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Doudna J.A.,Howard Hughes Medical Institute | Doudna J.A.,University of California at Berkeley | Doudna J.A.,Lawrence Berkeley National Laboratory | Charpentier E.,Helmholtz Center for Infection Research | And 2 more authors.
Science | Year: 2014

The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics. © 2014, American Association for the Advancement of Science. All rights reserved.

Agency: Cordis | Branch: H2020 | Program: RIA | Phase: INFRADEV-4-2014-2015 | Award Amount: 9.04M | Year: 2015

Marine (blue) biotechnology is the key to unlocking the huge economic potential of the unique biodiversity of marine organisms. This potential remains largely underexploited due to lack of connectivity between research services, practical and cultural difficulties in connecting science with industry, and high fragmentation of regional research, development and innovation (RDI) policies. To overcome these barriers, EMBRIC (European Marine Biological Resource Infrastructure Cluster) will link biological and social science research infrastructures (EMBRC, MIRRI, EU-OPENSCREEN, ELIXIR, AQUAEXCEL, RISIS) and will build inter-connectivity along three dimensions: science, industry and regions. The objectives of EMBRIC are to: (1) develop integrated workflows of high quality services for access to biological, analytical and data resources, and deploy common underpinning technologies and practices; (2) strengthen the connection of science with industry by engaging companies and by federating technology transfer (TT) services; (3) defragment RDI policies and involve maritime regions with the construction of EMBRIC. Acceleration of the pace of scientific discovery and innovation from marine bioresources will be achieved through: (i) establishment of multidisciplinary service-oriented technological workflows; (ii) joint development activities focusing on bioprospection for novel marine natural products, and marker-assisted selection in aquaculture; (iii) training and knowledge transfer; (iv) pilot transnational access to cluster facilities and services. EMBRIC will also connect TT officers from contrasted maritime regions to promote greater cohesion in TT practices. It will engage with policy-makers with the aim of consolidating a perennial pan-European virtual infrastructure cluster rooted in the maritime regions of Europe and underpinning the blue bioeconomy.

Bussow K.,Helmholtz Center for Infection Research
Current Opinion in Structural Biology | Year: 2015

The mammalian cell lines HEK293 and CHO have become important expression hosts in structural biology. Generating stable mammalian cell lines remains essential for studying the function and structure of recombinant proteins, despite the emergence of highly efficient transient transfection protocols. Production with stable cell lines can be scaled up easily and high volumetric product yield can be achieved. Protein structure reports of the past two years that used stable cell lines were surveyed for this review. Well-established techniques and novel approaches for generating stable cell lines and stable cell pools are presented, including cell sorting, site-specific recombination, transposons, the Lentivirus system and phage integrases. Host cell line optimization by endoglycosidase overexpression and sequence-specific genome engineering is highlighted. © 2015 Published by Elsevier Ltd.

A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification. High level production of HBsAg was carried out with recombinant Pichia pastoris using the methanol inducible AOX1 expression system. The recombinant vaccine was isolated in form of VLPs after several down-stream steps from detergent-treated cell lysates. Search for the intracellular localization of the antigen using electron microscopic studies in combination with immunogold labeling revealed the presence of HBsAg in an extended endoplasmic reticulum where it was found to assemble into defined multi-layered, lamellar structures. The distance between two layers was determined as ~6 nm indicating that these lamellas represent monolayers of well-ordered HBsAg subunits. We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell. It is concluded that high level production and intrinsic slow HBsAg VLP assembly kinetics are leading to retention and accumulation of the antigen in the endoplasmic reticulum where it assembles at least partly into defined lamellar structures. Further transport of HBsAg to the Golgi apparatus is impaired thus leading to secretory pathway disfunction and the formation of an extended endoplasmic reticulum which bulges into irregular cloud-shaped formations. As VLPs were not found within the cells it is concluded that the VLP assembly process must take place during down-stream processing after detergent-mediated disassembly of HBsAg lamellas and subsequent reassembly of HBsAg into spherical VLPs.

Crouse J.,ETH Zurich | Kalinke U.,Helmholtz Center for Infection Research | Oxenius A.,ETH Zurich
Nature Reviews Immunology | Year: 2015

Type I interferons (IFNs) are pro-inflammatory cytokines that are rapidly induced in different cell types during viral infections. The consequences of type I IFN signalling include direct antiviral activity, innate immune cell activation and regulation of adaptive immune responses. In this Review, we discuss recent conceptual advances in our understanding of indirect and direct regulation of T cell immunity by type I IFNs, which can either promote or inhibit T cell activation, proliferation, differentiation and survival. This regulation depends, to a large extent, on the timing of type I IFN exposure relative to T cell receptor signalling. Type I IFNs also provide activated T cells with resistance to natural killer cell-mediated elimination. © 2015 Macmillan Publishers Limited. All rights reserved.

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