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      New Flexon-based reagents for tissue-specific Auxin-Inducible Degradation and for characterizing Cre and Flp drivers in C. elegans

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      Author(s): 1 , § , 1
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      Journal: microPublication Biology
      Publisher: Caltech Library

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          Abstract

          A Flexon stop cassette interrupts translation of a coding region until it is excised by a recombinase to allow for gene expression. We have expanded options for Auxin-Inducible Degradation by generating Flexon-based transgenes for tissue-specific expression of the ubiquitin ligase substrate recognition component TIR1 or the variant TIR1(F79G) after excision of the Flexon by Cre recombinase. We also describe Flexon-based tester transgenes to facilitate gathering accurate information about the expression pattern of Cre and Flp recombinase drivers that can be used in conjunction with any conditional expression reagents that utilize these recombinases.

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          Single-copy insertion of transgenes in Caenorhabditis elegans.

          Christian Frøkjaer-Jensen, M Wayne Davis, Christopher E Hopkins … (2008)
          At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.
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            The auxin-inducible degradation (AID) system enables versatile conditional protein depletion in C. elegans

            Liangyu Zhang, Jordan D. Ward, Ze Cheng … (2015)
            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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              Random and targeted transgene insertion in C. elegans using a modified Mosl transposon

              Christian Frøkjær-Jensen, M. Wayne Davis, Mihail Sarov … (2014)
              We have generated a recombinant Mos1 transposon that can insert up to 45 kb transgenes into the C. elegans genome. The minimal Mos1 transposon (miniMos) is 550 bp long and inserts DNA into the genome at high frequency (~60% of injected animals). Genetic and antibiotic markers can be used for selection and the transposon is active in C. elegans isolates and C. briggsae. We have used the miniMos transposon to generate six universal MosSCI landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes. We have also generated two collections of strains: A set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.
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                Author and article information

                Journal
                Journal ID (nlm-ta): MicroPubl Biol
                Journal ID (iso-abbrev): MicroPubl Biol
                Title: microPublication Biology
                Publisher: Caltech Library
                ISSN (Electronic): 2578-9430
                Publication date (Electronic, pub): 19 August 2024
                Publication date (Electronic, collection): 2024
                Volume: 2024
                Electronic Location Identifier: 10.17912/micropub.biology.001315
                Affiliations
                [1 ] Dept. of Biological Sciences, Columbia University, New York, New York, USA
                Author notes
                [§ ] Correspondence to: Julia Wittes ( jw2327@ 123456columbia.edu )

                The authors declare that there are no conflicts of interest present.

                Article
                Accession ID: WBPaper00067148
                DOI: 10.17912/micropub.biology.001315
                PMC ID: 11369693
                PubMed ID: 39228994
                SO-VID: 5aa38c6c-79e2-4c48-8d48-53844b3830c9
                Copyright © Copyright: © 2024 by the authors
                License:

                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 : 5 August 2024
                Date revision received : 17 August 2024
                Date accepted : 17 August 2024
                Funding
                This work was supported by grants R35GM131746 from the National Institute of General Medical Sciences (to I.G.), F32GM140732 (to J.W.), and training grant 5T32DK007328.
                Categories
                Subject: Materials and Reagents
                Subject: Methods
                Subject: C. Elegans

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