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The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps - generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.

BackgroundMicrophthalmia, anophthalmia, and coloboma (MAC) spectrum disease encompasses a group of eye malformations which play a role in childhood visual impairment. Although the predominant cause of eye malformations is known to be heritable in nature, with 80% of cases displaying loss-of-function mutations in the ocular developmental genes OTX2 or SOX2, the genetic abnormalities underlying the remaining cases of MAC are incompletely understood. This study intended to identify the novel genes and pathways required for early eye development. Additionally, pathways involved in eye formation during embryogenesis are also incompletely understood. This study aims to identify the novel genes and pathways required for early eye development through systematic forward screening of the mammalian genome.ResultsQuery of the International Mouse Phenotyping Consortium (IMPC) database (data release 17.0, August 01, 2022) identified 74 unique knockout lines (genes) with genetically associated eye defects in mouse embryos. The vast majority of eye abnormalities were small or absent eyes, findings most relevant to MAC spectrum disease in humans. A literature search showed that 27 of the 74 lines had previously published knockout mouse models, of which only 15 had ocular defects identified in the original publications. These 12 previously published gene knockouts with no reported ocular abnormalities and the 47 unpublished knockouts with ocular abnormalities identified by the IMPC represent 59 genes not previously associated with early eye development in mice. Of these 59, we identified 19 genes with a reported human eye phenotype. Overall, mining of the IMPC data yielded 40 previously unimplicated genes linked to mammalian eye development. Bioinformatic analysis showed that several of the IMPC genes colocalized to several protein anabolic and pluripotency pathways in early eye development. Of note, our analysis suggests that the serine-glycine pathway producing glycine, a mitochondrial one-carbon donator to folate one-carbon metabolism (FOCM), is essential for eye formation.ConclusionsUsing genome-wide phenotype screening of single-gene knockout mouse lines, STRING analysis, and bioinformatic methods, this study identified genes heretofore unassociated with MAC phenotypes providing models to research novel molecular and cellular mechanisms involved in eye development. These findings have the potential to hasten the diagnosis and treatment of this congenital blinding disease.

AbstractIdentifying the genetic determinants of pain is a scientific imperative given the magnitude of the global health burden that pain causes. Here, we report a genetic screen for nociception, performed under the auspices of the International Mouse Phenotyping Consortium. A biased set of 110 single-gene knockout mouse strains was screened for 1 or more nociception and hypersensitivity assays, including chemical nociception (formalin) and mechanical and thermal nociception (von Frey filaments and Hargreaves tests, respectively), with or without an inflammatory agent (complete Freund's adjuvant). We identified 13 single-gene knockout strains with altered nocifensive behavior in 1 or more assays. All these novel mouse models are openly available to the scientific community to study gene function. Two of the 13 genes (Gria1 and Htr3a) have been previously reported with nociception-related phenotypes in genetically engineered mouse strains and represent useful benchmarking standards. One of the 13 genes (Cnrip1) is known from human studies to play a role in pain modulation and the knockout mouse reported herein can be used to explore this function further. The remaining 10 genes (Abhd13, Alg6, BC048562, Cgnl1, Cp, Mmp16, Oxa1l, Tecpr2, Trim14, and Trim2) reveal novel pathways involved in nociception and may provide new knowledge to better understand genetic mechanisms of inflammatory pain and to serve as models for therapeutic target validation and drug development.

