Mutant Mouse Lines Research Articles

TarGo: network based target gene selection system for human disease related mouse models

Genetically engineered mouse models are used in high-throughput phenotyping screens to understand genotype-phenotype associations and their relevance to human diseases. However, not all mutant mouse lines with detectable phenotypes are associated with human diseases. Here, we propose the “Target gene selection system for Genetically engineered mouse models” (TarGo). Using a combination of human disease descriptions, network topology, and genotype-phenotype correlations, novel genes that are potentially related to human diseases are suggested. We constructed a gene interaction network using protein-protein interactions, molecular pathways, and co-expression data. Several repositories for human disease signatures were used to obtain information on human disease-related genes. We calculated disease- or phenotype-specific gene ranks using network topology and disease signatures. In conclusion, TarGo provides many novel features for gene function prediction.

Soft windowing application to improve analysis of high-throughput phenotyping data.

MotivationHigh-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors.ResultsHere we introduce ‘soft windowing’, a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype–phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources.Availability and implementationThe method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin.Supplementary information Supplementary data are available at Bioinformatics online.

Nuclear envelope-localized torsinA-LAP1 complex regulates hepatic VLDL secretion and steatosis.

Deciphering novel pathways regulating liver lipid content has profound implications for understanding the pathophysiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Recent evidence suggests that the nuclear envelope is a site of regulation of lipid metabolism but there is limited appreciation of the responsible mechanisms and molecular components within this organelle. We showed that conditional hepatocyte deletion of the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1) caused defective VLDL secretion and steatosis, including intranuclear lipid accumulation. LAP1 binds to and activates torsinA, an AAA+ ATPase that resides in the perinuclear space and continuous main ER. Deletion of torsinA from mouse hepatocytes caused even greater reductions in VLDL secretion and profound steatosis. Both of these mutant mouse lines developed hepatic steatosis and subsequent steatohepatitis on a regular chow diet in the absence of whole-body insulin resistance or obesity. Our results establish an essential role for the nuclear envelope-localized torsinA-LAP1 complex in hepatic VLDL secretion and suggest that the torsinA pathway participates in the pathophysiology of nonalcoholic fatty liver disease.

Lrrc7 mutant mice model developmental emotional dysregulation that can be alleviated by mGluR5 allosteric modulation

LRRC7 has been identified as a candidate gene for severe childhood emotional dysregulation. Direct experimental evidence for a role of LRRC7 in the disease is needed, as is a better understanding of its impact on neuronal structure and signaling, and hence potential treatment targets. Here, we generated and analyzed an Lrrc7 mutant mouse line. Consistent with a critical role of LRRC7 in emotional regulation, mutant mice had inappropriate juvenile aggressive behavior and significant anxiety-like behavior and social dysfunction in adulthood. The pivotal role of mGluR5 signaling was demonstrated by rescue of behavioral defects with augmentation of mGluR5 receptor activity by 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB). Intra-peritoneal injection of CDPPB alleviated abnormal juvenile behavior, as well as anxiety-like behavior and hypersociability at adulthood. Furthermore, mutant primary neurons had impaired neurite outgrowth which was rescued by CDPPB treatment. In conclusion, Lrrc7 mutant mice provide a valuable tool to model childhood emotional dysregulation and persistent mental health comorbidities. Moreover, our data highlight an important role of LRRC7 in mGluR5 signaling, which is a potential new treatment target for anxiety and social dysfunction.

Genetic mutation of Frem3 does not causeFraser syndrome in mice.

QBRICK, FRAS1, and FREM2 compose a family of extracellular matrix proteins characterized by twelve consecutive CSPG repeats and single or multiple Calx-β motifs. Dysfunction of these proteins have been associated with Fraser syndrome, which is characterized by malformation of skin, eyes, digits, and kidneys. FREM3 is another member of the 12-CSPG protein family. However, it remains unknown whether genetic dysfunction of FREM3 also causes Fraser syndrome or another developmental disorder. Here we investigated a Frem3 mutant mouse line generated by CRISPR/Cas9-mediated genome editing. The FREM3 mutant homozygotes were born at the expected Mendelian ratio and did not possess any defects characteristic of Fraser syndrome. These results indicate that the dysfunction of FREM3 is not associated with Fraser syndrome.

