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Abstract Purpose Corneal dysmorphologies (CDs) are typically classified as either regressive degenerative corneal dystrophies (CDtrs) or defective growth and differentiation-driven corneal dysplasias (CDyps). Both eye disorders have multifactorial etiologies. While previous work has elucidated many aspects of CDs, such as presenting symptoms, epidemiology, and pathophysiology, the genetic mechanisms remain incompletely understood. The purpose of this study was to analyze phenotype data from 8,707 knockout mouse lines to identify new genes associated with the development of CDs in humans. Methods 8,707 knockout mouse lines phenotyped by the International Mouse Phenotyping Consortium were queried for genes associated with statistically significant (P

Abstract 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.

Despite a substantial body of research, we lack fundamental understanding of the pathophysiology of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) including pulmonary and cardiovascular outcomes, in part due to limitations of murine models. Most models use transgenic mice (K18) that express the human (h) angiotensin converting enzyme 2 (ACE2), ACE2 knock-in (KI) mice, or mouse-adapted strains of SARS-CoV-2. Further, many SARS-CoV-2 variants produce fatal neurologic disease in K18 mice and most murine studies focus only on acute disease in the first 14 days post inoculation (dpi). To better enable understanding of both acute (14 dpi) infection phases, we describe the development and characterization of a novel non-lethal KI mouse that expresses both the ACE2 and transmembrane serine protease 2 (TMPRSS2) genes (hACE2/hTMPRSS2). The human genes were engineered to replace the orthologous mouse gene loci but remain under control of their respective murine promoters, resulting in expression of ACE2 and TMPRSS2 instead of their murine counterparts. After intranasal inoculation with an omicron strain of SARS-CoV-2, hACE2/hTMPRSS2 KI mice transiently lost weight but recovered by 7 dpi. Infectious SARS-CoV-2 was detected in nasopharyngeal swabs 1-2 dpi and in lung tissues 2-6 dpi, peaking 4 dpi. These outcomes were similar to those in K18 mice that were inoculated in parallel. To determine the extent to which hACE2/hTMPRSS2 KI mice are suitable to model pulmonary and cardiovascular outcomes, physiological assessments measuring locomotion, behavior and reflexes, biomonitoring to measure cardiac activity and respiration, and micro computed tomography to assess lung function were conducted frequently to 6 months post inoculation. Male but not female SARS-CoV-2 inoculated hACE2/hTMPRSS2 KI mice showed a transient reduction in locomotion compared to control saline treated mice. No significant changes in respiration, oxygen saturation, heart rate variability, or conductivity were detected in SARS-CoV-2 inoculated mice of either sex. When re-inoculated 6 months after the first inoculation, hACE2/hTMPRSS2 KI became re-infected with disease signs similar to after the first inoculation. Together these data show that a newly generated hACE2/hTMPRSS2 KI mouse can be used to study mild COVID-19.

Abstract 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.

Abstract Topologically associating domain (TAD) boundaries partition the genome into distinct regulatory territories. Anecdotal evidence suggests that their disruption may interfere with normal gene expression and cause disease phenotypes1–3, but the overall extent to which this occurs remains unknown. Here we demonstrate that targeted deletions of TAD boundaries cause a range of disruptions to normal in vivo genome function and organismal development. We used CRISPR genome editing in mice to individually delete eight TAD boundaries (11–80 kb in size) from the genome. All deletions examined resulted in detectable molecular or organismal phenotypes, which included altered chromatin interactions or gene expression, reduced viability, and anatomical phenotypes. We observed changes in local 3D chromatin architecture in 7 of 8 (88%) cases, including the merging of TADs and altered contact frequencies within TADs adjacent to the deleted boundary. For 5 of 8 (63%) loci examined, boundary deletions were associated with increased embryonic lethality or other developmental phenotypes. For example, a TAD boundary deletion near Smad3/Smad6 caused complete embryonic lethality, while a deletion near Tbx5/Lhx5 resulted in a severe lung malformation. Our findings demonstrate the importance of TAD boundary sequences for in vivo genome function and reinforce the critical need to carefully consider the potential pathogenicity of noncoding deletions affecting TAD boundaries in clinical genetics screening.

Abstract Background Microphthalmia, 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. Results Query 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. Conclusions Using 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.

Abstract 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.

Abstract Background The 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. Methods Here 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. Results We 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. Conclusions Information 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.

Metabolites play an important role in physiology, yet the complexity of the metabolome and its interaction with disease and aging is poorly understood. Here the authors present a comprehensive atlas of the mouse brain metabolome and how it changes during aging.

