Mouse Models Of Human Disease Research Articles

A mathematical estimation of the physical forces driving podocyte detachment

Loss of podocytes, possibly through the detachment of viable cells, is a hallmark of progressive glomerular disease. Podocytes are exposed to considerable physical forces due to pressure and flow resulting in circumferential wall stress and tangential shear stress exerted on the podocyte cell body, which have been proposed to contribute to podocyte depletion. However, estimations of invivo alterations of physical forces in glomerular disease have been hampered by a lack of quantitative functional and morphological data. Here, we used ultra-resolution data and computational analyses in a mouse model of human disease, hereditary late-onset focal segmental glomerular sclerosis, to calculate increased mechanical stress upon podocyte injury. Transversal shear stress on the lateral walls of the foot processes was prominently increased during the initial stages of podocyte detachment. Thus, our study highlights the importance of targeting glomerular hemodynamics to treat glomerular disease.

Serum Antibodies to N-Glycolylneuraminic Acid Are Elevated in Duchenne Muscular Dystrophy and Correlate with Increased Disease Pathology in Cmah−/−mdx Mice

Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5'-monophospho-(CMP)-N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome-linked muscular dystrophy (mdx) model for DMD. Cmah-/-mdx mice can be induced to develop anti-Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah-/-mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats.

Histopathology is required to identify and characterize myopathies in high-throughput phenotype screening of genetically engineered mice.

The development of mouse models that replicate the genetic and pathological features of human disease is important in preclinical research because these types of models enable the completion of meaningful pharmacokinetic, safety, and efficacy studies. Numerous relevant mouse models of human disease have been discovered in high-throughput screening programs, but there are important specific phenotypes revealed by histopathology that are not reliably detected by any other physiological or behavioral screening tests. As part of comprehensive phenotypic analyses of over 4000 knockout (KO) mice, histopathology identified 12 lines of KO mice with lesions indicative of an autosomal recessive myopathy. This report includes a brief summary of histological and other findings in these 12 lines. Notably, the inverted screen test detected muscle weakness in only 4 of these 12 lines (Scyl1, Plpp7, Chkb, and Asnsd1), all 4 of which have been previously recognized and published. In contrast, 6 of 8 KO lines showing negative or inconclusive findings on the inverted screen test (Plppr2, Pnpla7, Tenm1, Srpk3, Sidt2, Yif1b, Mrs2, and Pnpla2) had not been previously identified as having myopathies. These findings support the need to include histopathology in phenotype screening protocols in order to identify novel genetic myopathies that are not clinically evident or not detected by the inverted screen test.

Saturation mutagenesis defines novel mouse models of severe spine deformity.

ABSTRACTEmbryonic formation and patterning of the vertebrate spinal column requires coordination of many molecular cues. After birth, the integrity of the spine is impacted by developmental abnormalities of the skeletal, muscular and nervous systems, which may result in deformities, such as kyphosis and scoliosis. We sought to identify novel genetic mouse models of severe spine deformity by implementing in vivo skeletal radiography as part of a high-throughput saturation mutagenesis screen. We report selected examples of genetic mouse models following radiographic screening of 54,497 mice from 1275 pedigrees. An estimated 30.44% of autosomal genes harbored predicted damaging alleles examined twice or more in the homozygous state. Of the 1275 pedigrees screened, 7.4% presented with severe spine deformity developing in multiple mice, and of these, meiotic mapping implicated N-ethyl-N-nitrosourea alleles in 21% of pedigrees. Our study provides proof of concept that saturation mutagenesis is capable of discovering novel mouse models of human disease, including conditions with skeletal, neural and neuromuscular pathologies. Furthermore, we report a mouse model of skeletal disease, including severe spine deformity, caused by recessive mutation in Scube3. By integrating results with a human clinical exome database, we identified a patient with undiagnosed skeletal disease who harbored recessive mutations in SCUBE3, and we demonstrated that disease-associated mutations are associated with reduced transactivation of Smad signaling in vitro. All radiographic results and mouse models are made publicly available through the Mutagenetix online database with the goal of advancing understanding of spine development and discovering novel mouse models of human disease.

