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      A Tet/Q Hybrid System for Robust and Versatile Control of Transgene Expression in C. elegans

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          Summary

          Binary gene regulatory tools such as the Tetracycline (Tet)-controlled transcription system have revolutionized genetic research in multiple organisms, but their applications to the worm remain very limited. Here we report that the canonical Tet system is largely inactive in the worm but can be adapted for the worm by introducing multiple modifications, a crucial one being the use of the transcription activation domain from the fungal Q binary system. The resultant Tet/Q hybrid system proves more robust and flexible than either of its precursors, enabling elaborate modes of transgene manipulation previously hard to achieve in the worm, including inducible intersectional regulation and, in combination with the Q system, independent control of distinct transgenes within the same cells. Furthermore, we demonstrated, as an example of its applications, that the hybrid system can tightly and efficiently control Cre expression. This study establishes Tet/Q as a premier binary system for worm genetic research.

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          Highlights

          • The popular Tet-controlled gene regulatory system proves inapplicable to the worm

          • The fungal Q binary gene regulatory system is moderately active in the worm

          • A hybrid Tet/Q system is capable of robust, rapid and tunable transgene induction

          • Further modifications enable sophisticated regulation previously hard to achieve

          Abstract

          Genetics; Techniques in Genetics; Model Organism

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          Most cited references35

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          Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.

          We describe a dominant behavioral marker, rol-6(su-1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double-strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single-stranded oligonucleotide was co-injected with the double-stranded DNA.
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            The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans

            Experimental manipulation of protein abundance in living cells or organisms is an essential strategy for investigation of biological regulatory mechanisms. Whereas powerful techniques for protein expression have been developed in Caenorhabditis elegans, existing tools for conditional disruption of protein function are far more limited. To address this, we have adapted the auxin-inducible degradation (AID) system discovered in plants to enable conditional protein depletion in C. elegans. We report that expression of a modified Arabidopsis TIR1 F-box protein mediates robust auxin-dependent depletion of degron-tagged targets. We document the effectiveness of this system for depletion of nuclear and cytoplasmic proteins in diverse somatic and germline tissues throughout development. Target proteins were depleted in as little as 20-30 min, and their expression could be re-established upon auxin removal. We have engineered strains expressing TIR1 under the control of various promoter and 3′ UTR sequences to drive tissue-specific or temporally regulated expression. The degron tag can be efficiently introduced by CRISPR/Cas9-based genome editing. We have harnessed this system to explore the roles of dynamically expressed nuclear hormone receptors in molting, and to analyze meiosis-specific roles for proteins required for germ line proliferation. Together, our results demonstrate that the AID system provides a powerful new tool for spatiotemporal regulation and analysis of protein function in a metazoan model organism.
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              The Q system: a repressible binary system for transgene expression, lineage tracing, and mosaic analysis.

              We describe a new repressible binary expression system based on the regulatory genes from the Neurospora qa gene cluster. This "Q system" offers attractive features for transgene expression in Drosophila and mammalian cells: low basal expression in the absence of the transcriptional activator QF, high QF-induced expression, and QF repression by its repressor QS. Additionally, feeding flies quinic acid can relieve QS repression. The Q system offers many applications, including (1) intersectional "logic gates" with the GAL4 system for manipulating transgene expression patterns, (2) GAL4-independent MARCM analysis, and (3) coupled MARCM analysis to independently visualize and genetically manipulate siblings from any cell division. We demonstrate the utility of the Q system in determining cell division patterns of a neuronal lineage and gene function in cell growth and proliferation, and in dissecting neurons responsible for olfactory attraction. The Q system can be expanded to other uses in Drosophila and to any organism conducive to transgenesis. 2010 Elsevier Inc. All rights reserved.
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                Author and article information

                Contributors
                Journal
                iScience
                iScience
                iScience
                Elsevier
                2589-0042
                27 December 2018
                25 January 2019
                27 December 2018
                : 11
                : 224-237
                Affiliations
                [1 ]School of Life Sciences and Technology, ShanghaiTech University, Shanghai, P.R. China
                [2 ]Department Immunobiology, Yale University School of Medicine, New Haven, CT, USA
                Author notes
                []Corresponding author chitian@ 123456shanghaitech.edu.cn
                [3]

                These authors contributed equally

                [4]

                Lead Contact

                Article
                S2589-0042(18)30253-0
                10.1016/j.isci.2018.12.023
                6327101
                30634168
                80a71387-c07a-497b-a6aa-e88bd575c953
                © 2018 The Author(s)

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                History
                : 24 July 2018
                : 11 October 2018
                : 20 December 2018
                Categories
                Article

                genetics,techniques in genetics,model organism
                genetics, techniques in genetics, model organism

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