Ericsson et al. show that different vendors (suppliers of mouse strains) harbor distinct microbiomes, which drive distinct behavioral phenotypes when the genetics are fixed. They specifically focus on changes relating to exploratory and anxiety-related behavior, physiological phenotypic parameters, glucose metabolism, and blood leukocytes. They conclude by emphasizing that supplier-origin fecal microbiomes represent potential sources of poor experimental reproducibility and suggest means to optimize experimentation with mice and their microbiomes.

Abstract We proteotyped blood plasma from 30 mouse knockout strains and corresponding wild-type mice from the International Mouse Phenotyping Consortium. We used targeted proteomics with internal standards to quantify 375 proteins in 218 samples. Our results provide insights into the manifested effects of each gene knockout at the plasma proteome level. We first investigated possible contamination by erythrocytes during sample preparation and labeled, in one case, up to 11 differential proteins as erythrocyte originated. Second, we showed that differences in baseline protein abundance between female and male mice were evident in all mice, emphasizing the necessity to include both sexes in basic research, target discovery, and preclinical effect and safety studies. Next, we identified the protein signature of each gene knockout and performed functional analyses for all knockout strains. Further, to demonstrate how proteome analysis identifies the effect of gene deficiency beyond traditional phenotyping tests, we provide in-depth analysis of two strains, C8a −/− and Npc2 +/− . The proteins encoded by these genes are well-characterized providing good validation of our method in homozygous and heterozygous knockout mice. Ig alpha chain C region, a poorly characterized protein, was among the differentiating proteins in C8a −/− . In Npc2 +/− mice, where histopathology and traditional tests failed to differentiate heterozygous from wild-type mice, our data showed significant difference in various lysosomal storage disease-related proteins. Our results demonstrate how to combine absolute quantitative proteomics with mouse gene knockout strategies to systematically study the effect of protein absence. The approach used here for blood plasma is applicable to all tissue protein extracts.

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.

Abstract Skeletal muscle anatomy and physiology are sexually dimorphic but molecular underpinnings and muscle‐specificity are not well‐established. Variances in metabolic health, fitness level, sedentary behavior, genetics, and age make it difficult to discern inherent sex effects in humans. Therefore, mice under well‐controlled conditions were used to determine female and male (n = 19/sex) skeletal muscle fiber type/size and capillarity in superficial and deep gastrocnemius (GA‐s, GA‐d), soleus (SOL), extensor digitorum longus (EDL), and plantaris (PLT), and transcriptome patterns were also determined (GA, SOL). Summed muscle weight strongly correlated with lean body mass (r2 = 0.67, p 6000 (GA) and >4000 (SOL) mRNAs differentially‐expressed by sex; only a minority of these were shared across GA and SOL. Pathway analyses revealed differences in ribosome biology, transcription, and RNA processing. Curation of sexually dimorphic muscle transcripts shared in GA and SOL, and literature datasets from mice and humans, identified 11 genes that we propose are canonical to innate sex differences in muscle: Xist, Kdm6a, Grb10, Oas2, Rps4x (higher, females) and Ddx3y, Kdm5d, Irx3, Wwp1, Aldh1a1, Cd24a (higher, males). These genes and those with the highest “sex‐biased” expression in our study do not contain estrogen‐response elements (exception, Greb1), but a subset are proposed to be regulated through androgen response elements. We hypothesize that innate muscle sexual dimorphism in mice and humans is triggered and then maintained by classic X inactivation (Xist, females) and Y activation (Ddx3y, males), with coincident engagement of X encoded (Kdm6a) and Y encoded (Kdm5d) demethylase epigenetic regulators that are complemented by modulation at some regions of the genome that respond to androgen.

Discovery of causal variants for monogenic disorders has been facilitated by whole exome and genome sequencing, but does not provide a diagnosis for all patients. Here, the authors propose a Full Spectrum of Intolerance to Loss-of-Function (FUSIL) categorization that integrates gene essentiality information to aid disease gene discovery.

Abstract 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.

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.

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.

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.

Circadian systems provide a fitness advantage to organisms by allowing them to adapt to daily changes of environmental cues, such as light/dark cycles. The molecular mechanism underlying the circadian clock has been well characterized. However, how internal circadian clocks are entrained with regular daily light/dark cycles remains unclear. By collecting and analyzing indirect calorimetry (IC) data from more than 2000 wild-type mice available from the International Mouse Phenotyping Consortium (IMPC), we show that the onset time and peak phase of activity and food intake rhythms are reliable parameters for screening defects of circadian misalignment. We developed a machine learning algorithm to quantify these two parameters in our misalignment screen (SyncScreener) with existing datasets and used it to screen 750 mutant mouse lines from five IMPC phenotyping centres. Mutants of five genes (Slc7a11, Rhbdl1, Spop, Ctc1 and Oxtr) were found to be associated with altered patterns of activity or food intake. By further studying the Slc7a11tm1a/tm1a mice, we confirmed its advanced activity phase phenotype in response to a simulated jetlag and skeleton photoperiod stimuli. Disruption of Slc7a11 affected the intercellular communication in the suprachiasmatic nucleus, suggesting a defect in synchronization of clock neurons. Our study has established a systematic phenotype analysis approach that can be used to uncover the mechanism of circadian entrainment in mice.

