MouseNet© database: digital management of a large-scale mutagenesis project

The Munich ENU Mouse Mutagenesis Screen is a large-scale mutant production, phenotyping, and mapping project. It encompasses two animal breeding facilities and a number of screening groups located in the general area of Munich. A central database is required to manage and process the immense amount of data generated by the mutagenesis project. This database, which we named MouseNet?, runs on a Sybase platform and will finally store and process all data from the entire project. In addition, the system comprises a portfolio of functions needed to support the workflow management of the core facility and the screening groups. MouseNet? will make all of the data available to the participating screening groups, and later to the international scientific community. MouseNet? will consist of three major software components: • Animal Management System (AMS) • Sample Tracking System (STS) • Result Documentation System (RDS) MouseNet? provides the following major advantages: • being accessible from different client platforms via the Internet • being a full-featured multi-user system (including access restriction and data locking mechanisms) • relying on a professional RDBMS (relational database management system) which runs on a UNIX server platform • supplying workflow functions and a variety of plausibility checks. Received: 20 December 1999 / Accepted: 20 December 1999     

Exploring the elephant: histopathology in high-throughput phenotyping of mutant mice

Recent advances in gene knockout techniques and the in vivo analysis of mutant mice, together with the advent of large-scale projects for systematic mouse mutagenesis and genome-wide phenotyping, have allowed the creation of platforms for the most complete and systematic analysis of gene function ever undertaken in a vertebrate. The development of high-throughput phenotyping pipelines for these and other large-scale projects allows investigators to search and integrate large amounts of directly comparable phenotype data from many mutants, on a genomic scale, to help develop and test new hypotheses about the origins of disease and the normal functions of genes in the organism. Histopathology has a venerable history in the understanding of the pathobiology of human and animal disease, and presents complementary advantages and challenges to in vivo phenotyping. In this review, we present evidence for the unique contribution that histopathology can make to a large-scale phenotyping effort, using examples from past and current programmes at Lexicon Pharmaceuticals and The Jackson Laboratory, and critically assess the role of histopathology analysis in high-throughput phenotyping pipelines.     

TOMATOMA: a novel tomato mutant database distributing Micro-Tom mutant collections

The tomato is an excellent model for studies of plants bearing berry-type fruits and for experimental studies of the Solanaceae family of plants due to its conserved genetic organization. In this study, a comprehensive mutant tomato population was generated in the background of Micro-Tom, a dwarf, rapid-growth variety. In this and previous studies, a family including 8,598 and 6,422 M(2) mutagenized lines was produced by ethylmethane sulfonate (EMS) mutagenesis and γ-ray irradiation, and this study developed and investigated these M(2) plants for alteration of visible phenotypes. A total of 9,183 independent M(2) families comprising 91,830 M(2) plants were inspected for phenotypic alteration, and 1,048 individual mutants were isolated. Subsequently, the observed mutant phenotypes were classified into 15 major categories and 48 subcategories. Overall, 1,819 phenotypic categories were found in 1,048 mutants. Of these mutants, 549 were pleiotropic, whereas 499 were non-pleiotropic. Multiple different mutant alleles per locus were found in the mutant libraries, suggesting that the mutagenized populations were nearly saturated. Additionally, genetic analysis of backcrosses indicated the successful inheritance of the mutations in BC(1)F(2) populations, confirming the reproducibility in the morphological phenotyping of the M(2) plants. To integrate and manage the visible phenotypes of mutants and other associated data, we developed the in silico database TOMATOMA, a relational system interfacing modules between mutant line names and phenotypic categories. TOMATOMA is a freely accessible database, and these mutant recourses are available through the TOMATOMA (http://tomatoma.nbrp.jp/index.jsp).     

Crop 3D—a LiDAR based platform for 3D high-throughput crop phenotyping

With the growing population and the reducing arable land, breeding has been considered as an effective way to solve the food crisis.As an important part in breeding, high-throughput phenotyping can accelerate the breeding process effectively. Light detection and ranging(LiDAR) is an active remote sensing technology that is capable of acquiring three-dimensional(3 D) data accurately,and has a great potential in crop phenotyping. Given that crop phenotyping based on LiDAR technology is not common in China,we developed a high-throughput crop phenotyping platform, named Crop 3 D, which integrated LiDAR sensor, high-resolution camera, thermal camera and hyperspectral imager. Compared with traditional crop phenotyping techniques, Crop 3 D can acquire multi-source phenotypic data in the whole crop growing period and extract plant height, plant width, leaf length, leaf width, leaf area, leaf inclination angle and other parameters for plant biology and genomics analysis. In this paper, we described the designs,functions and testing results of the Crop 3 D platform, and briefly discussed the potential applications and future development of the platform in phenotyping. We concluded that platforms integrating LiDAR and traditional remote sensing techniques might be the future trend of crop high-throughput phenotyping.     