Behavioural effects of cage systems on the G93A Superoxide Dismutase 1 transgenic mouse model for amyotrophic lateral sclerosis.

Environmental factors inherent to animal facilities can impact on the neuro-behavioural phenotype of laboratory mice and genetic mouse models for human diseases. Many facilities have upgraded from traditional 'open filter top' cages (FT) to individually ventilated cage (IVC) systems, which have been shown to modify various behavioural responses of laboratory mice. Importantly, the impact of IVC housing on the G93A superoxide dismutase 1 mouse model of amyotrophic lateral sclerosis (ALS) is currently unknown. Male and female wild type-like (WT) and heterozygous SOD1G93A mice were group-housed in FT or IVC systems from PND 30 ± 5 onwards. Body weight and motor function were assessed weekly from 15 weeks onward. Mice were also tested for cognitive abilities (i.e., fear conditioning and social recognition memory) and sensorimotor gating (i.e., prepulse inhibition: PPI). SOD1G93A mice lost body weight, and their motor function degenerated over time compared with control littermates. Motor impairments developed faster when SOD1G93A females were housed in IVCs. Context and cue freezing were increased in SOD1G93A females compared with controls, whereas all SOD1G93A mice exhibited lower acoustic startle and PPI than WT mice. IVC housing led to an increase in cue freezing in males and reduced the severity of PPI deficits in SOD1G93A females. Overall, IVC housing impacted moderately on the SOD1G93A phenotype but central behavioural deficits were still evident across housing conditions. Nonetheless, our findings indicate the importance of assessing the effect of cage system in genetic mouse models as these systems can modulate the magnitude and onset of genotypic differences.

CyTOF: An Emerging Technology for Single-Cell Proteomics in the Mouse.

The ability to analyze the proteome of single cells is critical for the advancement of studies of steady-state and pathological processes. Mass cytometry, or CyTOF, combines principles of mass spectrometry and flow cytometry to enable single-cell analysis of protein expression. CyTOF can simultaneously assess DNA content and proteins and has the capacity to measure 40 to 100 parameters in each cell. Applying this technology to tissues or cells on slides, termed imaging mass cytometry (IMC), allows for visualization of normal and diseased tissues in situ. The high-dimensional proteomic analysis that can be undertaken with CyTOF and IMC has the potential to enhance our understanding of complex and heterogeneous developmental and disease pathways. This article will describe the CyTOF experimental workflow, including reagent selection, sample preparation, and data analysis. CyTOF is compared to flow cytometry, focusing on the strengths and weaknesses of these powerful techniques. Importantly, we review key studies in mouse models of human disease that highlight the strength of CyTOF and IMC to drive discovery research and therapeutic advancement. © 2021 Wiley Periodicals LLC.

Convergence: Lactosylceramide-Centric Signaling Pathways Induce Inflammation, Oxidative Stress, and Other Phenotypic Outcomes.