In the original published version of the article, Valerie Vancollie was mistakenly omitted from the list of members of the International Mouse Phenotyping Consortium. In addition, recognition of funding from Wellcome Trust grant WT098051 was mistakenly omitted from the Acknowledgements.The errors have been corrected in both the PDF and HTML versions of the paper.

SUMMARY 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.

The Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines were developed to address the lack of reproducibility in biomedical animal studies and improve the communication of research findings. While intended to guide the preparation of peer-reviewed manuscripts, the principles of transparent reporting are also fundamental for in vivo databases. Here, we describe the benefits and challenges of applying the guidelines for the International Mouse Phenotyping Consortium (IMPC), whose goal is to produce and phenotype 20,000 knockout mouse strains in a reproducible manner across ten research centres. In addition to ensuring the transparency and reproducibility of the IMPC, the solutions to the challenges of applying the ARRIVE guidelines in the context of IMPC will provide a resource to help guide similar initiatives in the future.

Targeted mutations in mouse disrupt local chromatin structure and may lead to unanticipated local effects. We evaluated targeted gene promoter silencing in a group of six mutants carrying the tm1a Knockout Mouse Project allele containing both a LacZ reporter gene driven by the native promoter and a neo selection cassette. Messenger RNA levels of the reporter gene and targeted gene were assessed by qRT-PCR, and methylation of the promoter CpG islands and LacZ coding sequence were evaluated by sequencing of bisulfite-treated DNA. Mutants were stratified by LacZ staining into presumed Silenced and Expressed reporter genes. Silenced mutants had reduced relative quantities LacZ mRNA and greater CpG Island methylation compared with the Expressed mutant group. Within the silenced group, LacZ coding sequence methylation was significantly and positively correlated with CpG Island methylation, while promoter CpG methylation was only weakly correlated with LacZ gene mRNA. The results support the conclusion that there is promoter silencing in a subset of mutants carrying the tm1a allele. The features of targeted genes which promote local silencing when targeted remain unknown.

Marker assisted speed congenics technique is commonly used to facilitate backcrossing of mouse strains in nearly half the time it normally takes otherwise. Traditionally, the technique is performed by analyzing PCR amplified regions of simple sequence length polymorphism (SSLP) markers between the recipient and donor strains: offspring with the highest number of markers showing the recipient genome across all chromosomes is chosen for the next generation. Although there are well-defined panels of SSLP makers established between certain pairs of mice strains, they are incomplete for most strains. The availability of well-established marker sets for speed congenic screens would enable the scientific community to transfer mutations across strain backgrounds. In this study, we tested the suitability of over 400 SSLP marker sets among 10 mouse strains commonly used for generating genetically engineered models. The panel of markers presented here can readily identify the specified strains and will be quite useful in marker assisted speed congenic screens. Moreover, unlike newer single nucleotide polymorphism (SNP) array methods which require sophisticated equipment, the SSLP markers panel described here only uses PCR and agarose gel electrophoresis of amplified products; therefore it can be performed in most research laboratories.

Procedures for cryopreserving embryos vary considerably, each having its specific advantages and disadvantages in terms of technical feasibility, embryo survival yield, temperature permissibility and species- or strain-dependent applicability. Here we report a high osmolality vitrification (HOV) method that is advantageous in these respects. Cryopreservation by vitrification is generally very simple, but, unlike slow freezing, embryos should be kept at a supercooling temperature (below -130°C) to avoid cryodamage. We overcame this problem by using an HOV solution containing 42.5% (v/v) ethylene glycol, 17.3% (w/v) Ficoll and 1.0 M sucrose. This solution is more viscous than other cryopreservation solutions, but easy handling of embryos was assured by employing a less viscous equilibration solution before vitrification. Most (>80%) embryos cryopreserved in this solution survived at -80°C for at least 30 days. Normal mice were recovered even after intercontinental transportation in a conventional dry-ice package for 2-3 days, indicating that special containers such as dry shippers with liquid nitrogen vapor are unnecessary. The HOV solution could also be employed for long-term storage in liquid nitrogen, as with other conventional cryoprotectants. Finally, we confirmed that this new vitrification method could be applied successfully to embryos of all six strains of mice we have tested so far. Thus, our HOV method provides an efficient and reliable strategy for the routine cryopreservation of mouse embryos in animal facilities and biomedical laboratories, and for easy and cheap transportation.