Lauryl M. J. Nutter

High-throughput discovery of novel developmental phenotypes.

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.

Efficient Generation of Germ Line Transmitting Chimeras from C57BL/6N ES Cells by Aggregation with Outbred Host Embryos

Genetically modified mouse strains derived from embryonic stem (ES) cells have become essential tools for functional genomics and biomedical research. Large scale mutagenesis projects are producing libraries of mutant C57BL/6 (B6) ES cells to enable the functional annotation of every gene of the mouse genome. To realize the utility of these resources, efficient and accessible methods of generating mutant mice from these ES cells are necessary. Here, we describe a combination of ICR morula aggregation and a chemically-defined culture medium with widely available and accessible components for the high efficiency generation of germline transmitting chimeras from C57BL/6N ES cells. Together these methods will ease the access of the broader biomedical research community to the publicly available B6 ES cell resources.

Disease model discovery from 3,328 gene knockouts by The International Mouse Phenotyping Consortium.

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.

Chromosomal reinsertion of broken RSS ends during T cell development

The V(D)J recombinase catalyzes DNA transposition and translocation both in vitro and in vivo. Because lymphoid malignancies contain chromosomal translocations involving antigen receptor and protooncogene loci, it is critical to understand the types of “mistakes” made by the recombinase. Using a newly devised assay, we characterized 48 unique TCRβ recombination signal sequence (RSS) end insertions in murine thymocyte and splenocyte genomic DNA samples. Nearly half of these events targeted “cryptic” RSS-like elements. In no instance did we detect target-site duplications, which is a hallmark of recombinase-mediated transposition in vitro. Rather, these insertions were most likely caused by either V(D)J recombination between a bona fide RSS and a cryptic RSS or the insertion of signal circles into chromosomal loci via a V(D)J recombination-like mechanism. Although wild-type, p53, p53 x scid, H2Ax, and ATM mutant thymocytes all showed similar levels of RSS end insertions, core-RAG2 mutant thymocytes showed a sevenfold greater frequency of such events. Thus, the noncore domain of RAG2 serves to limit the extent to which the integrity of the genome is threatened by mistargeting of V(D)J recombination.

Therapeutic Potential of Spleen Tyrosine Kinase Inhibition for Treating High-Risk Precursor B Cell Acute Lymphoblastic Leukemia

Pre-BCR–independent activation of spleen tyrosine kinase plays a pathogenic role in high-risk B cell acute lymphoblastic leukemia. Targeting the “SYK-ness” in B-ALL Intensive chemotherapy in B cell acute lymphoblastic leukemia (B-ALL) provides improved outcomes for children and achieves remission in adults, but patients in both groups relapse, resulting in low survival rates. The discovery of aberrant signaling pathways in cancers has spurred development of targeted kinase inhibitors. In a mouse model of spontaneous B-ALL, Perova et al. demonstrate that aberrant activation of the spleen tyrosine kinase (SYK) was required for leukemic cell growth. SYK pathway activity and B-ALL cell survival were sensitive to two inhibitors of SYK signaling. Like the mouse model, the authors show that primary pediatric and adult human B-ALL samples exhibited basal SYK activation. Phosphorylation of SYK and its targets and leukemic cell proliferation were attenuated by in vitro treatment of the human leukemic cells with SYK inhibitors. Xenotransplantation of poor-prognosis primary human B-ALL samples into immunodeficient mice resulted in extensive bone marrow engraftment and dissemination to spleen, liver, and central nervous system. In vivo treatment of the transplanted animals with SYK inhibitors reduced human leukemia burden in these tissues. Thus, SYK activation regulates key signal transduction pathways of abnormal growth in multiple subtypes of B-ALL, suggesting that small-molecule SYK inhibitors may be promising agents for treating poor-prognosis and relapsed B-ALL. Intensified and central nervous system (CNS)–directed chemotherapy has improved outcomes for pediatric B cell acute lymphoblastic leukemia (B-ALL) but confers treatment-related morbidities. Moreover, many patients suffer relapses, underscoring the need to develop new molecular targeted B-ALL therapies. Using a mouse model, we show that leukemic B cells require pre–B cell receptor (pre-BCR)–independent spleen tyrosine kinase (SYK) signaling in vivo for survival and proliferation. In diagnostic samples from human pediatric and adult B-ALL patients, SYK and downstream targets were phosphorylated regardless of pre-BCR expression or genetic subtype. Two small-molecule SYK inhibitors, fostamatinib and BAY61-3606, attenuated the growth of 69 B-ALL samples in vitro, including high-risk (HR) subtypes. Orally administered fostamatinib reduced heavy disease burden after xenotransplantation of HR B-ALL samples into immunodeficient mice and decreased leukemia dissemination into spleen, liver, kidneys, and the CNS of recipient mice. Thus, SYK activation sustains the growth of multiple HR B-ALL subtypes, suggesting that SYK inhibitors may improve outcomes for HR and relapsed B-ALL.

p53-Independent apoptosis disrupts early organogenesis in embryos lacking both ataxia-telangiectasia mutated and Prkdc.