Lactosylceramide (LacCer), also known as CD17/CDw17, is a member of a large family of small molecular weight compounds known as glycosphingolipids. It plays a pivotal role in the biosynthesis of glycosphingolipids, primarily by way of serving as a precursor to the majority of its higher homolog sub-families such as gangliosides, sulfatides, fucosylated-glycosphingolipids and complex neutral glycosphingolipids—some of which confer “second-messenger” and receptor functions. LacCer is an integral component of the “lipid rafts,” serving as a conduit to transduce external stimuli into multiple phenotypes, which may contribute to mortality and morbidity in man and in mouse models of human disease. LacCer is synthesized by the action of LacCer synthase (β-1,4 galactosyltransferase), which transfers galactose from uridine diphosphate galactose (UDP-galactose) to glucosylceramide (GlcCer). The convergence of multiple physiologically relevant external stimuli/agonists—platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), stress, cigarette smoke/nicotine, tumor necrosis factor-α (TNF-α), and in particular, oxidized low-density lipoprotein (ox-LDL)—on β-1,4 galactosyltransferase results in its phosphorylation or activation, via a “turn-key” reaction, generating LacCer. This newly synthesized LacCer activates NADPH (nicotinamide adenine dihydrogen phosphate) oxidase to generate reactive oxygen species (ROS) and a highly “oxidative stress” environment, which trigger a cascade of signaling molecules and pathways and initiate diverse phenotypes like inflammation and atherosclerosis. For instance, LacCer activates an enzyme, cytosolic phospholipase A2 (cPLA2), which cleaves arachidonic acid from phosphatidylcholine. In turn, arachidonic acid serves as a precursor to eicosanoids and prostaglandin, which transduce a cascade of reactions leading to inflammation—a major phenotype underscoring the initiation and progression of several debilitating diseases such as atherosclerosis and cancer. Our aim here is to present an updated account of studies made in the field of LacCer metabolism and signaling using multiple animal models of human disease, human tissue, and cell-based studies. These advancements have led us to propose that previously unrelated phenotypes converge in a LacCer-centric manner. This LacCer synthase/LacCer-induced “oxidative stress” environment contributes to inflammation, atherosclerosis, skin conditions, hair greying, cardiovascular disease, and diabetes due to mitochondrial dysfunction. Thus, targeting LacCer synthase may well be the answer to remedy these pathologies.

Regulatory T Cell-Derived TRAIL Is Not Required for Peripheral Tolerance.

TRAIL (Tnfsf10/TRAIL/CD253/Apo2L) is an important immune molecule that mediates apoptosis. TRAIL can play key roles in regulating cell death in the tumor and autoimmune microenvironments. However, dissecting TRAIL function remains difficult because of the lack of optimal models. We have now generated a conditional knockout (Tnfsf10L/L) for cell type–specific analysis of TRAIL function on C57BL/6, BALB/c, and NOD backgrounds. Previous studies have suggested a role for TRAIL in regulatory T cell (Treg)–mediated suppression. We generated mice with a Treg-restricted Tnfsf10 deletion and surprisingly found no impact on tumor growth in C57BL/6 and BALB/c tumor models. Furthermore, we found no difference in the suppressive capacity of Tnfsf10-deficient Tregs and no change in function or proliferation of T cells in tumors. We also assessed the role of TRAIL on Tregs in two autoimmune mouse models: the NOD mouse model of autoimmune diabetes and the myelin oligodendrocyte glycoprotein (MOG) C57BL/6 model of experimental autoimmune encephalomyelitis. We found that deletion of Tnfsf10 on Tregs had no effect on disease progression in either model. We conclude that Tregs do not appear to be dependent on TRAIL exclusively as a mechanism of suppression in both the tumor and autoimmune microenvironments, although it remains possible that TRAIL may contribute in combination with other mechanisms and/or in different disease settings. Our Tnfsf10 conditional knockout mouse should prove to be a useful tool for the dissection of TRAIL function on different cell populations in multiple mouse models of human disease.

Conditioning Regimens in Long-Term Pre-Clinical Studies to Support Development of Ex Vivo Gene Therapy: Review of Nonproliferative and Proliferative Changes.

Hematopoietic stem cell gene therapy has become a successful therapeutic strategy for some inherited genetic disorders. Pre-clinical toxicity studies performed to support the human clinical trials using viral-mediated gene transfer and autologous hematopoietic stem and progenitor cell (HSPC) transplantation are complex and the use of mouse models of human diseases makes interpretation of the results challenging. In addition, they rely on the use of conditioning agents that must induce enough myeloablation to allow engraftment of transduced and transplanted HSPC. Busulfan and total body irradiation (TBI) are the most commonly used conditioning regimens in the mouse. Lenticular degeneration and atrophy of reproductive organs are expected histopathological changes. Proliferative and nonproliferative lesions can be observed with different incidence and distribution across strains and mouse models of diseases. The occurrence of these lesions can interfere with the interpretation of pre-clinical toxicity and tumorigenicity studies performed to support the human clinical studies. As such, it is important to be aware of the background incidence of lesions induced by different conditioning regimens. We review the histopathology results from seven long-term studies, five using TBI and two using busulfan.

