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      Liposome-based transfection enhances RNAi and CRISPR-mediated mutagenesis in non-model nematode systems

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          Abstract

          Nematodes belong to one of the most diverse animal phyla. However, functional genomic studies in nematodes, other than in a few species, have often been limited in their reliability and success. Here we report that by combining liposome-based technology with microinjection, we were able to establish a wide range of genomic techniques in the newly described nematode genus Auanema. The method also allowed heritable changes in dauer larvae of Auanema, despite the immaturity of the gonad at the time of the microinjection. As proof of concept for potential functional studies in other nematode species, we also induced RNAi in the free-living nematode Pristionchus pacificus and targeted the human parasite Strongyloides stercoralis.

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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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            Specific interference by ingested dsRNA.

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              Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome III.

              Genome sequencing projects generate a wealth of information; however, the ultimate goal of such projects is to accelerate the identification of the biological function of genes. This creates a need for comprehensive studies to fill the gap between sequence and function. Here we report the results of a functional genomic screen to identify genes required for cell division in Caenorhabditis elegans. We inhibited the expression of approximately 96% of the approximately 2,300 predicted open reading frames on chromosome III using RNA-mediated interference (RNAi). By using an in vivo time-lapse differential interference contrast microscopy assay, we identified 133 genes (approximately 6%) necessary for distinct cellular processes in early embryos. Our results indicate that these genes represent most of the genes on chromosome III that are required for proper cell division in C. elegans embryos. The complete data set, including sample time-lapse recordings, has been deposited in an open access database. We found that approximately 47% of the genes associated with a differential interference contrast phenotype have clear orthologues in other eukaryotes, indicating that this screen provides putative gene functions for other species as well.
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                Author and article information

                Contributors
                Andre.Pires@warwick.ac.uk
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                24 January 2019
                24 January 2019
                2019
                : 9
                : 483
                Affiliations
                [1 ]ISNI 0000 0000 8809 1613, GRID grid.7372.1, School of Life Sciences, , University of Warwick, ; Coventry, CV4 7AL UK
                [2 ]ISNI 0000 0004 1936 8972, GRID grid.25879.31, Department of Pathobiology, School of Veterinary Medicine, , University of Pennsylvania, ; Philadelphia, Pennsylvania United States of America
                Author information
                http://orcid.org/0000-0002-0741-7197
                Article
                37036
                10.1038/s41598-018-37036-1
                6345965
                30679624
                7a65f417-4fd0-4333-aea3-334e51857435
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                History
                : 11 January 2018
                : 28 November 2018
                Funding
                Funded by: FundRef https://doi.org/10.13039/501100000268, RCUK | Biotechnology and Biological Sciences Research Council (BBSRC);
                Award ID: BB/L019884/1
                Award ID: BB/L019884/1
                Award Recipient :
                Funded by: FundRef https://doi.org/10.13039/100000002, U.S. Department of Health & Human Services | National Institutes of Health (NIH);
                Award ID: AI50688
                Award ID: AI105856
                Award ID: OD P40-10939
                Award ID: AI50688
                Award ID: AI105856
                Award ID: OD P40-10939
                Award Recipient :
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