MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

Venken, Koen J. T.; Schulze, Karen L.; Haelterman, Nele A; Pan, Hongling; He, Yuchun; Evans-Holm, Martha; Carlson, Joseph W.; Levis, Robert; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.

doi:10.1038/nmeth.1662

Cited by 655 publications

(819 citation statements)

References 79 publications

(118 reference statements)

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“…1). This study adds useful resources to the collection of phiC31 lines currently available (29). We find that reporters in a wide range of chromatin domains are sensitive to 1360; thus, the influence of 1360 is not limited to repeat-rich, heterochromatic sites.…”

Section: Discussionmentioning

confidence: 83%

Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements

Sentmanat

Elgin

2012

Proc. Natl. Acad. Sci. U.S.A.

104

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A persistent question in biology is how cis-acting sequence elements influence trans-acting factors and the local chromatin environment to modulate gene expression. We reported previously that the DNA transposon 1360 can enhance silencing of a reporter in a heterochromatic domain of Drosophila melanogaster. We have now generated a collection of variegating phiC31 landingpad insertion lines containing 1360 and a heat-shock protein 70 (hsp70)-driven white reporter to explore the mechanism of 1360-sensitive silencing. Many 1360-sensitive sites were identified, some in apparently euchromatic domains, although all are close to heterochromatic masses. One such site (line 1198; insertion near the base of chromosome arm 2L) has been investigated in detail. ChIP analysis shows 1360-dependent Heterochromatin Protein 1a (HP1a) accumulation at this otherwise euchromatic site. The phiC31 landing pad system allows different 1360 constructs to be swapped with the full-length element at the same genomic site to identify the sequences that mediate 1360-sensitive silencing. Short deletions over sites with homology to PIWI-interacting RNAs (piRNAs) are sufficient to compromise 1360-sensitive silencing. Similar results were obtained on replacing 1360 with Invader4 (a retrotransposon), suggesting that this phenomenon likely applies to a broader set of transposable elements. Our results suggest a model in which piRNA sequence elements behave as cis-acting targets for heterochromatin assembly, likely in the early embryo, where piRNA pathway components are abundant, with the heterochromatic state subsequently propagated by chromatin modifiers present in somatic tissue.epigenetics | position-effect variegation T ransposable elements (TEs) are major structural features of nearly all eukaryotic genomes, and can play a role as cisacting regulatory features with profound influence over the gene regulatory networks of their host (1). However, if left unchecked, the deleterious effects of TE mobilization can become insurmountable for the system, damaging genome integrity. Thus, silencing mechanisms that prevent TE mobilization are fundamental to the faithful propagation of genetic information. Position-effect variegation (PEV), a mosaic expression pattern resulting from silencing in some cells that normally express a gene, has frequently been used as an indicator of heterochromatic environments (2, 3). We reported previously that the DNA transposon 1360 can enhance reporter silencing in repeat-rich heterochromatic regions (4). To gain insight into the mechanisms involved, we sought to identify sequence elements present in 1360 that contribute to 1360-sensitive PEV, using an adjacent heat-shock protein 70 (hsp70)-driven white gene as the reporter.We used the phiC31 landing pad system, where phiC31 integrase mediates recombination between a landing pad (inserted in the genome) containing phage attachment (attP) sites and a donor construct containing bacterial attachment (attB) sites (5). This system allows us to maintain a constant genomic cont...

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Section: Discussionmentioning

confidence: 83%

Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements

Sentmanat

Elgin

2012

Proc. Natl. Acad. Sci. U.S.A.

104

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“…Transposon mutagenesis, albeit suffering from insertion bias [72], allows for easy retrieval of positional information, and forms the basis for a downstream toolkit of genetic applications including imprecise excision knock-out, Gal4-UAS overexpression of flanking genes, or element replacement by targeting vectors, to name but a few [73][74][75][76][77]. Extensive libraries of P-element-based transposon insertions are available through stock centers, along with deletion and duplication lines [78][79][80][81]. Finally, targeted gene knock-out using optimized targeting plasmids in combination with CRISPR will greatly accelerate full KO coverage of the fly genome [82].…”

Section: Methods To Generate Immune Deficient Cells Tissues or Organmentioning

confidence: 99%

Methods to study Drosophila immunity

Neyen

Bretscher

Binggeli

et al. 2014

Methods

127

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a b s t r a c tInnate immune mechanisms are well conserved throughout evolution, and many theoretical concepts, molecular pathways and gene networks are applicable to invertebrate model organisms as much as vertebrate ones. Drosophila immunity research benefits from an easily manipulated genome, a fantastic international resource of transgenic tools and over a quarter century of accumulated techniques and approaches to study innate immunity. Here we present a short collection of ways to challenge the fruit fly immune system with various pathogens and parasites, as well as read-outs to assess its functions, including cellular and humoral immune responses. Our review covers techniques for assessing the kinetics and efficiency of immune responses quantitatively and qualitatively, such as survival analysis, bacterial persistence, antimicrobial peptide gene expression, phagocytosis and melanisation assays. Finally, we offer a toolkit of Drosophila strains available to the research community for current and future research.

