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  • Periodic Report Summary 2 - INFRAFRONTIER-I3 (Development of mouse mutant resources for functional analyses of human diseases - Enhancing the translation of research into innovation)


Project ID: 312325
Country: Germany

Periodic Report Summary 2 - INFRAFRONTIER-I3 (Development of mouse mutant resources for functional analyses of human diseases - Enhancing the translation of research into innovation)

Project Context and Objectives:
INFRAFRONTIER-I3 project objectives
The mouse shows great similarities in development, physiology and biochemistry to humans, which makes it a key model for research into human disease. The major challenges for mouse functional genomics in the 21st century are to 1) Develop a series of mutant alleles for every gene in the mouse genome, 2) Determine the phenotypic consequences of each mutation, and 3) Identify mouse models for the complete disease spectrum in humans. To further develop and exploit the emerging mouse mutant resource, mouse models must be preserved and made available to the European biomedical research community. To this effect, the INFRAFRONTIER-I3 project brings together the leading European centers for systemic phenotyping of mouse mutants and the European Mouse Mutant Archive network. The INFRAFRONTIER-I3 partners aim to meet the future challenges presented by phenotyping, archiving and disseminating mouse models in the ERA as follows:

• Contribute to resource development by archiving of 1215 new mouse mutant lines
• Provide Transnational Access (TA) to mouse production and 1st line phenotyping capacities
• Offer a specialized axenic service to produce, maintain and to distribute germ-free mice
• Provide user friendly accession to INFRAFRONTIER services, extensive manual data curation and cross referencing with other mouse databases
• Improve user services by developing novel phenotyping and cryopreservation SOPs and by refining innovative research instrumentation
• Engage with the user community using a wide range of PR activities, a dedicated user meeting and an industry liaison workshop
• Offer state of the art cryopreservation and phenotyping training courses
• Benchmark INFRAFRONTIER services with other major repositories

Project Results:
INFRAFRONTIER-I3 project - activities and results
During the first and second reporting periods the INFRAFRONTIER-I3 project made excellent progress. Major achievements are:

• Archiving of 1287 new mouse mutant lines by the EMMA repository
• Execution of 5 Transnational Access calls and allocation of all 52 user projects leading to the development and characterization of new mouse models of human diseases
• Innovation of Metabolic PhenoCages refining the measurement of energy balance regulation in mutant mice
• Technology development progress leading to 5 new SOPs, the publication of four methods papers, and the development of pilot multiplex-assays for biomarker phenotyping
• Launch of new INFRAFRONTIER web portal with user friendly accession to all INFRAFRONTIER resources and services
• Data integration with the International Mouse Phenotyping Consortium (IMPC)
• Extensive outreach covering an industry liaison meeting, a gnotobiology workshop, 60 presentations at scientific meetings, and 11 training courses for 124 students
• Deployment of Tableau business intelligence software and benchmarking of INFRAFRONTIER services

WP2 Communication and user interaction
A major achievement during the first reporting period was the re-launch of the INFRAFRONTIER web portal at, which is the major interface to the INFRAFRONTIER user community. The web portal provides background information on the organisation of INFRAFRONTIER and access to all resources and data. The web site traffic increased significantly and at present the INFRAFRONTIER portal has ~ 6000 unique users per month. Internal communication among all partners of the network is largely facilitated by general project meetings. During the first & second project period a total of six general network meetings were organized for up to 90 participants including network members, external guests and Advisory Board members. INFRAFRONTIER outreach activities cover ~ 60 presentations by network partners at scientific and policy meetings. Key contributions were presentations at a dedicated symposium on mouse phenotyping for clinical research at the 2015 European Society for Human Genetics Conference in Glasgow. Industry users were specifically targeted via an industry liaison meeting that was held in conjunction with the International Mouse Phenotyping Consortium (IMPC) annual meeting in November 2014. A major achievement in period 1 was the closing of an agreement with Genoway allowing industry scientists access to the EUCOMM resources. A major element of the INFRAFRONTIER outreach activities are hands on training courses. During periods 1 & 2 a total of 8 cryocourses for about 60 students from across Europe, and 3 phenotyping courses for 64 students were held.