The ataxia-telangiectasia mutated (ATM) protein and the nonhomologous end-joining (NHEJ) pathway play crucial roles in sensing and repairing DNA double-strand breaks in postnatal cells. However, each pathway is dispensable for early embryogenesis. Loss of both ATM and Prkdc/Ku is synthetically lethal, but neither the developmental processes perturbed nor the mechanisms of lethality have been determined by previous reports. Here, we show that ATM and Prkdc collaborate to maintain genomic stability during gastrulation and early organogenesis, a period of rapid proliferation and hypersensitivity to DNA damage. At E7.5 to E8.5, ATM−/−Prkdcscid/scid embryos displayed normal proliferation indices but exhibited excessive apoptosis and elevated expression of Ser15-phosphorylated p53. Thus, this crucial regulatory residue of p53 can be phosphorylated in the absence of ATM or Prkdc. However, loss of p53 did not abrogate or delay embryonic lethality, revealing that apoptosis is p53 independent in these in ATM−/−Prkdcscid/scid embryos. Because mice with combined disruptions of ATM and other NHEJ components (ligase IV, Artemis) are viable, our data suggest a novel NHEJ-independent function for Prkdc/Ku that is required to complete early embryogenesis in the absence of ATM. (Mol Cancer Res 2006;4(5):311–8)

Engineering the embryo

Generation of transgenic mice by pronuclear DNA injection into fertilized eggs has been well-established for over 30 y (1). However, it remains a rather crude approach: injected sequences randomly insert as concatamers, they can be mutagenic, and they have variable expression depending on the site of integration, a phenomenon called position effects. In PNAS, Tasic et al. (2) report that pronuclear injection of PhiC31 integrase can catalyze site-specific integration of single-copy transgenes in up to 40% of live pups. This approach can reduce variability because of position effects and provide improved control over transgene expression. This and other recent reports of site-specific integration in zygotes (2–6) reveal additional possibilities for targeted transgenesis in mice and species where ES cells are not available.

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction.

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function.The full extent of the genetic basis for hearing impairment is unknown. Here, as part of the International Mouse Phenotyping Consortium, the authors perform a hearing loss screen in 3006 mouse knockout strains and identify 52 new candidate genes for genetic hearing loss.

Identification of genetic elements in metabolism by high-throughput mouse phenotyping.

Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome.The genetic basis of metabolic diseases is incompletely understood. Here, by high-throughput phenotyping of 2,016 knockout mouse strains, Rozman and colleagues identify candidate metabolic genes, many of which are associated with unexplored regulatory gene networks and metabolic traits in human GWAS.

Response to “Unexpected mutations after CRISPR–Cas9 editing in vivo ”

untreated embryos from the same embryo pool. Without specific information about the genetic relationship between the control and F0 animals, one can attribute all the mutations observed to genetic drift between independently bred mouse colonies or commercial strains over time. There have been three other published studies using next-generation sequencing for the unbiased examination of Cas9 off-target mutations in mouse model production6-8. None of these studies identified significant off-target activity. Two of these studies, one using Cas9 nickase6 and one using Cas9 endonuclease8, carefully assessed SNVs, and neither study observed SNVs that could not be attributed to either the parental colony or genetic drift within the colony used for embryo production. Importantly, the number of indels that could be attributed to Cas9 was also very small, <1 per founder, on average7,8. Despite this overwhelming evidence to the contrary, Schaefer et al. offer no explanation of their conflicting outcome. The International Mouse Phenotyping Consortium (IMPC) agrees with the editors of Nature Methods9 that standardization of approaches to experimental design and quality control using Cas9, or indeed any genome-editing tool, are vitally important, both to the future clinical applications of Cas9 and to the production of mouse models using Cas9. Only with excellent quality assurance during production and quality control of derived mouse lines can we have confidence in our ability to generate reliable and reproducible data.