The DNA transposon Minos as a tool for transgenesis and functional genomic analysis in vertebrates and invertebrates

Pavlopoulos, Anastasios; Oehler, Stefan; Kapetanaki, Maria G.; Savakis, Charalambos

doi:10.1186/gb-2007-8-s1-s2

Cited by 42 publications

(43 citation statements)

References 60 publications

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“…Thus, iTRAC may serve as a genetic Swiss army knife, allowing the exploitation of gene traps in a virtually endless number of ways. The approach and the vectors presented here are likely to be applicable in a broad range of animal models, as all the constituents are known to work in widely divergent species: the Minos transposon is an excellent vector for gene trapping not only in arthropods, but also in mammals and in Ciona (Pavlopoulos et al, 2007;Sasakura et al, 2007); the fC31 integrase system has found application in Drosophila, zebrafish, Xenopus, mouse and human (Allen and Weeks, 2005;Groth et al, 2004;Groth et al, 2000;Lister, 2010), and we have shown here that it works very efficiently in Parhyale; the PhHsp70a element can mediate exon trapping in Parhyale and Drosophila and, given the wide conservation of splice acceptor sites, is likely to be useful more broadly. (A)The interplasmid assay for fC31 integrase-mediated recombination involves injecting plasmids carrying an attP or an attB site (red and green, respectively) with fC31 integrase mRNA into Parhyale embryos, and assaying recombination by PCR using primers F1, F2 and R. Each gel lane represents a single experiment, in which different combinations of plasmids and integrase mRNA were injected.…”

Section: Research Reportmentioning

confidence: 99%

A versatile strategy for gene trapping and trap conversion in emerging model organisms

et al. 2011

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SUMMARYGenetic model organisms such as Drosophila, C. elegans and the mouse provide formidable tools for studying mechanisms of development, physiology and behaviour. Established models alone, however, allow us to survey only a tiny fraction of the morphological and functional diversity present in the animal kingdom. Here, we present iTRAC, a versatile gene-trapping approach that combines the implementation of unbiased genetic screens with the generation of sophisticated genetic tools both in established and emerging model organisms. The approach utilises an exon-trapping transposon vector that carries an integrase docking site, allowing the targeted integration of new constructs into trapped loci. We provide proof of principle for iTRAC in the emerging model crustacean Parhyale hawaiensis: we generate traps that allow specific developmental and physiological processes to be visualised in unparalleled detail, we show that trapped genes can be easily cloned from an unsequenced genome, and we demonstrate targeting of new constructs into a trapped locus. Using this approach, gene traps can serve as platforms for generating diverse reporters, drivers for tissue-specific expression, gene knockdown and other genetic tools not yet imagined.

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Section: Research Reportmentioning

confidence: 99%

A versatile strategy for gene trapping and trap conversion in emerging model organisms

et al. 2011

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“…However, P-element activity is very host-restricted and appears to be inactive in non-Drosophilids [47]. In contrast, Minos elements have demonstrated a much wider host spectrum, including Drosophila and non-Drosophilid insects (for review, see [48]). …”

Section: Minosmentioning

confidence: 99%

“…In the fruit fly, the excision of Minos could leave precise conversion at the insertion sites or 6 bp characteristic footprints [48]. However, in mammalian systems, the footprints appear more complex, potentially due to differences in the host chromatidrepair machinery in these organisms [49,51].…”

Section: Minosmentioning

confidence: 99%

Transposon tools hopping in vertebrates

Ni¹,

Clark²,

Fahrenkrug³

et al. 2008

Briefings in Functional Genomics and Proteomics

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In the past decade, tools derived from DNA transposons have made major contributions to vertebrate genetic studies from gene delivery to gene discovery. Multiple, highly complementary systems have been developed, and many more are in the pipeline. Judging which DNA transposon element will work the best in diverse uses from zebrafish genetic manipulation to human gene therapy is currently a complex task. We have summarized the major transposon vector systems active in vertebrates, comparing and contrasting known critical biochemical and in vivo properties, for future tool design and new genetic applications.

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“…Third, most fly misexpression constructs have used the P-element vector, which is not able to provide complete coverage of the genome due to insertional biases Spradling et al 2011). This can be remedied by using the Minos transposon as a vector, since it has little insertion site specificity beyond a TA dinucleotide and thus provides access to P-element insertional "coldspots" (Metaxakis et al 2005;Pavlopoulos et al 2007;Bellen et al 2011).…”

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“…Hto combines targeted expression of endogenous genes with protein tagging and permits expression of stable C-terminal protein fragments, which have rarely been subjected to genetic screens in the past. Importantly, since Minos can transpose in a broad range of organisms, the Hto system could potentially be applied to other species (Pavlopoulos et al 2007;de Wit et al 2010;Hozumi et al 2010;Sasakura et al 2010).…”

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Inducible Protein Traps with Dominant Phenotypes for Functional Analysis of theDrosophilaGenome

et al. 2014

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The Drosophila melanogaster genome has been extensively characterized, but there remains a pressing need to associate gene products with phenotypes, subcellular localizations, and interaction partners. A multifunctional, Minos transposon-based protein trapping system called Hostile takeover (Hto) was developed to facilitate in vivo analyses of endogenous genes, including live imaging, purification of protein complexes, and mutagenesis. The Hto transposon features a UAS enhancer with a basal promoter, followed by an artificial exon 1 and a standard 59 splice site. Upon GAL4 induction, exon 1 can splice to the next exon downstream in the flanking genomic DNA, belonging to a random target gene. Exon 1 encodes a dual tag (FLAG epitope and mCherry red fluorescent protein), which becomes fused to the target protein. Hto was mobilized throughout the genome and then activated by eye-specific GAL4; an F 1 screen for abnormal eye phenotypes was used to identify inserts that express disruptive fusion proteins. Approximately 1.7% of new inserts cause eye phenotypes. Of the first 23 verified target genes, 21 can be described as regulators of cell biology and development. Most are transcription factor genes, including AP-2, CG17181, cut, klu, mamo, Sox102F, and sv. Other target genes [l(1)G0232, nuf, pum, and Syt4] make cytoplasmic proteins, and these lines produce diverse fluorescence localization patterns. Hto permits the expression of stable carboxy-terminal subfragments of proteins, which are rarely tested in conventional genetic screens. Some of these may disrupt specific cell pathways, as exemplified by truncated forms of Mastermind and Nuf.

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The DNA transposon Minos as a tool for transgenesis and functional genomic analysis in vertebrates and invertebrates

Cited by 42 publications

References 60 publications

A versatile strategy for gene trapping and trap conversion in emerging model organisms

A versatile strategy for gene trapping and trap conversion in emerging model organisms

Transposon tools hopping in vertebrates

Inducible Protein Traps with Dominant Phenotypes for Functional Analysis of theDrosophilaGenome

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