRB are expressed in largely distinct subsets of neurons and that ETHRA-but not ETHRB-expressing neurons are required for ecdysis at all developmental stages. However, both genetic and neuronal manipulations indicate an essential role for ETHRB at pupal and adult, but not larval, ecdysis. We also identify several functionally important subsets of ETHR-expressing neurons including one that coexpresses the peptide Leucokinin and regulates fluid balance to facilitate ecdysis at the pupal stage. The general strategy presented here of using a receptor gene as an entry point for genetic and neuronal manipulations should be useful in establishing patterns of functional connectivity in other hormonally regulated networks.

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Section: Molecular Biologymentioning

confidence: 99%

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The Splice Isoforms of the Drosophila Ecdysis Triggering Hormone Receptor Have Developmentally Distinct Roles

et al. 2015

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“…Several methods have been developed to swap transcription factor drivers between transgenic binary expression systems. However, this requires either de novo generation of new driver lines [integrase swappable in vivo targeting element (InSITE)] (Gohl et al 2011) or PhiC31-induced insertion of a transcription factor cassette into an existing minos mediated integration cassette (MiMIC) genomic insertion (Venken et al 2011;Diao et al 2015;Gnerer et al 2015).Here, we developed a method that converts existing transgenic GAL4 lines into QF2 lines with two simple genetic crosses. This method uses the CRISPR/Cas9 system to induce DSBs in transgenic GAL4, and HDR to drive conversion of GAL4 into a QF2-expressing line through the use of a single transgenic donor cassette in the genome.…”

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Editing Transgenic DNA Components by Inducible Gene Replacement in Drosophila melanogaster

Lin¹,

2016

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Gene conversions occur when genomic double-strand DNA breaks (DSBs) trigger unidirectional transfer of genetic material from a homologous template sequence. Exogenous or mutated sequence can be introduced through this homology-directed repair (HDR). We leveraged gene conversion to develop a method for genomic editing of existing transgenic insertions in Drosophila melanogaster. The clustered regularly-interspaced palindromic repeats (CRISPR)/Cas9 system is used in the homology assisted CRISPR knock-in (HACK) method to induce DSBs in a GAL4 transgene, which is repaired by a single-genomic transgenic construct containing GAL4 homologous sequences flanking a T2A-QF2 cassette. With two crosses, this technique converts existing GAL4 lines, including enhancer traps, into functional QF2 expressing lines. We used HACK to convert the most commonly-used GAL4 lines (labeling tissues such as neurons, fat, glia, muscle, and hemocytes) to QF2 lines. We also identified regions of the genome that exhibited differential efficiencies of HDR. The HACK technique is robust and readily adaptable for targeting and replacement of other genomic sequences, and could be a useful approach to repurpose existing transgenes as new genetic reagents become available.KEYWORDS gene conversion; CRISPR/Cas9; homology-directed repair; genomic engineering; updating transgenes D OUBLE-strand DNA breaks (DSBs) in genomic DNA are potentially lethal to the cell as they are substrates for genomic rearrangements. Two major endogenous repair mechanisms exist to resolve such DNA damage. Nonhomologous end-joining (NHEJ) involves ligation of the damaged ends and often results in small insertion/deletion (indel) mutations at the site of the DSB (Lieber 2010). This causes frameshifting and the expression of mutated or nonfunctional protein. In contrast, homologous recombination (HR) uses a homologous template for repair to preserve genomic integrity. Gene conversion is one form of HR and involves unidirectional transfer of genetic material from a donor sequence to a highly homologous target (Engels et al. 1990;Gloor et al. 1991;Chen et al. 2007). Donors can be from the sister chromosome (allelic gene conversion) or dispersed sequences not at the same locus (ectopic gene conversion).Current genomic engineering methods use a DSB induced at a target genomic locus to generate disrupting mutations, driven by NHEJ, or to introduce new genetic sequences, driven by homology-directed repair (HDR). Several techniques have been developed to introduce target-specific DSBs, including zincfinger nucleases, transcription-activator-like effecter nucleases, and, most recently, clustered regularly-interspaced palindromic repeats (CRISPR) (Jinek et al. 2012;Doudna and Sontheimer 2014). Cas9 is the endonuclease of the type II CRISPR system and creates a DSB at genomic locations directed by a guide RNA (gRNA). The CRISPR/Cas9 system is favored for its high efficiency and ability to use a small gRNA to specifically target most genomic DNA locations (Cong et al. 2013;Hsu et a...

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“…Moreover, some neurons were found to release more than one types of neurotransmitters. Therefore, improved genetic tools are required in order to obtain more accurate cell type categorization (Diao et al 2015).…”

Section: Discussionmentioning

confidence: 99%

A single-cell level and connectome-derived computational model of the Drosophila brain

Huang

Wang

et al. 2018

Preprint

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Computer simulations play an important role in testing hypotheses, integrating knowledge, and providing predictions of neural circuit functions. While considerable effort has been dedicated into simulating primate or rodent brains, the fruit fly (Drosophila melanogaster) is becoming a promising model animal in computational neuroscience for its small brain size, complex cognitive behavior, and abundancy of data available from genes to circuits.Moreover, several Drosophila connectome projects have generated a large number of neuronal images that account for a significant portion of the brain, making a systematic investigation of the whole brain circuit possible. Supported by FlyCircuit (http://www.flycircuit.tw), one of the largest Drosophila neuron image databases, we began a long-term project with the goal to construct a whole-brain spiking network model of the Drosophila brain. In this paper, we report the outcome of the first phase of the project. We developed the Flysim platform, which 1) identifies the polarity of each neuron arbor, 2) predicts connections between neurons, 3) translates morphology data from the database into physiology parameters for computational modeling, 4) reconstructs a brain-wide network model, which consists of 20,089 neurons and 1,044,020 synapses, and 5) performs computer simulations of the resting state. We compared the reconstructed brain network with a randomized brain network by shuffling the connections of each neuron. We found that the reconstructed brain can be easily stabilized by implementing synaptic short-term depression, while the randomized one exhibited seizure-like firing activity under the same treatment.Furthermore, the reconstructed Drosophila brain was structurally and dynamically more diverse than the randomized one and exhibited both Poisson-like and patterned firing activities. Despite being at its early stage of development, this single-cell level brain model allows us to study some of the fundamental properties of neural networks including network balance, critical behavior, long-term stability, and plasticity.All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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Plug-and-Play Genetic Access to Drosophila Cell Types using Exchangeable Exon Cassettes

Cited by 334 publications

References 51 publications

The Splice Isoforms of the Drosophila Ecdysis Triggering Hormone Receptor Have Developmentally Distinct Roles

The Splice Isoforms of the Drosophila Ecdysis Triggering Hormone Receptor Have Developmentally Distinct Roles

Editing Transgenic DNA Components by Inducible Gene Replacement in Drosophila melanogaster

A single-cell level and connectome-derived computational model of the Drosophila brain

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