Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes.

Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PAPERCLIP:

Combining ecological, eco-cultural, and environmental justice parameters to create Eco-EJ indicators to monitor cultural and environmental justices for diverse communities around contaminated sites.

Assessing environmental quality often requires selection of indicators that can be employed over large spatial scales and over long-time periods to assess the health and well-being of species, natural communities, and ecosystems, and to detect changes warranting intervention. Typically, the ecologic environment and the human environment are evaluated separately and selection of indicators and monitoring approaches are not integrated even though ecological indicators may also provide information on risk to human consumers from contaminants (e.g., eco-cultural indicators) or because of disease levels. This paper is a call for ecologists and managers to consider diverse cultural and environmental injustice disparities and health issues when selecting indicators for environmental assessment and monitoring. There is an opportunity for managers and community members to work together to preserve ecological and cultural resources and heritages. We propose a paradigm that selects indicators and monitoring approaches that lend themselves to the integration of human-diversity and uniqueness in the same manner that the selection of ecological indicators and monitoring approaches consider biological species diversity and uniqueness. The proposed paradigm builds on ecological risk assessment techniques, developing analogous endpoints for neighboring communities. For example, identification and protection of human communities, particularly culturally diverse and environmental justice communities, identification of contaminant corridors (e.g., through water or green corridors) into communities, and eco-monitoring of vulnerable communities are not routine at contaminated sites. Green corridors refers to a width of wild habitat (forest, grasslands) that connects other similar habitat paths (usually a corridor runs through an urban or suburban habitat). We coin the term Eco-EJ indicators for these endpoints, including examination of (1) unique cultural relationships to resources; (2) connectedness of on-site and off-site resources and habitats; (3) health of threatened, rare, and unique cultures and communities; and (4) linkages between ecological, eco-cultural, and public health for monitoring and assessment. We also propose that assessment and monitoring include these Eco-EJ indicators, especially for communities near facilities that have extensive chemical or radiological contamination.Developing these indicators to assess risk to culturally diverse and environmental justice communities would be an equivalent goal to reducing risk for significant ecological resources (e.g., endangered species, species of special concern). These Eco-EJ indicators are complementary to the usual human health-risk assessments, would include surveys of neighboring vulnerable communities, and require time and re-organization of current data and additional data collection at site boundaries and in adjacent communities, as well as rethinking the human component of indicators. This approach lends itself to addressing some diverse cultural and environmental justice issues with current indicator selection and biomonitoring, and helps identify specific hotspots of unique ecosystem risk and environmental justice community risk. We briefly discuss ecological and eco-cultural monitoring already on-going at three Department of Energy sites to illustrate how the addition of these indicators might work and add value to environmental management and to their relationships with surrounding communities. We recommend that managers of contaminated sites convene people from culturally diverse communities, environmental justice communities, local and federal government, Tribes, resource trustees, managers, and other stakeholders to develop appropriate site-specific indicators to address environmental inequities around contaminated facilities.

Risk to ecological resources following remediation can be due mainly to increased resource value of successful restoration: A case study from the Department of Energy's Hanford Site.