WP3 Innovation
The objective of the innovation work package is to combine a typical indirect calorimetry set-up with features of metabolic cages, allowing the sampling and quantification of urine and feces in parallel with gas exchange measurements. Feeding and drinking behavior will be monitored along with activity measurement. This innovation will refine the measurement of energy balance regulation in mutant mice and may facilitate the characterization of new animal models for human disorders. Towards this goal we evaluated and documented the current state of technology for Metabolic PhenoCage (MPC) systems as prerequisite for the development of a refined MPC module. Three key topics for improvement of components were identified: 1) separation quality of feces and urine, 2) functionality and animal welfare issues regarding water uptake, and 3) design and material of cage floor. These elements were assessed in detail as prerequisite for the overall design of the MPC. Very specific design options were compared in trials not using live mice to achieve highly standardized conditions. These trials focused on the choice of material (glass, plastic material), surface appearance (e.g. groove shape) and surface properties (e.g. coating with hydrophobic nanomaterials). In the second reporting period designs of the separation method based on a groove-shaped surface and using a special plastic material were evaluated and refined with extensive animal tests which are still ongoing. Different models of drinking nipples were tested for the purpose of facilitating the drinking process for the animals and making it more enjoyable (animal welfare). The bottom of cage floors was re-designed under animal welfare aspects and tests with animals are ongoing. A key innovative step during the development of refined MPCs will be the implementation of indirect calorimetry in combination with the collection of feces and urine. Currently, a set-up is being evaluated to allow air flows and circulation suitable for gas exchange measurements.

WP4 Cryopreservation of mouse mutant lines
A major activity of the INFRAFRONTIER-I3 project is the archiving of up to 1215 mouse mutant lines as either embryos or sperm. Archived lines are made available via the INFRAFRONTIER portal (, and the IMSR database at Overall, a total of 1636 mouse lines were submitted during the course of the first & second INFRAFRONTIER-I3 project periods, thus exceeding the projected milestone of 914 mouse lines by far. A total of 1287 mouse mutant lines are archived and quality controlled and available to the wider community. A total of 41% of the reported lines account for community contributed lines and the remainder of lines originate from project efforts such as EUCOMMtools. The median archiving time for embryo freezing was 3.1 months, for sperm freezing 2.8 months, respectively. Most submitted lines were targeted mutations, mainly KO mutations due to the contributions from large scale projects to the International Mouse Phenotyping Consortium (IMPC). Strain donations were made across Europe with major contributions from Germany, UK and France. Mouse mutant lines were donated by a total of 240 different submitters or institutions. The INFRAFRONTIER-I3 WP4 activities contributed significantly to the current EMMA archive of 5545 mouse mutant lines and firmly established EMMA as the third largest mouse repository worldwide.

WP5 Technology development and implementation / cryotechnologies
The first & second reporting periods have seen some excellent progress on developing novel protocols for transporting germplasm in the absence of LN2. These innovations have come about through the coordinated efforts of the INFRANFRONTIER partners in collaboration with colleagues in Japan and China. A simple, inexpensive and robust protocol has been developed that facilitates the transportation of frozen sperm on dry-ice. A second protocol describing the transportation of unfrozen sperm retained in the epididymides has been prepared. This protocol uses a proprietary tissue transportation medium (Lifor) that maintains the fertility of the sperm for >72 hours at refrigerated temperatures (4-8°C). Overall five SOPs were published on the INFRAFRONTIER website along with information sheets and training videos. Furthermore, four methods papers were published in ‘Current Protocols in Mouse Biology’. Significant progress has also been made with the ICSI program. We are able to consistently produce pups from mice on an inbred or mixed background using either freshly harvested sperm or frozen/thawed sperm. In addition, we initiated a new program to develop a robust embryo and oocyte verification protocol that is compatible with storage in plastic semen straws, rather than cryo-vials. We have developed a protocol that works extremely well for embryo freezing, but oocyte vitrification needs further optimization to improve IVF success rates. Complementary materials such as cool boxes and cooling plates were developed jointly with companies and are commercially available.