Unexpected Compensatory Increase in Shank3 Transcripts in Shank3 Knock-Out Mice Having Partial Deletions of Exons.

Genetic variants of the SH3 and multiple ankyrin repeat domains 3 (SHANK3) gene, which encodes excitatory postsynaptic core scaffolds cause numerous brain disorders. Several lines of Shank3 knock-out (KO) mice with deletions of different Shank3 exons have previously been generated and characterized. The different Shank3 KO mouse lines have both common and line-specific phenotypes. Shank3 isoform diversity is considered a mechanism underlying phenotypic heterogeneity, and compensatory changes through regulation of Shank3 expression may contribute to this heterogeneity. However, whether such compensatory changes occur in Shank3 KO mouse lines has not been investigated in detail. Using previously reported RNA-sequencing analyses, we identified an unexpected increase in Shank3 transcripts in two different Shank3 mutant mouse lines (Shank3B and Shank3ΔC) having partial deletions of Shank3 exons. We validated an increase in Shank3 transcripts in the hippocampus, cortex, and striatum, but not in the cerebellum, of Shank3B heterozygous (HET) and KO mice, using qRT-PCR analyses. In particular, expression of the N-terminal exons 1–12, but not the more C-terminal exons 19–22, was observed to increase in Shank3B mice with deletion of exons 13–16. This suggests a selective compensatory activation of upstream Shank3 promoters. Furthermore, using domain-specific Shank3 antibodies, we confirmed that the increased Shank3 transcripts in Shank3B KO mice produced a small Shank3 isoform that was not detected in wild-type mice. Taken together, our results illustrate another layer of complexity in the regulation of Shank3 expression in the brain, which may also contribute to the phenotypic heterogeneity of different Shank3 KO mouse lines.

Targeted Mutations in the Mouse via Embryonic Stem Cells.

Genetic modification of mouse embryonic stem (ES) cells is a powerful technology that enabled the generation of a plethora of mutant mouse lines to study gene function and mammalian biology. Here we describe ES cell culture and transfection techniques used to manipulate the ES cell genome to obtain targeted ES cell clones. We include the standard gene targeting approach as well as the application of the CRISPR/Cas9 system that can improve the efficiency of homologous recombination in ES cells by introducing a double-strand DNA break at the target site.

CBE1 is a manchette and mitochondria associated protein with a potential role in somatic cell proliferation

Ciliated bronchial epithelium 1 (CBE1) is a microtubule-associated protein localized to the manchette and developing flagellum during spermiogenesis and is associated with sperm maturation arrest in humans. It was hypothesized that CBE1 functions in microtubule-mediated transport mechanisms and sperm tail formation. To test this hypothesis, we analyzed Cbe1 expression and localization during spermiogenesis, and in mouse inner medullary collecting duct-3 (IMCD3) cells as a model of ciliogenesis. Furthermore, we generated and analyzed the fertility of a Cbe1 mutant mouse line. Mice containing a homozygous deletion in the long forms of Cbe1 were born at a lower frequency than predicted by Mendelian inheritance; however, adult male mice were fertile. An in-depth analysis of the Cbe1 gene revealed alternative transcript variants, which were not affected by the exon 2 mutation. To assess whether short variants compensate for the loss of long variants, exons 2 and 4 (which affect all variants) were individually mutated in IMCD3 cells and the effects on cell proliferation and ciliogenesis were analyzed. In wild-type IMCD3 cells, both variants were upregulated during cilia assembly. CBE1 protein was not a structural component of cilia; rather, CBE1 localized to the mitochondria and the contractile ring of dividing IMCD3 cells. Although IMCD3 cells carrying the mutation in long variants showed no phenotypic alterations, the mutation in exon 4 resulted in a significantly decreased proliferation rate. This study reveals that long isoforms of CBE1 are not essential for male fertility. Data, however, suggest that CBE1 is associated with intramanchette transport and midpiece formation of the sperm tail.