Many nations are faced with the need to remediate large contaminated sites following World War II, the Cold War, and abandoned industrial sites, and to return them to productive land uses. In the United States, the Department of Energy (DOE) has the largest cleanup challenge, and its Hanford Site in the state of Washington has the most extensive and most expensive cleanup task. Ideally, the risk to ecological resources on remediation sites is evaluated before, during, and after remediation, and the risk from, or damage to, ecological resources from contaminants should be lower following remediation. In this paper, we report the risk to ecological resources before, during, and as a consequence of remediation on contaminated units requiring cleanup, and then examine the causes for changes in risk by evaluating 56 cleanup evaluation units (EUs) at the Hanford Site. In this case, remediation includes a restoration phase. In general, the risk to ecological and eco-cultural resources is currently not discernible or low at most contaminated units, increases during remediation, and decreases thereafter. Remediation often causes physical disruption to ecosystems as it reduces the risk from exposure to contaminants. Most new remediation projects at the Hanford Site include ecological restoration. Ecological restoration results in the potential for the presence of higher quality resources after remediation than currently exists on these contaminated lands and facilities. Although counter-intuitive, our evaluation of the risk to ecological resources following remediation indicated that a significant percentage of units (61%) will be at increased risk in the post-remediation period. This increased risk is due to DOE's successful remediation and restoration that results in a higher percent of native vegetation and higher ecological value on the sites in the post-remediation period than before. These newly-created resources can then be at risk from post-remediation activities. Risks to these new higher quality resources include the potential for spread of invasive species and of noxious grasses used in previous cleanup actions, disruption of ecosystems (including those with state or federally listed species and unique ecosystems), compaction of soil, use of pesticides to control invasive species, and the eventual need for continued monitoring activities. Thus, by greatly improving the existing habitat and health of eco-receptors, and maintaining habitat corridors between high quality habitats, the ecological resources in the post-remediated units are at risk unless care is taken to protect them. Many of the negative effects of both remediation and future monitoring (or other future land uses) can be avoided by planning and management early in the remediation process. We suggest DOE and other agencies convene a panel of managers, remediation scientists, regulators, environmental and ecological scientists, Native Americans, economists, and the public to develop a generic list of performance metrics for the restoration phase of remediation, including evaluation of success, which could be applied across the DOE complex.

A paradigm for protecting ecological resources following remediation as a function of future land use designations: a case study for the Department of Energy's Hanford Site.

Since the late 1980s, there has been a US federal mandate to clean up contaminated sites remaining from the Second World War, the Cold War, and abandoned industries. One determinant of cleanup standards for remediation is future land use-how will the land be used and by whom? Land use decisions may be consensus documents developed by site owners, state and federal agencies, and local stakeholders. Often there are competing views and/or claims on how remediated sites should be used, including as open or green space. Large sites are likely to have more ecological heterogeneity within similar land use designations because of differences in climate, geology, topography, and history of human use. This paper uses the Department of Energy's (DOE) Hanford Site as a case study to examine how and whether future land use designations will protect species, species diversity, heterogeneity, and ecosystems once remediation is complete. The objective of this paper is to describe "future land use designations" on a large, complex site (DOE's Hanford Site) and to examine the following: (1) how future land use designations were made and have changed over time, (2) how land use designations included the value of ecological resources, (3) how risk evaluations of ecological resources from remediation were made, and (4) how future land use may affect the health and well-being of ecological resources on site in the post-remediation period. The paper provides a paradigm for integrating ecological protection into future land use designations such that rare and sensitive resources are protected throughout the process. The paradigm includes the following: (1) developing future land use designations, (2) defining resource levels (values), (3) relating resource levels to land use designations and management, (4) defining risk evaluations, (5) determining the likelihood that valuable resources will occur on each land use type after remediation, and (6) evaluating the potential risk to those resources that results from activities allowed under future land use designations. The paper discusses the importance of each step, the implications for protection of ecological resources, and the importance of land use designations in the assessment of risk to ecological resources from both continued monitoring and maintenance by DOE (or other land owners) and the activities permitted by the established future land use designations.

Evaluation of ecological resources at operating facilities at contaminated sites: The Department of Energy's Hanford Site as a case study.

