Expansion of the Gateway MultiSite Recombination Cloning Toolkit

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Abstract

Precise manipulation of transgene expression in genetic model organisms has led to advances in understanding fundamental mechanisms of development, physiology, and genetic disease. Transgene construction is, however, a precondition of transgene expression, and often limits the rate of experimental progress. Here we report an expansion of the modular Gateway MultiSite recombination-cloning platform for high efficiency transgene assembly. The expansion includes two additional destination vectors and entry clones for the LexA binary transcription system, among others. These new tools enhance the expression levels possible with Gateway MultiSite generated transgenes and make possible the generation of LexA drivers and reporters with Gateway MultiSite cloning. In vivo data from transgenic Drosophila functionally validating each novel component are presented and include neuronal LexA drivers, LexAop2 red and green fluorescent synaptic vesicle reporters, TDC2 and TRH LexA, GAL4, and QF drivers, and LexAop2, UAS, and QUAS channelrhodopsin2 T159C reporters.

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Most cited references 18

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P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.

Koen J T Venken, Yuchun He, Roger A. Hoskins … (2006)

We describe a transgenesis platform for Drosophila melanogaster that integrates three recently developed technologies: a conditionally amplifiable bacterial artificial chromosome (BAC), recombineering, and bacteriophage PhiC31-mediated transgenesis. The BAC is maintained at low copy number, facilitating plasmid maintenance and recombineering, but is induced to high copy number for plasmid isolation. Recombineering allows gap repair and mutagenesis in bacteria. Gap repair efficiently retrieves DNA fragments up to 133 kilobases long from P1 or BAC clones. PhiC31-mediated transgenesis integrates these large DNA fragments at specific sites in the genome, allowing the rescue of lethal mutations in the corresponding genes. This transgenesis platform should greatly facilitate structure/function analyses of most Drosophila genes.

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Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects.

S. Sweeney, K Broadie, J. Keane … (1995)

Tetanus toxin cleaves the synaptic vesicle protein synaptobrevin, and the ensuing loss of neurotransmitter exocytosis has implicated synaptobrevin in this process. To further the study of synaptic function in a genetically tractable organism and to generate a tool to disable neuronal communication for behavioural studies, we have expressed a gene encoding tetanus toxin light chain in Drosophila. Toxin expression in embryonic neurons removes detectable synaptobrevin and eliminates evoked, but not spontaneous, synaptic vesicle release. No other developmental or morphological defects are detected. Correspondingly, only synaptobrevin (n-syb), but not the ubiquitously expressed syb protein, is cleaved by tetanus toxin in vitro. Targeted expression of toxin can produce specific behavioral defects; in one case, the olfactory escape response is reduced.

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Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons.

T Kitamoto (2001)

Behavior is a manifestation of temporally and spatially defined neuronal activities. To understand how behavior is controlled by the nervous system, it is important to identify the neuronal substrates responsible for these activities, and to elucidate how they are integrated into a functional circuit. I introduce a novel and general method to conditionally perturb anatomically defined neurons in intact Drosophila. In this method, a temperature-sensitive allele of shibire (shi(ts1)) is overexpressed in neuronal subsets using the GAL4/UAS system. Because the shi gene product is essential for synaptic vesicle recycling, and shi(ts1) is semidominant, a simple temperature shift should lead to fast and reversible effects on synaptic transmission of shi(ts1) expressing neurons. When shi(ts1) expression was directed to cholinergic neurons, adult flies showed a dramatic response to the restrictive temperature, becoming motionless within 2 min at 30 degrees C. This temperature-induced paralysis was reversible. After being shifted back to the permissive temperature, they readily regained their activity and started to walk in 1 min. When shi(ts1) was expressed in photoreceptor cells, adults and larvae exhibited temperature-dependent blindness. These observations show that the GAL4/UAS system can be used to express shi(ts1) in a specific subset of neurons to cause temperature-dependent changes in behavior. Because this method allows perturbation of the neuronal activities rapidly and reversibly in a spatially and temporally restricted manner, it will be useful to study the functional significance of particular neuronal subsets in the behavior of intact animals. Copyright 2001 John Wiley & Sons, Inc.

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Author and article information

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Brian D. McCabe: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, USA )

ISSN (Electronic): 1932-6203

Publication date Collection: 2013

Publication date (Electronic): 18 October 2013

Volume: 8

Issue: 10

Electronic Location Identifier: e77724

Affiliations

[1]Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana, United States of America

Columbia University, United States of America

Author notes

* E-mail: sstowers@ 123456montana.edu

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: RSS. Performed the experiments: RSS HKS ARD CDK. Analyzed the data: RSS HKS. Contributed reagents/materials/analysis tools: RSS HKS ARD CDK. Wrote the manuscript: RSS.

Article

Publisher ID: PONE-D-13-29700

DOI: 10.1371/journal.pone.0077724

PMC ID: 3799639

PubMed ID: 24204935

SO-VID: 376c41e3-361f-4f96-9d16-33ab487e417d

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This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 18 July 2013

Date accepted : 3 September 2013

Funding

This work received financial support from the Whitehall Foundation ( http://www.whitehall.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Expansion of the Gateway MultiSite Recombination Cloning Toolkit

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Most cited references 18

P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.

Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects.

Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons.

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