A platform for soybean molecular breeding: the utilization of core collections for food security

Soybean is an important crop not only for human consumption but also for its addition of nitrogen to the soil during crop rotation. China has the largest collection of cultivated soybeans (Glycine max) and wild soybeans (Glycine soja) all over the world. The platform of soybean core, mini core and integrated applied core collections has been developed in the past decade based on systematic researches which included the sampling strategies, statistical methods, phenotypic data and SSR markers. Meanwhile, intergrated applied core collections including accessions with single or integrated favorite traits are being developed in order to meet the demand of soybean breeding. These kinds of core collections provide powerful materials for evaluation of germplasm, identification of trait-specific accessions, gene discovery, allele mining, genomic study, maker development, and molecular breeding. Some successful cases have proved the usefulness and efficiency of this platform. The platform is helpful for enhancing utilization of soybean genetic resources in sustainable crop improvement for food security. The efficient utilization of this platform in the future is relying on accurate phenotyping methods, abundant functional markers, high-throughput genotyping platforms, and effective breeding programs.     

Resources for image-based high-throughput phenotyping in crops and data sharing challenges

High-throughput phenotyping (HTP) platforms are capable of monitoring the phenotypic variation of plants through multiple types of sensors, such as red green and blue (RGB) cameras, hyperspectral sensors, and computed tomography, which can be associated with environmental and genotypic data. Because of the wide range of information provided, HTP datasets represent a valuable asset to characterize crop phenotypes. As HTP becomes widely employed with more tools and data being released, it is important that researchers are aware of these resources and how they can be applied to accelerate crop improvement. Researchers may exploit these datasets either for phenotype comparison or employ them as a benchmark to assess tool performance and to support the development of tools that are better at generalizing between different crops and environments. In this review, we describe the use of image-based HTP for yield prediction, root phenotyping, development of climate-resilient crops, detecting pathogen and pest infestation, and quantitative trait measurement. We emphasize the need for researchers to share phenotypic data, and offer a comprehensive list of available datasets to assist crop breeders and tool developers to leverage these resources in order to accelerate crop breeding.

Various approaches are used to analyze high-throughput phenotyping data and tools can be developed and assessed using available image-based datasets.     

Systematic Analysis of Experimental Phenotype Data Reveals Gene Functions

High-throughput phenotyping projects in model organisms have the potential to improve our understanding of gene functions and their role in living organisms. We have developed a computational, knowledge-based approach to automatically infer gene functions from phenotypic manifestations and applied this approach to yeast (Saccharomyces cerevisiae), nematode worm (Caenorhabditis elegans), zebrafish (Danio rerio), fruitfly (Drosophila melanogaster) and mouse (Mus musculus) phenotypes. Our approach is based on the assumption that, if a mutation in a gene leads to a phenotypic abnormality in a process , then must have been involved in , either directly or indirectly. We systematically analyze recorded phenotypes in animal models using the formal definitions created for phenotype ontologies. We evaluate the validity of the inferred functions manually and by demonstrating a significant improvement in predicting genetic interactions and protein-protein interactions based on functional similarity. Our knowledge-based approach is generally applicable to phenotypes recorded in model organism databases, including phenotypes from large-scale, high throughput community projects whose primary mode of dissemination is direct publication on-line rather than in the literature.     

Understanding mammalian genetic systems: the challenge of phenotyping in the mouse

Understanding mammalian genetic systems is predicated on the determination of the relationship between genetic variation and phenotype. Several international programmes are under way to deliver mutations in every gene in the mouse genome. The challenge for mouse geneticists is to develop approaches that will provide comprehensive phenotype datasets for these mouse mutant libraries. Several factors are critical to success in this endeavour. It will be important to catalogue assay and environment and where possible to adopt standardised procedures for phenotyping tests along with common environmental conditions to ensure comparable datasets of phenotypes. Moreover, the scale of the task underlines the need to invest in technological development improving both the speed and cost of phenotyping platforms. In addition, it will be necessary to develop new informatics standards that capture the phenotype assay as well as other factors, genetic and environmental, that impinge upon phenotype outcome.     

piggyBac transposon somatic mutagenesis with an activated reporter and tracker (PB-SMART) for genetic screens in mice