The U.S. and other developed nations are faced with many contaminated sites remaining from World War II, the Cold War, and abandoned industries, that require remediation and restoration to allow future land uses with minimum acceptable risk to humans and ecological resources. For large Department of Energy (DOE) sites with massive remediation tasks remaining, it is important for managers to be able to assure regulators, Tribal Nations, and the public that human and ecological health are protected. Hanford Site has the largest and most expensive cleanup task within the DOE complex; cleanup will continue beyond 2090. Cleanup involves the use of operating facilities, which also may present a risk to humans or ecological resources. We present a brief description of a methodology to evaluate risks to ecological receptors at the Hanford Site from remaining remediation tasks, and evaluate the risk to ecological resources that operating facilities present currently, during active cleanup of these facilities, and during the post cleanup period. Operating facilities include current, active operations that are located on the site and aid in site cleanup, including both storage and treatment operations. At the Hanford Site, they include waste treatment plants, sludge basins, waste trenches, Central Waste Complex, storage facilities, and disposal facilities, among others. Risk ratings for ecological resources are highest during the remediation phase. Risk ratings for the operating facilities at the Hanford Site range from not discernible to medium currently, from not discernible (ND) to high during active cleanup, and from not discernible to medium following cleanup. The highest ratings are for the Waste Treatment and Immobilization Plant that is being constructed to stabilize radioactive and chemical wastes, and the Liquid Effluent Retention and Treatment Facility that removes and deactivates hazardous contaminants from waste water. Higher ratings in the post-cleanup period are largely due to restoration of ecological resources during cleanup, which increases the potential for injury (if these resources are harmed) because a site will then have higher quality resources after cleanup than it did before. Assessing the value of ecological resources, and determining potential consequences during active remediation and after remediation is essential for compliance with state and federal laws. Understanding the risks to ecological resources from now until clean-up is completed at these facilities is important because of the potential for ecological resources of high value to be degraded, and because cleanup completion is not expected until 2090 or later. The methodology can be applied to any contaminated site requiring a rapid method of assessing potential damages to ecological resources from proposed management actions.

A Methodology to Evaluate Ecological Resources and Risk Using Two Case Studies at the Department of Energy's Hanford Site.

An assessment of the potential risks to ecological resources from remediation activities or other perturbations should involve a quantitative evaluation of resources on the remediation site and in the surrounding environment. We developed a risk methodology to rapidly evaluate potential impact on ecological resources for the U.S. Department of Energy's Hanford Site in southcentral Washington State. We describe the application of the risk evaluation for two case studies to illustrate its applicability. The ecological assessment involves examining previous sources of information for the site, defining different resource levels from 0 to 5. We also developed a risk rating scale from non-discernable to very high. Field assessment is the critical step to determine resource levels or to determine if current conditions are the same as previously evaluated. We provide a rapid assessment method for current ecological conditions that can be compared to previous site-specific data, or that can be used to assess resource value on other sites where ecological information is not generally available. The method is applicable to other Department of Energy's sites, where its development may involve a range of state regulators, resource trustees, Tribes and other stakeholders. Achieving consistency across Department of Energy's sites for valuation of ecological resources on remediation sites will assure Congress and the public that funds and personnel are being deployed appropriately.

Functional remediation components: A conceptual method of evaluating the effects of remediation on risks to ecological receptors.

Governmental agencies, regulators, health professionals, tribal leaders, and the public are faced with understanding and evaluating the effects of cleanup activities on species, populations, and ecosystems. While engineers and managers understand the processes involved in different remediation types such as capping, pump and treat, and natural attenuation, there is often a disconnect between (1) how ecologists view the influence of different types of remediation, (2) how the public perceives them, and (3) how engineers understand them. The overall goal of the present investigation was to define the components of remediation types (= functional remediation). Objectives were to (1) define and describe functional components of remediation, regardless of the remediation type, (2) provide examples of each functional remediation component, and (3) explore potential effects of functional remediation components in the post-cleanup phase that may involve continued monitoring and assessment. Functional remediation components include types, numbers, and intensity of people, trucks, heavy equipment, pipes, and drill holes, among others. Several components may be involved in each remediation type, and each results in ecological effects, ranging from trampling of plants, to spreading invasive species, to disturbing rare species, and to creating fragmented habitats. In some cases remediation may exert a greater effect on ecological receptors than leaving the limited contamination in place. A goal of this conceptualization is to break down functional components of remediation such that managers, regulators, and the public might assess the effects of timing, extent, and duration of different remediation options on ecological systems.