Relevance of the Materno-Fetal Interface for the Induction of Antigen-Specific Immune Tolerance.

In humans, maternal IgGs are transferred to the fetus from the second trimester of pregnancy onwards. The transplacental delivery of maternal IgG is mediated by its binding to the neonatal Fc receptor (FcRn) after endocytosis by the syncytiotrophoblast. IgGs present in the maternal milk are also transferred to the newborn through the digestive epithelium upon binding to the FcRn. Importantly, the binding of IgGs to the FcRn is also responsible for the recycling of circulating IgGs that confers them with a long half-life. Maternally delivered IgG provides passive immunity to the newborn, for instance by conferring protective anti-flu or anti-pertussis toxin IgGs. It may, however, lead to the development of autoimmune manifestations when pathological autoantibodies from the mother cross the placenta and reach the circulation of the fetus. In recent years, strategies that exploit the transplacental delivery of antigen/IgG complexes or of Fc-fused proteins have been validated in mouse models of human diseases to impose antigen-specific tolerance, particularly in the case of Fc-fused factor VIII (FVIII) domains in hemophilia A mice or pre-pro-insulin (PPI) in the case of preclinical models of type 1 diabetes (T1D). The present review summarizes the mechanisms underlying the FcRn-mediated transcytosis of IgGs, the physiopathological relevance of this phenomenon, and the repercussion for drug delivery and shaping of the immune system during its ontogeny.

Effects of Colonization of Gnotobiotic Swiss Webster Mice with Helicobacter bilis.

Helicobacter bilis (Hb) causes hepatitis in some strains of inbred mice. The current study confirmed that Hb directly causes portal hepatitis in outbred gnotobiotic Swiss Webster (SW) mice, as we previously reported for conventional SW mice. Hbmonoassociated SW mice also developed mild enterocolitis, expanded gut-associated lymphoid tissue (GALT), and tertiary lymphoid tissue in the lower bowel. At 1 and 10 mo after infection, Hb-induced GALT hyperplasia exhibited well-organized, ectopic germinal centers with increased mononuclear cell apoptosis, MHC class II antigen presentation, and pronounced endothelial venule formation, consistent with features of tertiary lymphoid tissue. In the lower bowel, Hb induced mainly B220+ cells as well as CD4+ IL17+, CD4+ IFNγ+, and CD4+ FoxP3+ regulatory T cells and significantly increased IL10 mRNA expression. This gnotobiotic model confirmed that Hb causes portal hepatitis in outbred SW mice but stimulated GALT with an antiinflammatory bias. Because Hb had both anti- and proinflammatory effects on GALT, it should be considered a 'pathosymbiont provocateur' and merits further evaluation in mouse models of human disease.

Canine Models of Inherited Musculoskeletal and Neurodegenerative Diseases.

Mouse models of human disease remain the bread and butter of modern biology and therapeutic discovery. Nonetheless, more often than not mouse models do not reproduce the pathophysiology of the human conditions they are designed to mimic. Naturally occurring large animal models have predominantly been found in companion animals or livestock because of their emotional or economic value to modern society and, unlike mice, often recapitulate the human disease state. In particular, numerous models have been discovered in dogs and have a fundamental role in bridging proof of concept studies in mice to human clinical trials. The present article is a review that highlights current canine models of human diseases, including Alzheimer's disease, degenerative myelopathy, neuronal ceroid lipofuscinosis, globoid cell leukodystrophy, Duchenne muscular dystrophy, mucopolysaccharidosis, and fucosidosis. The goal of the review is to discuss canine and human neurodegenerative pathophysiologic similarities, introduce the animal models, and shed light on the ability of canine models to facilitate current and future treatment trials.