BackgroundThe diagnostic rate of Mendelian disorders in sequencing studies continues to increase, along with the pace of novel disease gene discovery. However, variant interpretation in novel genes not currently associated with disease is particularly challenging and strategies combining gene functional evidence with approaches that evaluate the phenotypic similarities between patients and model organisms have proven successful. A full spectrum of intolerance to loss-of-function variation has been previously described, providing evidence that gene essentiality should not be considered as a simple and fixed binary property.MethodsHere we further dissected this spectrum by assessing the embryonic stage at which homozygous loss-of-function results in lethality in mice from the International Mouse Phenotyping Consortium, classifying the set of lethal genes into one of three windows of lethality: early, mid, or late gestation lethal. We studied the correlation between these windows of lethality and various gene features including expression across development, paralogy and constraint metrics together with human disease phenotypes. We explored a gene similarity approach for novel gene discovery and investigated unsolved cases from the 100,000 Genomes Project.ResultsWe found that genes in the early gestation lethal category have distinct characteristics and are enriched for genes linked with recessive forms of inherited metabolic disease. We identified several genes sharing multiple features with known biallelic forms of inborn errors of the metabolism and found signs of enrichment of biallelic predicted pathogenic variants among early gestation lethal genes in patients recruited under this disease category. We highlight two novel gene candidates with phenotypic overlap between the patients and the mouse knockouts.ConclusionsInformation on the developmental period at which embryonic lethality occurs in the knockout mouse may be used for novel disease gene discovery that helps to prioritise variants in unsolved rare disease cases.

We searched a database of single-gene knockout (KO) mice produced by the International Mouse Phenotyping Consortium (IMPC) to identify candidate ciliopathy genes. We first screened for phenotypes in mouse lines with both ocular and renal or reproductive trait abnormalities. The STRING protein interaction tool was used to identify interactions between known cilia gene products and those encoded by the genes in individual knockout mouse strains in order to generate a list of "candidate ciliopathy genes." From this list, 32 genes encoded proteins predicted to interact with known ciliopathy proteins. Of these, 25 had no previously described roles in ciliary pathobiology. Histological and morphological evidence of phenotypes found in ciliopathies in knockout mouse lines are presented as examples (genes Abi2, Wdr62, Ap4e1, Dync1li1, and Prkab1). Phenotyping data and descriptions generated on IMPC mouse line are useful for mechanistic studies, target discovery, rare disease diagnosis, and preclinical therapeutic development trials. Here we demonstrate the effective use of the IMPC phenotype data to uncover genes with no previous role in ciliary biology, which may be clinically relevant for identification of novel disease genes implicated in ciliopathies.

The identification of causal variants in sequencing studies remains a considerable challenge that can be partially addressed by new gene-specific knowledge. Here, we integrate measures of how essential a gene is to supporting life, as inferred from viability and phenotyping screens performed on knockout mice by the International Mouse Phenotyping Consortium and essentiality screens carried out on human cell lines. We propose a cross-species gene classification across the Full Spectrum of Intolerance to Loss-of-function (FUSIL) and demonstrate that genes in five mutually exclusive FUSIL categories have differing biological properties. Most notably, Mendelian disease genes, particularly those associated with developmental disorders, are highly overrepresented among genes non-essential for cell survival but required for organism development. After screening developmental disorder cases from three independent disease sequencing consortia, we identify potentially pathogenic variants in genes not previously associated with rare diseases. We therefore propose FUSIL as an efficient approach for disease gene discovery.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.

[This corrects the article DOI: 10.1038/s42003-018-0226-0.].

[This corrects the article DOI: 10.1038/s42003-018-0226-0.].

Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.

This corrects the article DOI: 10.1038/nature19356.

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.

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.

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans.

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.

Too many racial/ethnic minorities do not reach their full potential for a healthy and rewarding life. This paper addresses the social determinants that impact, either directly or indirectly, child and adolescent health disparities. Understanding the role social determinants play in the life course of health status can help guide educational interventions and public policies in reducing racial/ethnic health disparities. We can no longer place the responsibility for adverse health outcomes solely on individuals and expect programs based primarily on individual health behaviors to eradicate the considerable racial/ethnic health care disparities that continue to exist in the United States today. (Contains 3 figures and 1 table.)