Large-scale neuroanatomical study uncovers 198 gene associations in mouse brain morphogenesis

Brain morphogenesis is an important process contributing to higher-order cognition, however our knowledge about its biological basis is largely incomplete. Here we analyze 118 neuroanatomical parameters in 1,566 mutant mouse lines and identify 198 genes whose disruptions yield NeuroAnatomical Phenotypes (NAPs), mostly affecting structures implicated in brain connectivity. Groups of functionally similar NAP genes participate in pathways involving the cytoskeleton, the cell cycle and the synapse, display distinct fetal and postnatal brain expression dynamics and importantly, their disruption can yield convergent phenotypic patterns. 17% of human unique orthologues of mouse NAP genes are known loci for cognitive dysfunction. The remaining 83% constitute a vast pool of genes newly implicated in brain architecture, providing the largest study of mouse NAP genes and pathways. This offers a complementary resource to human genetic studies and predict that many more genes could be involved in mammalian brain morphogenesis.

Loss of function mutation of Eftud2, the gene responsible for mandibulofacial dysostosis with microcephaly (MFDM), leads to pre-implantation arrest in mouse.

Mutations in EFTUD2 are responsible for the autosomal dominant syndrome named MFDM (mandibulofacial dysostosis with microcephaly). However, it is not clear how reduced levels of EFTUD2 cause abnormalities associated with this syndrome. To determine if the mouse can serve as a model for uncovering the etiology of abnormalities found in MFDM patients, we used in situ hybridization to characterize expression of Eftud2 during mouse development, and used CRISPR/Cas9 to generate a mutant mouse line with deletion of exon 2 of the mouse gene. We found that Eftud2 was expressed throughout embryonic development, though its expression was enriched in the developing head and craniofacial regions. Additionally, Eftud2 heterozygous mutant embryos had reduced EFTUD2 mRNA and protein levels. Moreover, Eftud2 heterozygous embryos were born at the expected Mendelian frequency, and were viable and fertile despite being developmentally delayed. In contrast, Eftud2 homozygous mutant embryos were not found post-implantation but were present at the expected Mendelian frequency at embryonic day (E) 3.5. Furthermore, only wild-type and heterozygous E3.5 embryos survived ex vivo culture. Our data indicate that Eftud2 expression is enriched in the precusor of structures affected in MFDM patients and show that heterozygous mice carrying deletion of exon 2 do not model MFDM. In addition, we uncovered a requirement for normal levels of Eftud2 for survival of pre-implantation zygotes.

Common and distinct transcriptional signatures of mammalian embryonic lethality

The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4–36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.

Novel mutant mouse line emphasizes the importance of protein kinase C theta for CD4+ T lymphocyte activation