PATHBIO: an international training program for precision mouse phenotyping.

Design and production of genetically engineered mouse strains by individual research laboratories, research teams, large-scale consortia, and the biopharmaceutical industry have magnified the need for qualified personnel to identify, annotate, and validate (phenotype) these potentially new mouse models of human disease. The PATHBIO project has been recently established and funded by the European Union's ERASMUS+ Knowledge Alliance program to address the current shortfall in formally trained personnel. A series of teaching workshops will be given by experts on anatomy, histology, embryology, imaging, and comparative pathology to increase the availability of individuals with formal training to contribute to this important niche of Europe's biomedical research enterprise. These didactic and hands-on workshops are organized into three modules: (1) embryology, anatomy, histology, and the anatomical basis of imaging, (2) image-based phenotyping, and (3) pathology. The workshops are open to all levels of participants from recent graduates to Ph.D., M.D., and veterinary scientists. Participation is available on a competitive basis at no cost for attending. The first series of Workshop Modules was held in 2019 and these will continue for the next 2years.

Homeostatic and pathogenic roles of PI3Kδ in the human immune system.

Phosphoinositide 3-kinase delta (PI3Kδ) mediates signaling transduction downstream of diverse immune receptors, including the T cell receptor (TCR), the B cell receptor (BCR), costimulatory molecules and cytokine receptors. Our understanding of the role of PI3Kδ in the immune system comes primarily from mice, and especially from the consequences of pharmacological inhibition of PI3Kδ in mouse models of human disease as well as the consequences of genetic manipulation, resulting in hyperactivation or loss of PI3Kδ function. In case of humans, in vitro studies with PI3Kδ-specific inhibitors, the consequences of treatment of hematologic malignancies with the PI3Kδ-specific inhibitor idelalisib and primary immunodeficiency disorders due to germline variants hyper- or underactivating PI3Kδ provide most of our knowledge on the role of PI3Kδ in immunity and immune regulation. In this review, we summarize the physiological and pathophysiological roles of PI3Kδ in the human immune system, focusing on immunodeficiency due to defects in PI3Kδ signaling and especially on the recently reported cases with mutations resulting in loss of PI3Kδ activity.

Replacing murine insulin 1 with human insulin protects NOD mice from diabetes.

Type 1, or autoimmune, diabetes is caused by the T-cell mediated destruction of the insulin-producing pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes akin to human type 1 diabetes. For this reason, the NOD mouse has been the preeminent murine model for human type 1 diabetes research for several decades. However, humanized mouse models are highly sought after because they offer both the experimental tractability of a mouse model and the clinical relevance of human-based research. Autoimmune T-cell responses against insulin, and its precursor proinsulin, play central roles in the autoimmune responses against pancreatic beta cells in both humans and NOD mice. As a first step towards developing a murine model of the human autoimmune response against pancreatic beta cells we set out to replace the murine insulin 1 gene (Ins1) with the human insulin gene (Ins) using CRISPR/Cas9. Here we describe a NOD mouse strain that expresses human insulin in place of murine insulin 1, referred to as HuPI. HuPI mice express human insulin, and C-peptide, in their serum and pancreata and have normal glucose tolerance. Compared with wild type NOD mice, the incidence of diabetes is much lower in HuPI mice. Only 15-20% of HuPI mice developed diabetes after 300 days, compared to more than 60% of unmodified NOD mice. Immune-cell infiltration into the pancreatic islets of HuPI mice was not detectable at 100 days but was clearly evident by 300 days. This work highlights the feasibility of using CRISPR/Cas9 to create mouse models of human diseases that express proteins pivotal to the human disease. Furthermore, it reveals that even subtle changes in proinsulin protect NOD mice from diabetes.

