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The HIV‐1 often evades a robust antiretroviral‐mediated immune response, leading to persistent infection within anatomically privileged sites including the CNS. Continuous low‐level infection occurs in the presence of effective antiretroviral therapy (ART) in CD4+ T cells and mononuclear phagocytes (MP; monocytes, macrophages, microglia, and dendritic cells). Within the CNS, productive viral infection is found exclusively in microglia and meningeal, perivascular, and choroidal macrophages. MPs serve as the principal viral CNS reservoir. Animal models have been developed to recapitulate natural human HIV‐1 infection. These include nonhuman primates, humanized mice, EcoHIV, and transgenic rodent models. These models have been used to study disease pathobiology, antiretroviral and immune modulatory agents, viral reservoirs, and eradication strategies. However, each of these models are limited to specific component(s) of human disease. Indeed, HIV‐1 species specificity must drive therapeutic and cure studies. These have been studied in several model systems reflective of latent infections, specifically in MP (myeloid, monocyte, macrophages, microglia, and histiocyte cell) populations. Therefore, additional small animal models that allow productive viral replication to enable viral carriage into the brain and the virus‐susceptible MPs are needed. To this end, this review serves to outline animal models currently available to study myeloid brain reservoirs and highlight areas that are lacking and require future research to more effectively study disease‐specific events that could be useful for viral eradication studies both in and outside the CNS. Review on recent rodent models that contain both human myeloid and T cells for future therapeutics and viral elimination investigations in CNS.

The International Mouse Phenotyping Consortium reports the generation of new mouse mutant strains for more than 5,000 genes, including 2,850 novel null, 2,987 novel conditional-ready and 4,433 novel reporter alleles.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXYindividuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.

CRISPR/Cas9 technology has transformed mouse genome editing with unprecedented precision, efficiency, and ease; however, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains rate-limiting. We thus developed CRISPR ribonucleoprotein (RNP) electroporation of zygotes (CRISPR-EZ), an electroporation-based technology that outperforms pronuclear and cytoplasmic microinjection in efficiency, simplicity, cost, and throughput. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/single-guide RNA (sgRNA) RNPs, facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. In a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline, CRISPR-EZ consistently outperformed microinjection. Here, we provide an optimized protocol covering sgRNA synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher with basic embryo-manipulation skills can obtain genetically modified mice in 6 weeks. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mammalian species.

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard–Soulier, Bardet–Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

Model organism (MO) research provides a basic understanding of biology and disease due to the evolutionary conservation of the molecular and cellular language of life. MOs have been used to identify and understand the function of orthologous genes, proteins, cells and tissues involved in biological processes, to develop and evaluate techniques and methods, and to perform whole-organism-based chemical screens to test drug efficacy and toxicity. However, a growing richness of datasets and the rising power of computation raise an important question: How do we maximize the value of MOs? In-depth discussions in over 50 virtual presentations organized by the National Institutes of Health across more than 10 weeks yielded important suggestions for improving the rigor, validation, reproducibility and translatability of MO research. The effort clarified challenges and opportunities for developing and integrating tools and resources. Maintenance of critical existing infrastructure and the implementation of suggested improvements will play important roles in maintaining productivity and facilitating the validation of animal models of human biology and disease.

Retinitis pigmentosa (RP), a retinal degenerative disease, is the leading cause of heritable blindness. Previously, we described that Arap1−/− mice develop a similar pattern of photoreceptor degeneration. Arap1 is an Arf-directed GTPase-activating protein shown to modulate actin cytoskeletal dynamics. Curiously, Arap1 expression was detected in Müller glia and retinal pigment epithelium (RPE), but not the photoreceptors themselves. In this study, we generated conditional knockout mice for Müller glia/RPE, Müller glia and RPE via targeting Rlbp1, Glast and Vmd2 promoters, respectively, to drive Cre recombinase expression to knock out Arap1. Vmd2-Cre Arap1tm1c/tm1c and Rlbp1-Cre Arap1tm1c/tm1c mice, but not Glast-Cre Arap1tm1c/tm1c mice, recapitulated the phenotype originally observed in germline Arap1−/− mice. Mass spectrometry analysis of human ARAP1 co-immunoprecipitation identified candidate binding partners of ARAP1, revealing potential interactants involved in phagocytosis, cytoskeletal composition, intracellular trafficking and endocytosis. Quantification of outer segment phagocytosis in vivo demonstrated a clear phagocytic defect in Arap1−/− mice compared to Arap1+/+ controls. We conclude that Arap1 expression in RPE is necessary for photoreceptor survival due to its indispensable function in RPE phagocytosis. This article has an associated First Person interview with the first author of the paper.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein sestrin-2 (SESN2) as a repressor of PDGFRβ signalling, and PDGFRβ signalling as an upstream regulator of alveolar maintenance programmes. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke-induced pulmonary emphysema by upregulating PDGFRβ expression via a selective accumulation of intracellular superoxide anions (O2−). We also show that SESN2 is overexpressed and PDGFRβ downregulated in the emphysematous lungs of individuals with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2-PDGFRβ interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD.

Background: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

This corrects the article DOI: 10.1038/nature19356

The research community can have confidence in data from well-executed phenotyping projects.