NSAIDs are commonly used among athletes for a variety of reasons. The purpose of this research is to gain insight on Division III athletes 'view and opinions of NSAIDs. A survey was developed incorporating the Theory of Planned Behavior and distributed to all winter and spring athletic teams of one Midwestern Dill University. By utilizing the Theory of Planned Behavior, this study found attitude toward behavior as the strongest predictor of behavioral intention (p < .001), while both Perceived Behavioral control (p < .001) and Intention (p < .001) were statistically significant predictors of behavior. Another finding from the study was that athletes perceived less than 25% of their teammates as taking NSAIDs. Displaying the importance of an athlete's own persona! values and opinions of NSAID use was the strongest predictor of intentions. Further assessment should include more athletes to get a better representation of the athletic department., Nonsteroidal anti-inflammatory drugs (NSAIDs) are among one of the most commonly purchased over the counter (OTC) medications. NSAIDs are used for a variety of reasons including: treatment of pain, soft [...]

This study assessed the Integrated Behavioral Model's (IBM) utility in explaining high-risk drinking among college students. A total of 356 participants completed a four-page questionnaire based on the (IBM) theory and their drinking behavior. The results from a path analysis revealed three significant constructs (p<0.05) which predicted intentions to engage in high-risk drinking: experiential attitude (0.34), injunctive norms (0.23), and self-efficacy (-0.28). The IBM explained approximately 45% and 26% of variance in intentions and high-risk drinking, respectively. Although limited in its use thus far, the IBM shows promise in its application regarding high-risk drinking prevention among college students.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.01.005 Byline: Ming Zhao (a), Jan Rohozinski (b), Manju Sharma (c), Jun Ju (a), Robert E. Braun (c), Colin E. Bishop (b)(d), Marvin L. Meistrich (a) Keywords: Spermatogenesis; Retroposons; Retrotransposition; Utp14; Juvenile spermatogonial depletion; X-chromosome; Spermatocytes; Spermatogonia; Male germ cells Abstract: The mouse retrogene Utp14b is essential for male fertility, and a mutation in its sequence results in the sterile juvenile spermatogonial depletion (jsd) phenotype. It is a retrotransposed copy of the Utp14a gene, which is located on the X chromosome, and is inserted within an intron of the autosomal acyl-CoA synthetase long-chain family member 3 (Acsl3) gene. To elucidate the roles of the Utp14 genes in normal spermatogenic cell development as a basis for understanding the defects that result in the jsd phenotype, we analyzed the various mRNAs produced from the Utp14b retrogene and their expression in different cell types. Two classes of transcripts were identified: variant 1, a transcript driven by the host gene promoter, that is predominantly found in germ cells but is ubiquitously expressed at low levels; and variants 2-5, a group of alternatively spliced transcripts containing some unique untranslated exons that are transcribed from a novel promoter that is germ-cell-specific. Utp14b (predominantly variant 1) is expressed at moderately high levels in pachytene spermatocytes, the developmental stage at which the expression of the X-linked Utp14a is suppressed. The levels of both classes of Utp14b transcripts were highest in round spermatids despite the transcription of Utp14a in these cells. We propose that when Utp14b initially inserted into Acsl3, it utilized the Acsl3 promoter to drive expression in pachytene spermatocytes to compensate for inactivation of Utp14a expression. The novel cell-type-specific promoter for Utp14b likely evolved later, as the protein may have acquired a germ cell-specific function in spermatid development. Author Affiliation: (a) Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, Box 066, 1515 Holcombe Blvd, Houston, TX 77030, USA (b) Department of Obstetrics and Gynecology, Baylor College of Medicine, 1709 Dryden Road, Houston, TX 77030, USA (c) Department of Genome Sciences, University of Washington School of Medicine, Box 357730, 1705 N.E. Pacific Street, Seattle, WA 98195, USA (d) Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA Article History: Received 28 September 2006; Revised 9 December 2006; Accepted 3 January 2007

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.11.016 Byline: Geraldine Seydoux (1), Robert E. Braun (2) Abstract: Oocytes and sperm are some of the most differentiated cells in our bodies, yet they generate all cell types after fertilization. Accumulating evidence suggests that this extraordinary potential is conferred to germ cells from the time of their formation during embryogenesis. In this Review, we describe common themes emerging from the study of germ cells in vertebrates and invertebrates. Transcriptional repression, chromatin remodeling, and an emphasis on posttranscriptional gene regulation preserve the totipotent genome of germ cells through generations. Author Affiliation: (1) Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA (2) Department of Genome Sciences, University of Washington School of Medicine, 1705 N.E. Pacific Street, Seattle, WA 98195, USA