The Tissue Factor Pathway Inhibitor Gamma Knockout Mouse Is a Better Model Than the Wild Type Mouse for Studying Tissue Factor-Related Human Diseases

BACKGROUND. Tissue factor (TF) is critical for hemostasis. However, TF also plays a pathogenic role in a multitude of diseases, including: thrombosis, coagulation-associated inflammation, multi-organ failure associated with infections, tumor growth, angiogenesis and metastasis. Tissue Factor Pathway Inhibitor (TFPI), the regulator of TF actions, produces FXa-dependent feedback inhibition of the FVIIa/TF catalytic complex, directly inhibits FXa, and suppresses cellular signaling by FVIIa/TF/FXa complexes. In man, TFPI is expressed as isoforms TFPIα and TFPIβ. The pathophysiologic impacts of the individual α and β isoforms have yet to be elucidated. Mouse models have been used extensively to provide insight into human physiology and disease, including abnormalities concerning hemostasis and TF-associated diseases. Like man, mice express TFPI α and β, but unlike man, mice express a third transcript encoding TFPIγ. In fact, we recently reported that the vast majority (98%) of TFPI in mouse plasma is derived from TFPIγ. We also reported that anti-TFPI antibodies enhance in vitro thrombin generation in hemophilia mouse plasma that contains only the TFPIγ isoform, which demonstrated that TFPIγ possesses anticoagulant activity. RESULTS. We report herein that, while complete TFPI gene deletion is 100% embryonic lethal, TFPIγ only mice [TFPIαβ(-/-)] are viable and reproduce. Offspring genotypes from parents with intact TFPIγ(+/+) exons and TFPIαβ(+/-) were 33 TFPIαβ(+/+), 57 TFPIαβ(+/-) and 34 TFPIαβ(-/-) at weaning. Also, TFPIγ only mice produced similar numbers of pups per litter as wild type mice. The viability of TFPIγ only mice proves endogenous mouse TFPIγ possesses substantial functional activity. TFPIα is considered to be a much more potent anticoagulant than TFPIγ. Our preliminary thrombin generation assay data suggests that the sum total anticoagulant activity of the endogenous low level (0.07 nM) of TFPIα is similar to the sum total anticoagulant activity of the endogenous high level (3.5 nM) of TFPIγ. The presence of active TFPIγ in mice, not found in man, confounds attempts to translate results from wild type mouse studies to the human condition. The extents to which TFPIγ influences the various wild type mouse models of human diseases are unknown. The presence of mouse TFPIγ also precludes our ability to properly distinguish the roles of TFPI α and β isoforms independently. Deletion of the TFPIγ exon eliminates this confounder. Fortunately, in TFPIαβ mice the levels, properties and location of TFPIα and TFPIβ in mice reflect those in man. Importantly, we can now report that our CRISPR-Cas9-generated TFPIγ KO strains - TFPIαβ mice, TFPIα only mice, and TFPIβ only mice are viable and reproduce. To illustrate the potential influence of TFPIγ in a mouse model, we used a tail vein-rebleeding assay to assess bleeding in TFPI isoform-specific mice in a FVIII KO background. Reported as mean number of clots per 15 minutes, results were: 4.8 for hemophilia controls; 25.4 for controls with FVIII replacement; 4.2 for TFPIα KOs; and 13.5 for TFPIβγ KOs. (The S.E.M. of each group was <1.0 ). We find that deletion of TFPIα (including in platelets!) does not affect bleeding (P>0.5 vs controls), whereas deletion of TFPIβ and γ improves hemostasis ( P<0.001 vs controls). In hemophilia mice, suppressing TFPIα was without effect. We don't expect this observation to translate to man. Rather, suppressing TFPIα in man is anticipated to enhance coagulation in hemophilia patients. (Note, multiple trials show that TFPI antibodies that inhibit all TFPI forms enhance coagulation). Moreover, in the absence of the confounding influence of TFPIγ, we anticipate suppression of TFPIα will improve coagulation and reduce bleeding in TFPIγ deleted hemophilia mice. Further studies in our TFPIγ deleted strains are necessary to determine if this prediction is correct and could influence future anti-TFPI approaches for man. CONCLUSIONS. 1.) Endogenous mouse TFPIγ possesses significant functional activity. 2.) TFPIγ confounds the extrapolation of results in wild type mouse models to human diseases. 3.) The TFPIγ KO mouse possesses a TFPIαβ profile that is similar to man. 4.) For essentially all studies of human TF-associated diseases in mouse models, it would be prudent to delete TFPIγ. Disclosures No relevant conflicts of interest to declare.

