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Essletzbichler P.,Haplogen GmbH | Konopka T.,Austrian Academy of Sciences | Santoro F.,Haplogen GmbH | Santoro F.,Karolinska Institutet | And 8 more authors.
Genome Research | Year: 2014

Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second gene copy. However, no completely haploid human cell line has been described, hampering the genetic accessibility of a subset of genes. The near-haploid human cell line HAP1 contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15. This large fragment encompasses 330 genes and is integrated on the long arm of Chromosome 19. Here, we employ a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state. Importantly, spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping revealed that engineered-HAPloid (eHAP) cells are fully haploid with no gross chromosomal aberrations induced by Cas9. Furthermore, whole-genome sequence and transcriptome analysis of the parental HAP1 and an eHAP cell line showed that transcriptional changes are limited to the excised Chromosome 15 fragment. Together, we demonstrate the feasibility of efficiently engineering megabase deletions with the CRISPR/Cas9 technology and report the first fully haploid human cell line. © 2014 Essletzbichler et al.


Schmid B.,University of Heidelberg | Rinas M.,German Cancer Research Center | Rinas M.,University of Heidelberg | Ruggieri A.,University of Heidelberg | And 13 more authors.
PLoS Pathogens | Year: 2015

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells. © 2015 Schmid et al.


Burckstummer T.,Haplogen GmbH | Banning C.,Haplogen GmbH | Hainzl P.,Haplogen GmbH | Hainzl P.,Austrian Academy of Sciences | And 37 more authors.
Nature Methods | Year: 2013

Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-α, TGF-β, IFN-γ and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.


Durnberger G.,Austrian Academy of Sciences | Durnberger G.,IMP Research Institute of Molecular Pathology | Burckstummer T.,Austrian Academy of Sciences | Burckstummer T.,Haplogen GmbH | And 15 more authors.
Genome Biology | Year: 2013

Background: The interactions between proteins and nucleic acids have a fundamental function in many biological processes, including gene transcription, RNA homeostasis, protein translation and pathogen sensing for innate immunity. While our knowledge of the ensemble of proteins that bind individual mRNAs in mammalian cells has been greatly augmented by recent surveys, no systematic study on the non-sequence-specific engagement of native human proteins with various types of nucleic acids has been reported.Results: We designed an experimental approach to achieve broad coverage of the non-sequence-specific RNA and DNA binding space, including methylated cytosine, and tested for interaction potential with the human proteome. We used 25 rationally designed nucleic acid probes in an affinity purification mass spectrometry and bioinformatics workflow to identify proteins from whole cell extracts of three different human cell lines. The proteins were profiled for their binding preferences to the different general types of nucleic acids. The study identified 746 high-confidence direct binders, 139 of which were novel and 237 devoid of previous experimental evidence. We could assign specific affinities for sub-types of nucleic acid probes to 219 distinct proteins and individual domains. The evolutionarily conserved protein YB-1, previously associated with cancer and drug resistance, was shown to bind methylated cytosine preferentially, potentially conferring upon YB-1 an epigenetics-related function. Conclusions: The dataset described here represents a rich resource of experimentally determined nucleic acid-binding proteins, and our methodology has great potential for further exploration of the interface between the protein and nucleic acid realms. © 2013 Dürnberger et al.; licensee BioMed Central Ltd.


Fischl W.,Haplogen GmbH | Bartenschlager R.,University of Heidelberg
Methods in Molecular Biology | Year: 2013

Large-scale antiviral drug discovery and systematic identification of host factors promoting or restricting virus replication require robust, scalable, and cost-effective assay systems that allow for high throughput and automation. Here we describe the construction and application of full-length infectious dengue virus reporter genomes for such screening applications. © 2013 Springer Science+Business Media, LLC.


Sieghart D.,Medical University of Vienna | Sieghart D.,Institute for Rheumatology and Balneology | Liszt M.,Institute for Rheumatology and Balneology | Liszt M.,Haplogen GmbH | And 6 more authors.
Journal of Cellular and Molecular Medicine | Year: 2015

Balneotherapy employing sulphurous thermal water is still applied to patients suffering from diseases of musculoskeletal system like osteoarthritis (OA) but evidence for its clinical effectiveness is scarce. Since the gasotransmitter hydrogen sulphide (H2S) seems to affect cells involved in degenerative joint diseases, it was the objective of this study to investigate the effects of exogenous H2S on fibroblast-like synoviocytes (FLS), which are key players in OA pathogenesis being capable of producing pro-inflammatory cytokines and matrix degrading enzymes. To address this issue primary FLS derived from OA patients were stimulated with IL-1β and treated with the H2S donor NaHS. Cellular responses were analysed by ELISA, quantitative real-time PCR, phospho-MAPkinase array and Western blotting. Treatment-induced effects on cellular structure and synovial architecture were investigated in three-dimensional extracellular matrix micromasses. NaHS treatment reduced both spontaneous and IL-1β-induced secretion of IL-6, IL-8 and RANTES in different experimental settings. In addition, NaHS treatment reduced the expression of matrix metallo-proteinases MMP-2 and MMP-14. IL-1β induced the phosphorylation of several MAPkinases. NaHS treatment partially reduced IL-1β-induced activation of several MAPK whereas it increased phosphorylation of pro-survival factor Akt1/2. When cultured in spherical micromasses, FLS intentionally established a synovial lining layer-like structure; stimulation with IL-1β altered the architecture of micromasses leading to hyperplasia of the lining layer which was completely inhibited by concomitant exposure to NaHS. These data suggest that H2S partially antagonizes IL-1β stimulation via selective manipulation of the MAPkinase and the PI3K/Akt pathways which may encourage development of novel drugs for treatment of OA. © 2015 John Wiley & Sons Ltd.


PubMed | Haplogen GmbH
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2013

Large-scale antiviral drug discovery and systematic identification of host factors promoting or restricting virus replication require robust, scalable, and cost-effective assay systems that allow for high throughput and automation. Here we describe the construction and application of full-length infectious dengue virus reporter genomes for such screening applications.

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