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“…Several methods have been developed to swap transcription factor drivers between transgenic binary expression systems. However, this requires either de novo generation of new driver lines [integrase swappable in vivo targeting element (InSITE)] (Gohl et al 2011) or PhiC31-induced insertion of a transcription factor cassette into an existing minos mediated integration cassette (MiMIC) genomic insertion (Venken et al 2011;Diao et al 2015;Gnerer et al 2015).Here, we developed a method that converts existing transgenic GAL4 lines into QF2 lines with two simple genetic crosses. This method uses the CRISPR/Cas9 system to induce DSBs in transgenic GAL4, and HDR to drive conversion of GAL4 into a QF2-expressing line through the use of a single transgenic donor cassette in the genome.…”

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confidence: 99%

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Editing Transgenic DNA Components by Inducible Gene Replacement in Drosophila melanogaster

Lin¹,

2016

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Gene conversions occur when genomic double-strand DNA breaks (DSBs) trigger unidirectional transfer of genetic material from a homologous template sequence. Exogenous or mutated sequence can be introduced through this homology-directed repair (HDR). We leveraged gene conversion to develop a method for genomic editing of existing transgenic insertions in Drosophila melanogaster. The clustered regularly-interspaced palindromic repeats (CRISPR)/Cas9 system is used in the homology assisted CRISPR knock-in (HACK) method to induce DSBs in a GAL4 transgene, which is repaired by a single-genomic transgenic construct containing GAL4 homologous sequences flanking a T2A-QF2 cassette. With two crosses, this technique converts existing GAL4 lines, including enhancer traps, into functional QF2 expressing lines. We used HACK to convert the most commonly-used GAL4 lines (labeling tissues such as neurons, fat, glia, muscle, and hemocytes) to QF2 lines. We also identified regions of the genome that exhibited differential efficiencies of HDR. The HACK technique is robust and readily adaptable for targeting and replacement of other genomic sequences, and could be a useful approach to repurpose existing transgenes as new genetic reagents become available.KEYWORDS gene conversion; CRISPR/Cas9; homology-directed repair; genomic engineering; updating transgenes D OUBLE-strand DNA breaks (DSBs) in genomic DNA are potentially lethal to the cell as they are substrates for genomic rearrangements. Two major endogenous repair mechanisms exist to resolve such DNA damage. Nonhomologous end-joining (NHEJ) involves ligation of the damaged ends and often results in small insertion/deletion (indel) mutations at the site of the DSB (Lieber 2010). This causes frameshifting and the expression of mutated or nonfunctional protein. In contrast, homologous recombination (HR) uses a homologous template for repair to preserve genomic integrity. Gene conversion is one form of HR and involves unidirectional transfer of genetic material from a donor sequence to a highly homologous target (Engels et al. 1990;Gloor et al. 1991;Chen et al. 2007). Donors can be from the sister chromosome (allelic gene conversion) or dispersed sequences not at the same locus (ectopic gene conversion).Current genomic engineering methods use a DSB induced at a target genomic locus to generate disrupting mutations, driven by NHEJ, or to introduce new genetic sequences, driven by homology-directed repair (HDR). Several techniques have been developed to introduce target-specific DSBs, including zincfinger nucleases, transcription-activator-like effecter nucleases, and, most recently, clustered regularly-interspaced palindromic repeats (CRISPR) (Jinek et al. 2012;Doudna and Sontheimer 2014). Cas9 is the endonuclease of the type II CRISPR system and creates a DSB at genomic locations directed by a guide RNA (gRNA). The CRISPR/Cas9 system is favored for its high efficiency and ability to use a small gRNA to specifically target most genomic DNA locations (Cong et al. 2013;Hsu et a...

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MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes

Cited by 655 publications

References 79 publications

Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements

Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements

Methods to study Drosophila immunity

Editing Transgenic DNA Components by Inducible Gene Replacement in Drosophila melanogaster

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