Rapid conversion of EUCOMM/KOMP-CSD alleles in mouse embryos using a cell-permeable Cre recombinase.

We describe here use of a cell-permeable Cre to efficiently convert the EUCOMM/KOMP-CSD tm1a allele to the tm1b form in preimplantation mouse embryos in a high-throughput manner, consistent with the requirements of the International Mouse Phenotyping Consortium-affiliated NIH KOMP2 project. This method results in rapid allele conversion and minimizes the use of experimental animals when compared to conventional Cre transgenic mouse breeding, resulting in a significant reduction in costs and time with increased welfare benefits.

Genomic analysis of a novel spontaneous albino C57BL/6N mouse strain.

We report an albino C57BL/6N mouse strain carrying a spontaneous mutation in the tyrosinase gene (C57BL/6N-Tyr(cWTSI)). Deep whole genome sequencing of founder mice revealed very little divergence from C57BL/6NJ and C57BL/6N (Taconic). This coisogenic strain will be of great utility for the International Mouse Phenotyping Consortium (IMPC), which uses the EUCOMM/KOMP targeted C57BL/6N ES cell resource, and other investigators wishing to work on a defined C57BL/6N background.

Molecular characterization of mutant mouse strains generated from the EUCOMM/KOMP-CSD ES cell resource.

The Sanger Mouse Genetics Project generates knockout mice strains using the EUCOMM/KOMP-CSD embryonic stem (ES) cell collection and characterizes the consequences of the mutations using a high-throughput primary phenotyping screen. Upon achieving germline transmission, new strains are subject to a panel of quality control (QC) PCR- and qPCR-based assays to confirm the correct targeting, cassette structure, and the presence of the 3' LoxP site (required for the potential conditionality of the allele). We report that over 86 % of the 731 strains studied showed the correct targeting and cassette structure, of which 97 % retained the 3' LoxP site. We discuss the characteristics of the lines that failed QC and postulate that the majority of these may be due to mixed ES cell populations which were not detectable with the original screening techniques employed when creating the ES cell resource.

The costs of delaying remediation on human, ecological, and eco-cultural resources: Considerations for the Department of Energy: A methodological framework.

Remediation and restoration of the Nation's nuclear legacy of radiological and chemical contaminated areas is an ongoing and costly challenge for the U.S. Department of Energy (DOE). For large sites, such as the Hanford and Savannah River Sites, successful remediation involves complex decisions related to remedies, end-states, timing, and sequencing of cleanup of separate and related contaminated units within a site. Hanford Site cannot clean up every unit simultaneously due to limits in funding, personnel, and technology. This paper addresses one of the major considerations - the consequences of delaying remediation of a unit on different receptors (e.g. people, ecological, and eco-cultural resources), using the DOE Hanford Site as a case study. We develop a list of attributes that managers should consider for successful remediation, examine how delaying remediation could affect workers, the public and ecological resources (including water resources), and use some examples to illustrate potential effects of delays. The factors to consider when deciding whether and how long to delay remediation of a unit include personnel, information and data, funding, equipment, structural integrity, contaminant source, and resource vulnerability. Each of these factors affects receptors differently. Any remediation task may be dependent on other remediation projects, on the availability of transport, containers, interim storage and ultimate disposition decisions, or the availability of trained personnel. Delaying remediation may have consequences for people (e.g. workers, site neighbors), plants, animals, ecosystems, and eco-cultural resources (i.e. those cultural values that depend upon ecological resources). The risks, benefits, and uncertainties for evaluating the consequences of delaying remediation are described and discussed. Assessing the advantages and disadvantages of delaying remediation is important for health professionals, ecologists, resource trustees, regulators, Tribal members, recreationists, fishermen, hunters, conservationists, and a wide range of other stakeholders.