WP5 Technology development and implementation / phenotyping
The objectives of the phenotyping technology development efforts are to carry out technology evaluation and piloting of new assays that will facilitate high-throughput analyses, enhance user services for new types of data unmet by the classical assays, reduce the cost of services and reduce the number of animals used in research. The goal of a first task is the piloting of high-throughput µCT imaging in order to identify relevant phenotypes in adult tissues as well as development defects during embryogenesis through 3-D image reconstruction. μCT is a preferred technology platform since it has very high resolution, is non-invasive and supports 3-D reconstruction. Using contrast agents, it is now possible to achieve excellent soft tissue detail. In an extensive series of validation experiments the optimal conditions for the µCT imaging of E18.5 mouse embryos using Lugol as a contrast agent were determined, and this led to the development of a SOP. The imaging of adult mice focused on the validation of μCT covering different tissues such as lung, liver and the CNS. The µCT imaging was successfully used to visualize and monitor in longitudinal studies the health or disease status of the lungs and airways. Using µCT imaging of liver tumors and iodinated α-tocopherol nano-emulsions as contrast agent allowed us to discriminate the tumor from the same tissue and to monitor longitudinally the progress of the disease by quantifying the liver tumor volume at several time-points. Under development is a pipeline for automated and fast anatomical phenotyping of mouse brains using µCT. A further major task of the phenotyping technology development efforts is biomarker phenotyping and the evaluation of multiplex assays for profiling and discovery. Goal of this task is to evaluate multiplex assays for profiling biomarkers in adult mice to deliver a novel multiplex series with small sample volume requirements that is amenable to automation and suitable for biomarker detection in various fluids / tissues. Towards this end the work package partners compiled a comprehensive list of markers covering a range of physiological systems, including the skeletal, cardiac, renal, metabolic, and immune system. Subsequently, the top 3 biomarkers from each area were used for the development and analysis of pilot panels. Two comprehensive 5- and 4-plex trial assays were undertaken by 3 mouse clinics in collaboration with the German biotech company AYOXXA, comparing their innovative LUNARIS technology platform to established technologies. Data analysis was still underway by the end of period 2.

WP7 Axenic service
The overall objective of this work package is to provide wild type and genetically modified animals raised in germ-free (GF) conditions to interested INFRAFRONTIER / EMMA customers. During the 1st and 2nd reporting 6 requests for the derivation of germ-free mice were successfully concluded, and 2 clients were provided germ-free mice that are kept on shelf. Furthermore, the Gulbenkian Institute providing this service joined the EC-Gnoto initiative, a European consortium dedicated exclusively to mice axenization and gnotobiology. A Gnotobiology Workshop covering technological and scientific issues was attended by ~ 80 participants on March 26-27 at the Instituto Gulbenkian de Ciência. Thus, by the end of period 2 all project deliverables are fulfilled.

WP6 Informatics
INFRAFRONTIER informatics is an overarching RTD activity with the objective to coordinate and integrate informatics activities necessary to continue development of the database, curation of mutant mouse strains, to improve user experience, and to develop data analysis tools to control and improve INFRAFRONTIER services. A first key milestone was the release of the INFRAFRONTIER portal which has been comprehensively described in the NAR database issues 2015. The manual curation of the database is critical to ensure mutant model data accuracy and consistency and their correct public and internal access. At the end of the second reporting period of the INFRAFRONTIER-I3 project 2556 lines in total had their background, transgene / genes / alleles and strain names / symbols manually reviewed and curated, with 326 more lines curated during the 2nd reporting period. We have continued to improve the EMMA database schema to better integrate with EMMA partners and to facilitate transfer of information about mutant mouse strains generated by INFRAFRONTIER partners and archived with the EMMA resource. This is critical as initiatives such as the International Mouse Phenotyping Consortium (IMPC) and the EUCOMMTools project are generating thousands of new mutant mouse strains in Europe. In the second reporting period this includes specifically major modifications to import mouse mutant lines generated using genome editing technologies such as CRISPR/Cas9. In conjunction with automated methods for importing strain data, we have also enhanced data submission by means of a new online data submission form guiding submitters through an 11-step process to record all the necessary data needed to deposit a strain. In addition to mouse strains, INFRAFRONTIER is now aiding in the distribution of a unique archive of embryonic stem cells (ESCs) derived from validated genetically engineered mouse models of cancer. The INFRAFONTIER partner Netherlands Cancer Institute (NKI) is developing this resource and INFRAFRONTIER informatics was involved in providing an online resource to allow users to obtain information and order these ESC clones. In the 2nd reporting period we have added additional clones, allele maps, protocols and links to publications describing the resource. Informatics provided further support for a benchmark analysis of INFRAFRONTIER services and the submission of reports to the INFRAFRONTIER-I3 management. Such analysis requires access to different data types within the EMMA resource. Towards this we have deployed Tableau analysis software on the EMMA resource and have provided access to it to INFRAFRONTIER partners.