BackgroundThe protein kinase C theta (PKCθ) has an important and non-redundant function downstream of the antigen receptor and co-receptor complex in T lymphocytes. PKCθ is not only essential for activation of NF-κB, AP-1 and NFAT and subsequent interleukin-2 expression, but also critical for positive selection and development of regulatory T lymphocytes in the thymus. Several domains regulate its activity, such as a pseudosubstrate sequence mediating an auto-inhibitory intramolecular interaction, the tandem C1 domains binding diacylglycerol, and phosphorylation at conserved tyrosine, threonine as well as serine residues throughout the whole length of the protein. To address the importance of the variable domain V1 at the very N-terminus, which is encoded by exon 2, a mutated version of PKCθ was analyzed for its ability to stimulate T lymphocyte activation.MethodsT cell responses were analyzed with promoter luciferase reporter assays in Jurkat T cells transfected with PKCθ expression constructs. A mouse line expressing mutated instead of wild type PKCθ was analyzed in comparison to PKCθ-deficient and wild type mice for thymic development and T cell subsets by flow cytometry and T cell activation by quantitative RT-PCR, luminex analysis and flow cytometry.ResultsIn cell lines, the exon 2-replacing mutation impaired the transactivation of interleukin-2 expression by constitutively active mutant form of PKCθ. Moreover, analysis of a newly generated exon 2-mutant mouse line (PKCθ-E2mut) revealed that the N-terminal replacement mutation results in an hypomorph mutant of PKCθ combined with reduced PKCθ protein levels in CD4+ T lymphocytes. Thus, PKCθ-dependent functions in T lymphocytes were affected resulting in impaired thymic development of single positive T lymphocytes in vivo. In particular, there was diminished generation of regulatory T lymphocytes. Furthermore, early activation responses such as interleukin-2 expression of CD4+ T lymphocytes were significantly reduced even though cell viability was not affected. Thus, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice.ConclusionTaken together, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. Both our in vitro T cell culture experiments and ex vivo analyses of a PKCθ-E2-mutant mouse line independently validate the importance of PKCθ downstream of the antigen-receptor complex for activation of CD4+ T lymphocytes.

SUN-473 The Contribution of Bmal1 Expression in the Suprachiasmatic Nucleus to Female Fertility

The preovulatory gonadotropin-releasing hormone (GnRH) and resulting luteinizing hormone (LH) surge is gated to the onset of the active period in mice by a circadian and steroid signal. The temporal signal that initiates this process is believed to arrive from the suprachiasmatic nucleus (SCN), the hypothalamic circadian pacemaker. The SCN may contribute to reproduction through two discrete projections: vasoactive inhibitory peptide (VIP) neurons in the SCN project to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area, and arginine vasopressin (AVP) neurons in the SCN project to kisspeptin (Kiss1) neurons in the anteroventral periventricular (AVPV). Genomic deletion of the critical circadian clock component, Bmal1, results in infertility in females. We sought to determine whether Bmal1 was critical in the SCN VIP and AVP neurons by using cre/lox technology to create three lines of mutant mice: AVP-Bmal1-/-, VIP-Bmal1-/-, and AVP-VIP-Bmal1-/-. We found no significant difference in LH levels following a kisspeptin challenge (2 mg/kg) between AVP-Bmal1-/- and Bmal1flox/flox (617.4 ± 216.9 vs 629.6 ± 110.6 pg/mL) or GnRH challenge (10 μg/kg) (1029.0 ± 246.2 vs 712.7 ± 142.6 pg/mL). Preliminary findings of an induced LH surge found AVP-Bmal1-/- and VIP-Bmal1-/- mice are able to mount a surge at the time of lights off (Bmal1flox/flox: 3 of 5; AVP-Bmal1-/-: 1 of 2, VIP-Bmal1:-/- 4 of 4; AVP-VIP-Bmal1-/- 0 of 2). Additionally, we were able to detect an endogenous surge at the time of lights off in both AVP-Bmal1-/-, VIP-Bmal1-/- mice, but not AVP-VIP-Bmal1-/-. However, AVP-Bmal1-/-, VIP-Bmal1-/-, and AVP-VIP-Bmal1-/- females carried litters to term and show no differences in the time to first litter compared to Bmal1flox/flox control (AVP-Bmal1-/-: 48 ± 4.8 vs 44.3 ± 0.3 days; VIP-Bmal1-/-: 29.6 ± 7.7 vs 31.0 ± 4.2 days; AVP-VIP-Bmal1-/-;27.7 ± 7 vs 23 ± 0 days); number of litters in 100 days (AVP-Bmal1-/-: 3.7 ±0.6 vs 4 ± 0.6; VIP-Bmal1-/-: 3.0 ± 1.0 vs 3.0 ± 0.0; AVP-VIP-Bmal1-/- 3.5 ± 0.7 vs 3.5 ± 0.7 days); or pups per litter (AVP-Bmal1-/-: 7.2 ± 0.7 vs 7.3 ± 0.7; VIP-Bmal1-/-: 6.2 ± 1.6 vs 7.6 ± 2.9; AVP-VIP-Bmal1-/- 8.1 ± 0.9 vs 9 ± 1.4 pups). Overall, these findings suggest that Bmal1 expression in the SCN AVP or VIP neurons is not required for fertility in a 12:12 light:dark cycle.

