Red Flour Beetle (<i>Tribolium</i>) Germline Transformation and Insertional Mutagenesis

Berghammer, Andreas J.; Weber, Markus; Trauner, Jochen; Klingler, Martin

doi:10.1101/pdb.prot5259

Cited by 30 publications

(24 citation statements)

References 4 publications

(1 reference statement)

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“…Tribolium embryos were injected at the syncytial blastoderm stage and handled as described previously (Berghammer et al, 2009). The injected concentrations of Cas9, gRNA and knock-in templates are indicated in Table 1.…”

Section: Tribolium Injection and Molecular Analysismentioning

confidence: 99%

“…Starting with classic genetic screens conducted in the 1980s (Beeman et al, 1989;Sulston and Anderson, 1996), the Tribolium research community has grown, bringing a plethora of genetic tools and approaches to this species. These approaches include transgenesis (Berghammer et al, 1999(Berghammer et al, , 2009Lorenzen et al, 2003;Pavlopoulos et al, 2004), RNAimediated knockdown (Bucher et al, 2002;Posnien et al, 2009), enhancer trapping (Lorenzen et al, 2003;Trauner et al, 2009), heatshock and GAL4/UAS-mediated mis-expression (Schinko et al, 2012(Schinko et al, , 2010 and live imaging (Benton et al, 2013;Sarrazin et al, 2012;Strobl and Stelzer, 2014). Moreover, the Tribolium genome is sequenced and well annotated (Kim et al, 2010;Richards et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

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Efficient CRISPR-mediated gene targeting and transgene replacement in the beetleTribolium castaneum

2015

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Gene-editing techniques are revolutionizing the way we conduct genetics in many organisms. The CRISPR/Cas nuclease has emerged as a highly versatile, efficient and affordable tool for targeting chosen sites in the genome. Beyond its applications in established model organisms, CRISPR technology provides a platform for genetic intervention in a wide range of species, limited only by our ability to deliver it to cells and to select mutations efficiently. Here, we test the CRISPR technology in an emerging insect model and pest, the beetle Tribolium castaneum. We use simple assays to test CRISPR/Cas activity, we demonstrate efficient expression of guide RNAs and Cas9 from Tribolium U6 and hsp68 promoters and we test the efficiency of knockout and knock-in approaches in Tribolium. We find that 55-80% of injected individuals carry mutations (indels) generated by non-homologous end joining, including mosaic bi-allelic knockouts; 71-100% carry such mutations in their germ line and transmit them to the next generation. We show that CRISPR-mediated gene knockout of the Tribolium E-cadherin gene causes defects in dorsal closure, which is consistent with RNAi-induced phenotypes. Homology-directed knock-in of marker transgenes was observed in 14% of injected individuals and transmitted to the next generation by 6% of injected individuals. Previous work in Tribolium mapped a large number of transgene insertions associated with developmental phenotypes and enhancer traps. We present an efficient method for re-purposing these insertions, via CRISPR-mediated replacement of these transgenes by new constructs.

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Section: Tribolium Injection and Molecular Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Efficient CRISPR-mediated gene targeting and transgene replacement in the beetleTribolium castaneum

2015

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“…Second, Tribolium provides many tools for functional studies. It possesses a particularly strong systemic RNAi response -which was the basis for a recent genome-wide RNAi screen -and techniques for forward genetic screens, transgenesis and live imaging are available (Benton et al, 2013;Berghammer et al, 1999Berghammer et al, , 2009Koelzer et al, 2014;Sarrazin et al, 2012). Therefore, we have chosen Tribolium for unbiased genomic approaches like ChIP-seq (D. Stappert, Two novel, complementary next generation sequencing approaches to reveal the dorso-ventral gene regulatory network of Tribolium castaneum.…”

Section: Introductionmentioning

confidence: 99%

Genome wide identification ofTriboliumdorsoventral patterning genes

et al. 2016

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The gene regulatory network controlling dorsoventral axis formation in insects has undergone drastic evolutionary changes. In Drosophila, a stable long-range gradient of Toll signalling specifies ventral cell fates and restricts BMP signalling to the dorsal half of the embryo. In Tribolium, however, Toll signalling is transient and only indirectly controls BMP signalling. In order to gain unbiased insights into the Tribolium network, we performed comparative transcriptome analyses of embryos with various dorsoventral pattering defects produced by parental RNAi for Toll and BMP signalling components. We also included embryos lacking the mesoderm ( produced by Tctwist RNAi) and characterized similarities and differences between Drosophila and Tribolium twist loss-of-function phenotypes. Using stringent conditions, we identified over 750 differentially expressed genes and analysed a subset with altered expression in more than one knockdown condition. We found new genes with localized expression and showed that conserved genes frequently possess earlier and stronger phenotypes than their Drosophila orthologues. For example, the leucine-rich repeat (LRR) protein Tartan, which has only a minor influence on nervous system development in Drosophila, is essential for early neurogenesis in Tribolium and the Tc-zinc-finger homeodomain protein 1 (Tc-zfh1), the orthologue of which plays a minor role in Drosophila muscle development, is essential for maintaining early Tc-twist expression, indicating an important function for mesoderm specification.

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“…Information on these lines is accessible at http://www.geku-base.uni-goettingen.de/, and lines are available from the members of the consortium. For details on insertional mutagenesis in Tribolium, see Red Flour Beetle (Tribolium) Germline Transformation and Insertional Mutagenesis (Berghammer et al 2009). www.cshprotocols.org…”

Section: Insertional Mutagenesismentioning

confidence: 99%

“…Again, the use of native Tribolium promoters should enhance the efficiency of these types of experiments. For details on creating transgenic lines of Tribolium, see Red Flour Beetle (Tribolium) Germline Transformation and Insertional Mutagenesis (Berghammer et al 2009). …”

Section: Transgenicsmentioning

confidence: 99%

The Red Flour Beetle, Tribolium castaneum (Coleoptera): A Model for Studies of Development and Pest Biology: Figure 1.

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INTRODUCTIONTribolium castaneum is a small, low-maintenance beetle that has emerged as a sophisticated model system for studying the evolution of development and that complements (in some cases, even rivals) Drosophila for functional genetic analysis of basic biological questions. Although Tribolium and Drosophila are both holometabolous insects, they differ fundamentally in larval and adult morphology. Even generally conserved developmental features, such as body segmentation, are achieved by quite different means. Thus, comparison of developmental mechanisms between these two insects can address many interesting questions concerning the evolution of morphology and other characters. Genetic tools available for Tribolium include genetic maps for visible and molecular markers, chromosomal rearrangements that enable lethal mutations to be balanced in true-breeding stocks, transposon-based transformation systems, a completed and annotated genome sequence, and systemic RNA interference (RNAi), which makes it possible to knock down any given gene and even particular splice variants in the offspring or in any tissue of the injected animal. Inactivating gene functions at various developmental stages provides new opportunities to investigate post-embryonic development, as well as larval and adult physiology, including hormonal control, host-parasite interactions, and pesticide resistance.

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Red Flour Beetle (Tribolium) Germline Transformation and Insertional Mutagenesis

Cited by 30 publications

References 4 publications

Efficient CRISPR-mediated gene targeting and transgene replacement in the beetleTribolium castaneum

Efficient CRISPR-mediated gene targeting and transgene replacement in the beetleTribolium castaneum

Genome wide identification ofTriboliumdorsoventral patterning genes

The Red Flour Beetle, Tribolium castaneum (Coleoptera): A Model for Studies of Development and Pest Biology: Figure 1.

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