Transnational Access activities are a major element of the INFRAFRONTIER-I3 project covering mouse production (WP8) and first line phenotyping services (WP9). As a first step towards implementation a common process to promote and evaluate TA applications was developed that involves an external Evaluation Committee. The Committee was actively involved in defining the evaluation process and selection criteria. During the first reporting period a total of 3 mouse production TA calls were published and fully executed, resulting in the assignment of all available 30 TA slots to successful applicants. Overall, a total of 130 project proposals from applicants across 17 eligible countries were submitted and evaluated. Successful project proposals cover the production of new mouse models for cancer research, motoneuron diseases, type 1 diabetes, metabolic diseases, liver diseases and mitochondrial diseases, amongst others. Excellent progress has been made in period 2 and for all 30 projects chimeric mice were generated, and for a total of 24 projects heterozygous mice were produced and shipped to selected applicants. All developed mutant lines will be archived in the EMMA repository to facilitate sharing and long term availability of these valuable new mouse models of human diseases. During the first reporting period a total of 2 mouse phenotyping TA calls were published and fully executed resulting in the assignment of 25 mouse phenotyping projects to successful applicants. Overall, a total of 76 project proposals from applicants across 14 eligible countries were submitted and evaluated. Successful project proposals cover e.g. a new spinocerebellar ataxia 7 (SCA7) mouse model, Down Syndrome models, and a G-protein coupled receptor GPR85 KO line and potential schizophrenia model, amongst others. Excellent progress has been made so far and for a total of 19 projects cohorts are being bred, for 14 projects phenotyping has started, and 9 phenotyping projects are completed.

Project management (WP1) underpins all INFRAFRONTIER-I3 activities. Its specific objectives are to set up an effective management framework for the INFRAFRONTIER-I3 project to ensure progress towards its planned objectives and contractual commitments. Major management activities were the design and implementation of the INFRAFRONTIER web portal (with WP6) and a benchmark analysis. As a first step, key performance indicators (KPIs) were defined as basis for the benchmark study. Following the implementation of the Tableau business intelligence software, a comprehensive set of reports for the defined KPIs and beyond were prepared and are now available at any time with current data. A report with INFRAFRONTIER data was compiled and sent to other major repositories such as CMMR, RIKEN and APN who engaged in this study and shared their performance data. The recorded INFRAFRONTIER data allow also an internal benchmark analysis as a basis to identify best practices and to improve overall performance of the network.

Potential Impact:
Expected project impact
The INFRAFRONTIER-I3 project contributed a large number of new mouse models to the EMMA repository. The projected further growth and development of the EMMA repository in the coming years will be essential to support the transition to a sustainable Research Infrastructure. INFRAFRONTIER-I3 and complementary efforts will lead to the EMMA repository holding more than 5500 mouse mutant lines in 2016. For many of these lines a comprehensive first line phenotypic screen will be undertaken and the data will be made freely available. This comprehensive and unique physical and data resource will support biomedical research in Europe by offering the opportunity to decipher molecular disease mechanisms and aiding the development of therapeutic strategies. Thus, the INFRAFRONTIER network will play a critical role in ensuring Europe maintains its leading role in the development of resources and knowledge of medically relevant mouse models by providing a user driven, sustainable platform offering access to unparalleled services and physical as well as data resources. The INFRAFRONTIER-I3 activities will further lead to significant advances in the operation and user services of the Research Infrastructure. Knowledge generated by the project will be widely disseminated by the website, presentations, scientific publications and SOPs and will contribute to standard development. Overall, the INFRAFRONTIER-I3 project will contribute to the further development of the European Research Area (ERA), and will support and interact with global initiatives such as the International Mouse Phenotyping Consortium (IMPC) and the Federation of International Mouse Resources (FIMRe).

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