Mouse models of hypoplastic left heart syndrome (HLHS) reveal new targets for potential intervention

Hypoplastic left heart syndrome (HLHS) is one of the most lethal congenital heart disease (CHD), with a 30% 5‐year mortality rate after surgical palliation. Patients can succumb to heart failure and among surviving patients, neurodevelopmental delay and neurobehavioral and neurocognitive disabilities are observed at a high prevalence. While hypoxia and hemodynamic disturbance related to the structural heart defect and from cardiopulmonary bypass surgical intervention are suggested as factors driving these poor outcomes, studies over the past decade indicate they are largely attributable to as yet unidentified patient intrinsic factors. The recent recovery of mouse models of HLHS has provided some insights into the nature of such intrinsic factors. Analysis of the HLHS mutant mice have revealed HLHS has a complex genetic architecture and is profoundly genetically heterogeneous. Detailed investigation of the Ohia HLHS mutant mouse line showed HLHS arises combinatorially from mutations in two genes, Sap130 – a chromatin modifying protein in the Sin3A HDAC repressor complex, and PchdhA9 ‐ a protocadherin mediating cell‐cell adhesion. While Sap130 drives the LV hypoplasia, Pcdha9 drives the aortic valve defects and can cause bicuspid aortic valve. These findings have been validated with the production and analysis of CRISPR gene targeted mice and zebrafish models.Analyses of CHD defects in Ohia mutant mice and the emergence of HLHS during embryonic/fetal development indicated the LV hypoplasia is not hemodynamically driven, but instead is associated with cell intrinsic defects involving mitotic cell cycle arrest. Interestingly, the Ohia mutant mice also showed forebrain abnormalities involving hypoplasia of the cerebral cortex, and dysplasia of the hippocampus, corpus callosum and cerebellum, brain structures also observed to be impacted in HLHS patients. Further analysis of a floxed Sap130 allele with Emx1‐Cre targeted forebrain‐specific deletion of Sap130 yielded viable adult mice with normal heart structure but with brain abnormalities that included microcephaly, similar to findings in the Ohia mutant fetuses. This was associated with cell intrinsic defects involving loss of neural progenitors. Brain MRI and neuronal fiber tractography with diffusion tensor imaging revealed extensive alterations in neural network connectivity. Behavioral assessments further showed learning and memory deficits. Together these findings suggest a shared genetic etiology for the heart and brain defects in HLHS. This likely is mediated by cell intrinsic defects, suggesting the need to shift the focus of therapeutic intervention from hemodynamic modulation to targeting the cell intrinsic deficits associated with HLHS. Only then will it be possible to achieve substantive improvement in cardiac and neurodevelopmental outcome for HLHS.Support or Funding InformationFunded by NIH Grants HL142788 and HL132024This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nine genes abundantly expressed in the epididymis are not essential for male fecundity in mice.

