An efficient strategy for TALEN-mediated genome engineering in Drosophila

In reverse genetics, a gene's function is elucidated through targeted modifications in the coding region or associated DNA cis-regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila. We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F1 generatio

An intrinsic tumour eviction mechanism in Drosophila mediated by steroid hormone signalling

Polycomb group proteins are epigenetic regulators maintaining transcriptional memory during cellular proliferation. In Drosophila larvae, malfunction of Polyhomeotic (Ph), a member of the PRC1 silencing complex, results in neoplastic growth. Here, we report an intrinsic tumour suppression mechanism mediated by the steroid hormone ecdysone during metamorphosis. Ecdysone alters neoplastic growth into a nontumorigenic state of the mutant ph cells which then become eliminated during adult stage. We demonstrate that ecdysone exerts this function by inducing a heterochronic network encompassing the activation of the microRNA lethal-7, which suppresses its target gene chronologically inappropriate morphogenesis. This pathway can also promote remission of brain tumours formed in brain tumour mutants, revealing a restraining of neoplastic growth in different tumour types. Given the conserved role of let-7, the identification and molecular characterization of this innate tumour eviction mechanism in flies might provide important clues towards the exploitation of related pathways for human tumour therapy.ISSN:2041-172

An efficient strategy for TALEN-mediated genome engineering in Drosophila

In reverse genetics, a gene’s function is elucidated through targeted modifications in the coding region or associated DNA cis -regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila . We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F 1 generation.ISSN:1362-4962ISSN:0301-561

A Deterministic Analysis of Genome Integrity during Neoplastic Growth in <i>Drosophila</i>

<div><p>The development of cancer has been associated with the gradual acquisition of genetic alterations leading to a progressive increase in malignancy. In various cancer types this process is enabled and accelerated by genome instability. While genome sequencing-based analysis of tumor genomes becomes increasingly a standard procedure in human cancer research, the potential necessity of genome instability for tumorigenesis in <i>Drosophila melanogaster</i> has, to our knowledge, never been determined at DNA sequence level. Therefore, we induced formation of tumors by depletion of the <i>Drosophila</i> tumor suppressor Polyhomeotic and subjected them to genome sequencing. To achieve a highly resolved delineation of the genome structure we developed the Deterministic Structural Variation Detection (DSVD) algorithm, which identifies structural variations (SVs) with high accuracy and at single base resolution. The employment of long overlapping paired-end reads enables DSVD to perform a deterministic, i.e. fragment size distribution independent, identification of a large size spectrum of SVs. Application of DSVD and other algorithms to our sequencing data reveals substantial genetic variation with respect to the reference genome reflecting temporal separation of the reference and laboratory strains. The majority of SVs, constituted by small insertions/deletions, is potentially caused by erroneous replication or transposition of mobile elements. Nevertheless, the tumor did not depict a loss of genome integrity compared to the control. Altogether, our results demonstrate that genome stability is not affected inevitably during sustained tumor growth in <i>Drosophila</i> implying that tumorigenesis, in this model organism, can occur irrespective of genome instability and the accumulation of specific genetic alterations.</p></div

Summary of PCR-based validation experiments.

<p>SVs are considered to be confirmed whenever at least one aberrant allele was detected either within <i>ph</i>-RNAi induced tumors or the parental strains irrespective of the zygosity. The size range of a tandem duplication corresponds to a single duplication event. n =  Number of tested events; Het  =  expected to be heterozygous; Hom  =  expected to be homozygous; Small ins.  =  Small insertions; Tan. dupl.  =  tandem duplications.</p

Engineering fibronectin-templated multi-component fibrillar extracellular matrices to modulate tissue-specific cell response