Somatic forward genetic screens have the power to interrogate thousands of genes in a single animal. Retroviral and transposon mutagenesis systems in mice have been designed and deployed in somatic tissues for surveying hematopoietic and solid tumor formation. In the context of cancer, the ability to visually mark mutant cells would present tremendous advantages for identifying tumor formation, monitoring tumor growth over time, and tracking tumor infiltrations and metastases into wild-type tissues. Furthermore, locating mutant clones is a prerequisite for screening and analyzing most other somatic phenotypes. For this purpose, we developed a system using the piggyBac (PB) transposon for somatic mutagenesis with an activated reporter and tracker, called PB-SMART. The PB-SMART mouse genetic screening system can simultaneously induce somatic mutations and mark mutated cells using bioluminescence or fluorescence. The marking of mutant cells enable analyses that are not possible with current somatic mutagenesis systems, such as tracking cell proliferation and tumor growth, detecting tumor cell infiltrations, and reporting tissue mutagenesis levels by a simple ex vivo visual readout. We demonstrate that PB-SMART is highly mutagenic, capable of tumor induction with low copy transposons, which facilitates the mapping and identification of causative insertions. We further integrated a conditional transposase with the PB-SMART system, permitting tissue-specific mutagenesis with a single cross to any available Cre line. Targeting the germline, the system could also be used to conduct F1 screens. With these features, PB-SMART provides an integrated platform for individual investigators to harness the power of somatic mutagenesis and phenotypic screens to decipher the genetic basis of mammalian biology and disease.     

A rice phenomics study—phenotype scoring and seed propagation of a T-DNA insertion-induced rice mutant population

With the completion of the rice genome sequencing project, the next major challenge is the large-scale determination of gene function. As an important crop and a model organism, rice provides major insights into gene functions important for crop growth or production. Phenomics with detailed information about tagged populations provides a good tool for functional genomics analysis. By a T-DNA insertional mutagenesis approach, we have generated a rice mutant population containing 55,000 promoter trap and gene activation or knockout lines. Approximately 20,000 of these lines have known integration sites. The T0 and T1 plants were grown in net “houses” for two cropping seasons each year since 2003, with the mutant phenotypes recorded. Detailed data describing growth and development of these plants, in 11 categories and 65 subcategories, over the entire four-month growing season are available in a searchable database, along with the genetic segregation information and flanking sequence data. With the detailed data from more than 20,000 T1 lines and 12 plants per line, we estimated the mutation rates of the T1 population, as well the frequency of the dominant T0 mutants. The correlations among different mutation phenotypes are also calculated. Together, the information about mutant lines, their integration sites, and the phenotypes make this collection, the Taiwan Rice Insertion Mutants (TRIM), a good resource for rice phenomics study. Ten T2 seeds per line can be distributed to researchers upon request. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chyr-Guan Chern, Ming-Jen Fan, and Su-May Yu have contributed equally to this work.     

Mouse phenotyping

Model organisms like the mouse are important tools to learn more about gene function in man. Within the last 20 years many mutant mouse lines have been generated by different methods such as ENU mutagenesis, constitutive and conditional knock-out approaches, knock-down, introduction of human genes, and knock-in techniques, thus creating models which mimic human conditions. Due to pleiotropic effects, one gene may have different functions in different organ systems or time points during development. Therefore mutant mouse lines have to be phenotyped comprehensively in a highly standardized manner to enable the detection of phenotypes which might otherwise remain hidden. The German Mouse Clinic (GMC) has been established at the Helmholtz Zentrum München as a phenotyping platform with open access to the scientific community (www.mousclinic.de; [1]). The GMC is a member of the EUMODIC consortium which created the European standard workflow EMPReSSslim for the systemic phenotyping of mouse models (http://www.eumodic.org/ [2]).     

Integrating mouse anatomy and pathology ontologies into a phenotyping database: Tools for data capture and training

The Mouse Disease Information System (MoDIS) is a data capture system for pathology data from laboratory mice designed to support phenotyping studies. The system integrates the mouse anatomy (MA) and mouse pathology (MPATH) ontologies into a Microsoft Access database facilitating the coding of organ, tissue, and disease process to recognized semantic standards. Grading of disease severity provides scores for all lesions that can then be used for quantitative trait locus (QTL) analyses and haplotype association gene mapping. Direct linkage to the Pathbase online database provides reference definitions for disease terms and access to photomicrographic images of similar diagnoses in other mutant mice. MoDIS is an open source and freely available program (). This provides a valuable tool for setting up a mouse pathology phenotyping program.     