BackgroundSpermatozoa become competent for fertilization during transit through the epididymis. As spermatozoa from the proximal caudal epididymis can fertilize eggs, proteins from the caput and corpus epididymis are required for sperm maturation.ObjectivesMicroarray analysis identified that more than 17,000 genes are expressed in the epididymis; however, few of these genes demonstrate expression restricted to the epididymis. To analyze epididymis‐enriched gene function in vivo, we generated knockout (KO) mutations in nine genes that are abundantly expressed in the caput and corpus region of the epididymis.Materials and methods KO mice were generated using the CRISPR/Cas9 system. The histology of the epididymis was observed with hematoxylin and eosin staining. KO males were caged with wild‐type females for 3–6 months to check fertility.ResultsWe generated individual mutant mouse lines having indel mutations in Pate1, Pate2, or Pate3. We also deleted the coding regions of Clpsl2, Epp13, and Rnase13, independently. Finally, the 150 kb region encoding Gm1110, Glb1l2, and Glb1l3 was deleted to generate a triple KO mouse line. Histology of the epididymis and sperm morphology of all KO lines were comparable to control males. The females mated with these KO males delivered pups at comparable numbers as control males.Discussion and conclusionWe revealed that nine genes abundantly expressed in the caput and corpus epididymis are dispensable for sperm function and male fecundity. CRISPR/Cas9‐mediated KO mice generation accelerates the screening of epididymis‐enriched genes for potential functions in reproduction.

Neural crest-derived neurons invade the ovary but not the testis during mouse gonad development

Testes and ovaries undergo sex-specific morphogenetic changes and adopt strikingly different morphologies, despite the fact that both arise from a common precursor, the bipotential gonad. Previous studies showed that recruitment of vasculature is critical for testis patterning. However, vasculature is not recruited into the early ovary. Peripheral innervation is involved in patterning development of many organs but has been given little attention in gonad development. In this study, we show that while innervation in the male reproductive complex is restricted to the epididymis and vas deferens and never invades the interior of the testis, neural crest-derived innervation invades the interior of the ovary around E16.5. Individual neural crest cells colonize the ovary, differentiate into neurons and glia, and form a dense neural network within the ovarian medulla. Using a sex-reversing mutant mouse line, we show that innervation is specific to ovary development, is not dependent on the genetic sex of gonadal or neural crest cells, and may be blocked by repressive guidance signals elevated in the male pathway. This study reveals another aspect of sexually dimorphic gonad development, establishes a precise timeline and structure of ovarian innervation, and raises many questions for future research.

Styk1 is specifically expressed in NK1.1+ lymphocytes including NK, γδ T, and iNKT cells in mice, but is dispensable for their ontogeny and function.

Innate Tcells, NK cells, and innate-like lymphocytes (ILCs) share transcriptional signatures that translate into overlapping developmental and functional programs. A prominent example for genes that are highly expressed in NK cells but not in ILCs is serine-threonine-tyrosine kinase 1 (Styk1 encoded by Styk1). We found Styk1 to be specifically expressed in lymphocytes positive for Killer cell lectin-like receptor subfamily B, member 1, also known as CD161 or NK1.1, i.e. in NK cell, αβ iNKT, and γδ NKTcell lineages. To investigate the role of Styk1 in the development and function of NK1.1+ innate T-cell subsets, we generated and analyzed a novel Styk1null mutant mouse line. Furthermore, we validated Styk1 expression in γδ NKTcells and in thymic, but not in peripheral invariant αβ iNKTcells through ex vivo analysis of a concomitantly generated transgenic Styk1 reporter mouse line. Despite the very specific expression of Styk1 in NK cells, γδ NKTcells, and thymic αβ iNKT, its absence did not alter homeostasis and function of these lineages. Thus, Styk1 expression is specific for NK cells and selected NK-like innate T-cell subsets, but dispensable for their development and function.

Rapid and high-efficient generation of mutant mice using freeze-thawed embryos of the C57BL/6J strain