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.02.013 Byline: Christopher Payne, Robert E. Braun Keywords: Histone modification; Germ cells; Spermatogonia; Epigenetics; Stem cells; Transcriptional repression Abstract: Chromatin structure plays an important role in the regulation of gene expression. Methylation of lysine residues on histone tails is an epigenetic mark that influences chromatin repression when specifically imparted on lysines 9 and 27 of histone H3, and on lysine 20 of H4. Histone lysines can be mono-, di-, and trimethylated, and all three modification states have been identified in different nuclear domains. Correlation of these methylated histone states to different stages of cell differentiation, however, is not extensive. Mammalian spermatogenesis is a developmental process ideal for studying the epigenetic control of differentiation. Maintenance of spermatogonial stem cells requires the transcriptional repressor Plzf, but a role for histone methylation has not been established. Here we show that Plzf-expressing spermatogonia completely lack monomethyl-H3-K27 and monomethyl-H4-K20, and contain very little monomethyl-H3-K9. Dimethylated H3-K27 and H4-K20 are detected as punctate foci in Plzf-positive cells, but dimethyl-H3-K9 is absent. Trimethylated H3-K9 and H4-K20 exhibit a unique perinuclear distribution that coincides with Plzf expression, localizing to punctate foci in more differentiated spermatogonia. Loss of Plzf correlates with increased punctate distribution of trimethylated H3-K9 and H4-K20 at the expense of perinuclear localization. These data signify the possible importance of different histone lysine methylation states in the epigenetic control of spermatogenesis. Author Affiliation: Department of Genome Sciences, University of Washington School of Medicine, Box 357730, 1705 N.E. Pacific Street, Seattle, WA 98195, USA Article History: Received 22 September 2005; Revised 26 January 2006; Accepted 13 February 2006

Within the mammalian testis, specialized tight junctions between somatic Sertoli cells create basal and apical polarity within the cells, restrict movement of molecules between cells, and separate the seminiferous epithelium into basal and adluminal compartments. These tight junctions form the basis of the blood--testis barrier, a structure whose function and dynamic regulation is poorly understood. In this study, we used microarray gene expression profiling to identify genes with altered transcript levels in a mouse model for conditional androgen insensitivity. We show that testosterone, acting through its receptor expressed in Sertoli cells, regulates the expression of claudin 3, which encodes a transient component of newly formed tight junctions. Sertoli cell-specific ablation of androgen receptor results in increased permeability of the blood--testis barrier to biotin, suggesting claudin 3 regulates the movement of small molecules across the Sertoli cell tight junctions. These results suggest that androgen action in Sertoli cells regulates germ cell differentiation, in part by controlling the microenvironment of the seminiferous epithelium. Our studies also indicate that hormonal strategies for male contraception may interfere with the blood--testis barrier. androgen receptor | testosterone | tight junctions

Gametes rely heavily on posttranscriptional control for their differentiation. Translational control, alternative splicing, and alternative processing of the 3' end of mRNAs are all common during spermatogenesis. Tent, which encodes a highly conserved 72-kDa protein, is expressed solely in germ ceils of the testis from the mid-pachytene stage until the elongating spennatid stage. TENR contains a double-stranded RNA binding domain, is localized to the nucleus, and is phylogenetically related to a family of adenosine deaminases involved in RNA editing. We show here that targeted mutation of the Tenr gene causes male sterility. Tenr mutant males have a reduced sperm count, and [Ten.sup.-/-] sperm show a decrease in motility and an increase in malformed heads. Despite their sterility, some epididymal sperm from Tenr mutants have normal morphology. The ability of Tenr mutant sperm to fertilize zona pellucida-free oocytes and to bind to, but not fertilize, zona pellucida-intact oocytes suggests that the normal-appearing sperm are not able to penetrate the zona pellucida. These data suggest that TENR plays an essential function in spermatid morphogenesis. Kerywords: Spermatogenesis; Posttranscriptional control; RNA editing