GSD: a genetic screen database

The systematic assignment of gene function to a sequenced genome is one of the outstanding challenges in the post-genomic era. Large-scale systematic mutagenesis screens are important tools for reaching this goal. Here we describe GSD, a software package that allows storage and integration of data from genetic screens. GSD was initially developed for a large-scale F3 mutagenesis screen for developmental mutants of medaka (Oryzias latipes). The version presented here supports a wide range of different screens (mutagenesis, RNAi, morpholinos, transgenesis and others) using different organisms. Data are stored in a relational database and can be made accessible through web interfaces. Researchers can enter data describing their screened embryos: They can track statistics, submit images and describe the resulting phenotypes using a phenotype classification ontology. We developed a fish phenotype classification ontology of medaka and zebrafish for this software package and made it available to the public. In addition, a list of genetic lines resulting from each screen can be generated. These lines (mutant alleles, transgenic lines) can be described and categorized in the same ways as the screened individuals. Raw data from the screen can be integrated to describe these lines. A query module that searches this list can be used to publish the screen results on the Internet. A test version is available at and the software can be downloaded from this site.     

Large-scale mutational analysis for the annotation of the mouse genome

After sequencing the human and mouse genomes, the annotation of these sequences with biological functions is an important challenge in genomic research. A major tool to analyse gene function on the organismal level is the analysis of mutant phenotypes. Because of its genetic and physiological similarity to man, the mouse has become the model organism of choice for the study of genetic diseases. In addition, there is at the moment no other vertebrate for which versatile techniques to manipulate the genome are as well developed. Several mouse mutagenesis projects have provided the proof-of-principle that a systematic and comprehensive mutagenesis of every gene in the mammalian genome will be feasible. An exhaustive functional annotation of the mammalian genome can only be achieved in a combination of phenotype- and gene-driven approaches in large- and small-scale academic and private projects. Major challenges will be to develop standardised phenotyping protocols for the clinical and pathological characterisation of mouse mutants, the improvement of mutation detection methods and the dissemination of resources and data. Beyond gene annotation, it will be necessary to understand how gene functions are integrated into the complex network of regulatory interactions in the cell.     

Informatics for mutagenesis: the design of Mutabase—a distributed data recording system for animal husbandry, mutagenesis, and phenotypic analysis

The increasing use of high-throughput methods for the production of biologically important information and the increasing diversity of that information pose considerable bioinformatics challenges. These challenges will be met by implementing electronic data management systems not only to capture the data, but increasingly to provide a platform for data integration and mining as we enter the post-genomic era. We discuss the design and implementation of such a data capture system, `Mutabase', as a model of how such electronic systems might be designed and implemented. Mutabase was created in support of a large-scale, phenotype-driven mouse mutagenesis program at MRC Mammalian Genetics Unit, Harwell, in collaboration with SmithKline Beecham Pharmaceuticals, Queen Mary and Westfield College, London, and Imperial College of Science, Technology and Medicine, London. The aim of this mutagenesis project is to make a significant contribution to the existing mouse mutant resource, closing the phenotype gap and providing many more models for fundamental research and disease modeling. Mutabase records experimental details at the `point of generation' and provides a number of dissemination and analysis tools for the experimental data, as well as providing a means of assessing various aspects of progress of the program. Mutabase uses a hypertext-based interface to provide interaction between a number of intranet-based client workstations and a central industrial strength database. Mutabase utilizes a variety of techniques in order to implement the user interface system including Perl/CGI, Java Servlets, and an experimental CORBA server. We discuss the relative merits of these methods in the context of the need to provide sound informatics approaches for the support of systematic mutagenesis programs. Received: 16 December 1999 / Accepted: 17 December 1999     

A novel 3D mouse embryo atlas based on micro-CT

The goal of the International Mouse Phenotyping Consortium (IMPC) is to phenotype targeted knockout mouse strains throughout the whole mouse genome (23,000 genes) by 2021. A significant percentage of the generated mice will be embryonic lethal; therefore, phenotyping methods tuned to the mouse embryo are needed. Methods that are robust, quantitative, automated and high-throughput are attractive owing to the numbers of mice involved. Three-dimensional (3D) imaging is a useful method for characterizing morphological phenotypes. However, tools to automatically quantify morphological information of mouse embryos from 3D imaging have not been fully developed. We present a representative mouse embryo average 3D atlas comprising micro-CT images of 35 individual C57BL/6J mouse embryos at 15.5 days post-coitum. The 35 micro-CT images were registered into a consensus average image with our automated image registration software and 48 anatomical structures were segmented manually. We report the mean and variation in volumes for each of the 48 segmented structures. Mouse organ volumes vary by 2.6-4.2% on a linear scale when normalized to whole body volume. A power analysis of the volume data reports that a 9-14% volume difference can be detected between two classes of mice with sample sizes of eight. This resource will be crucial in establishing baseline anatomical phenotypic measurements for the assessment of mutant mouse phenotypes, as any future mutant embryo image can be registered to the atlas and subsequent organ volumes calculated automatically.