Cells assemble fibronectin, the major extracellular matrix (ECM) protein, into fibrillar matrices, which serve as 3D architectural scaffolds to provide, together with other ECM proteins tissue-specific environments. Although recent approaches enable to bioengineer 3D fibrillar fibronectin matrices in vitro, it remains elusive how fibronectin can be co-assembled with other ECM proteins into complex 3D fibrillar matrices that recapitulate tissue-specific compositions and cellular responses. Here, we introduce the engineering of fibrillar fibronectin-templated 3D matrices that can be complemented with other ECM proteins, including vitronectin, collagen, and laminin to resemble ECM architectures observed in vivo. For the co-assembly of different ECM proteins, we employed their innate fibrillogenic mechanisms including shear forces, pH-dependent electrostatic interactions, or specific binding domains. Through recapitulating various tissue-specific ECM compositions and morphologies, the large scale multi-composite 3D fibrillar ECM matrices can guide fibroblast adhesion, 3D fibroblast tissue formation, or tissue morphogenesis of epithelial cells. In other examples, we customize multi-composite 3D fibrillar matrices to support the growth of signal propagating neuronal networks and of human brain organoids. We envision that these 3D fibrillar ECM matrices can be tailored in scale and composition to modulate tissue-specific responses across various biological length scales and systems, and thus to advance manyfold studies of cell biological systems.ISSN:1878-5905ISSN:0142-961

Coding sequences are less susceptible to SV accumulation.

<p>(A) Genome browser view depicting the concordant and discordant coverage of the control (blue) and the tumor (red) samples across two protein-coding genes, and identified SVs therein. The detected insertions and deletions localize outside of coding sequences, and affect introns, intergenic spaces and UTRs. (B) Genome-wide breakpoint distribution across distinct functional compartments. Different subsets of the genome were selected according to following characteristics: <i>genome</i> corresponds to the full-length genome; the <i>unique genes</i> do not share common positions with any other gene; <i>overlapping genes</i> are non-unique genes; <i>exonic</i> regions, containing <i>3′UTRs, 5′UTRs</i> and coding sequences (<i>CDS</i>) were obtained from the unique genes in order to avoid ambiguity; In addition, <i>intronic</i> and <i>intergenic</i> regions as well as donor/acceptor splice sites (<i>splice sites</i>) were considered. For each subset the number of contained breakpoints was computed and normalized to the total length.</p

Depletion of <i>polyhomeotic (ph)</i> induces neoplastic tumors.

<p>A) Ph expression in the normal wing disc of third instar larvae (left) expressing the reporter <i>en-GAL4 </i> UAS-<i>myr-RFP</i>, UAS-<i>Dicer2</i>, <i>NRE:EGFP</i> (right). B) Downregulation of Ph induced by the RNAi reporter observed in the posterior compartment. Posterior compartment (red RFP) shows overproliferation phenotype (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087090#pone.0087090-Saj1" target="_blank">[26]</a>). C) Schematic outline of the experimental workflow. Eggs of the same genotype were developed at different temperatures. The tumor suppressor <i>ph</i> is specifically depleted (RNAi) at 25°C within the posterior compartment (p, RFP signal in red) of wing imaginal discs, leading to the formation of large tumors (upper). To allow for the accumulation of SVs, tumors are transplanted for a period of four weeks. At 18°C depletion of Ph is not sufficient to drive tumorigenesis (lower) and corresponding wing imaginal discs were used as control. Genomic DNA from both samples was isolated and subjected to paired-end sequencing. Notch-dependent EGFP expression (green) marks the boundary of the dorsal and ventral compartments. The white dashed outline marks the remnant anterior compartment (a) with normal Notch signaling along the dorsal/ventral boundary, while the grey dashed outline labels the haltere disc (hd).</p

Genomic context analysis can indicate mutational mechanisms causing SVs.

<p>(A) DNA sequence and insertion frequency of the 10 most commonly inserted sequences identified within the control genome. For the tumor the tenth most frequently inserted sequence corresponds to CA with 897 insertions. For the sake of a clear representation the eleventh most frequently inserted sequence (AAA, 894 insertions) is shown. (B) The fraction of single base insertions within simple repeats consisting of the same base type, computed with respect to all single base insertions. Simple repeats of a minimum length of 4 were considered. (C) A genome browser view of a genomic locus containing two insertions (I/V), two deletions (II/IV) and one tandem duplication (III). As indicated by the discordant coverage and horizontal bars, these high-confidence SVs are both identified within the tumor and the control genomes, and have therefore been inherited from the parental strains.</p