BackgroundThe CRISPR/Cas9 technique has undergone many modifications to decrease the effort and shorten the time needed for efficient production of mutant mice. The use of fresh embryos consumes time and effort during oocytes preparation and fertilization before every experiment, and freeze-thawed embryos overcome this limitation. However, cryopreservation of 1-cell embryos is challenging. New methodWe introduce a protocol that combines a modified method for cryopreserving 1-cell C57BL/6J embryos with optimized electroporation conditions that were used to deliver CRISPR reagents into embryos, 1 h after thawing. ResultsFreeze-thawed 1-cell embryos showed similar survival rates and surprisingly high developmental rates compared to fresh embryos. Using our protocol, we generated several lines of mutant mice: knockout mice via non-homologous end joining (NHEJ) and knock-in mice via homology-directed repair (HDR) with high-efficient mutation rates (100%, 75% respectively) and a low mosaic rate within 4 weeks. Comparison with existing method (s)Our protocol associates the use of freeze-thawed embryos from an inbred strain and electroporation, and can be performed by laboratory personnel with basic training in embryo manipulation to generate mutant mice within short time periods. ConclusionWe developed a simple, economic, and robust protocol facilitating the generation of genetically modified mice, bypassing the need of backcrossing, with a high efficiency and a low mosaic rate. It makes the preparation of mouse models of human diseases a simple task with unprecedented ease, pace, and efficiency.

Folic acid modifies the shape of epithelial cells during morphogenesis via a Folr1 and MLCK dependent mechanism.

ABSTRACTFolic acid supplementation can prevent neural tube defects, but the specific molecular mechanisms by which it does have not been elucidated. During neural plate morphogenesis, epithelial cell apical constriction cooperates with other events to drive tissue-bending, and when defective, can result in neural tube defects. A Rho-kinase deficient binding mutant of the apical constriction regulating protein, Shroom3 (Shroom3R1838C), is one of only a handful of mouse mutant lines with neural tube defects that can be rescued by folic acid supplementation. This provided a unique opportunity to probe the functional rescue of a protein linked to neural tube development by folic acid. Utilizing an epithelial cell culture model of apical constriction, it was observed that treatment with exogenous folic acid, as well as co-expression of the folic acid receptor Folr1, can rescue the function of the Rho-kinase binding deficient mutant of Shroom3 in vitro. It was also determined that the rescuing ability of folic acid is RhoA and Rho-kinase independent but myosin light chain kinase (MLCK) and Src-kinase dependent. Inhibition of Rho-kinase-dependent apical constriction in chick embryo neural epithelium was also observed to be rescued by exogenous folic acid and that treatment with folic acid is accompanied by elevated activated myosin light chain and MLCK. Furthermore, doubly heterozygous mouse embryos lacking one copy each of Shroom3 and Folr1 exhibit a low rate of neural tube defects and also have lower levels of activated myosin light chain and MLCK. These studies suggest a novel mechanism by which folic acid modifies epithelial cell shape during morphogenesis, shedding light onto how folic acid may prevent neural tube defects.

Epigenetic dysregulation of Oxtr in Tet1-deficient mice has implications for neuropsychiatric disorders.

OXTR modulates a variety of behaviors in mammals, including social memory and recognition. Genetic and epigenetic dysregulation of OXTR has been suggested to be implicated in neuropsychiatric disorders, including autism spectrum disorder (ASD). While the involvement of DNA methylation is suggested, the mechanism underlying epigenetic regulation of OXTR is largely unknown. This has hampered the experimental design and interpretation of the results of epigenetic studies of OXTR in neuropsychiatric disorders. From the generation and characterization of a new line of Tet1 mutant mice - by deleting the largest coding exon 4 (Tet1Δe4) - we discovered for the first time to our knowledge that Oxtr has an array of mRNA isoforms and a complex transcriptional regulation. Select isoforms of Oxtr are significantly reduced in the brain of Tet1Δe4-/- mice. Accordingly, CpG islands of Oxtr are hypermethylated during early development and persist into adulthood. Consistent with the reduced express of OXTR, Tet1Δe4-/- mice display impaired maternal care, social behavior, and synaptic responses to oxytocin stimulation. Our findings elucidate a mechanism mediated by TET1 protein in regulating Oxtr expression by preventing DNA hypermethylation of Oxtr. The discovery of epigenetic dysregulation of Oxtr in TET1-deficient mouse brain supports the necessity of a reassessment of existing findings and a value of future studies of OXTR in neuropsychiatric disorders.