Genes essential for embryonic stem cells are associated with neurodevelopmental disorders.

Mouse embryonic stem cells (mESCs) are key components in generating mouse models for human diseases and performing basic research on pluripotency, yet the number of genes essential for mESCs is still unknown. We performed a genome-wide screen for essential genes in mESCs and compared it to screens in human cells. We found that essential genes are enriched for basic cellular functions, are highly expressed in mESCs, and tend to lack paralog genes. We discovered that genes that are essential specifically in mESCs play a role in pathways associated with their pluripotent state. We show that 29.5% of human genes intolerant to loss-of-function mutations are essential in mouse or human ESCs, and that the human phenotypes most significantly associated with genes essential for ESCs are neurodevelopmental. Our results provide insights into essential genes in the mouse, the pathways which govern pluripotency, and suggest that many genes associated with neurodevelopmental disorders are essential at very early embryonic stages.

An Altered Splicing Registry Explains the Differential ExSpeU1-Mediated Rescue of Splicing Mutations Causing Haemophilia A.

The exon recognition and removal of introns (splicing) from pre-mRNA is a crucial step in the gene expression flow. The process is very complex and therefore susceptible to derangements. Not surprisingly, a significant and still underestimated proportion of disease-causing mutations affects splicing, with those occurring at the 5’ splice site (5’ss) being the most severe ones. This led to the development of a correction approach based on variants of the spliceosomal U1snRNA, which has been proven on splicing mutations in several cellular and mouse models of human disease. Since the alternative splicing mechanisms are strictly related to the sequence context of the exon, we challenged the U1snRNA-mediated strategy in the singular model of the exon 5 of coagulation factor (F)VIII gene (F8) in which the authentic 5’ss is surrounded by various cryptic 5’ss. This scenario is further complicated in the presence of nucleotide changes associated with FVIII deficiency (Haemophilia A), which weaken the authentic 5’ss and create/strengthen cryptic 5’ss. We focused on the splicing mutations (c.602-32A > G, c.602-10T > G, c.602G > A, c.655G > A, c.667G > A, c.669A > G, c.669A > T, c.670G > T, c.670+1G > T, c.670+1G > A, c.670+2T > G, c.670+5G > A, and c.670+6T > C) found in patients with severe to mild Haemophilia A. Minigenes expression studies demonstrated that all mutations occurring within the 5’ss, both intronic or exonic, lead to aberrant transcripts arising from the usage of two cryptic intronic 5’ss at positions c.670+64 and c.670+176. For most of them, the observed proportion of correct transcripts is in accordance with the coagulation phenotype of patients. In co-transfection experiments, we identified a U1snRNA variant targeting an intronic region downstream of the defective exon (Exon Specific U1snRNA, U1sh7) capable to re-direct usage of the proper 5’ss (∼80%) for several mutations. However, deep investigation of rescued transcripts from +1 and +2 variants revealed only the usage of adjacent cryptic 5’ss, leading to frameshifted transcript forms. These data demonstrate that a single ExSpeU1 can efficiently rescue different mutations in the F8 exon 5, and provide the first evidence of the applicability of the U1snRNA-based approach to Haemophilia A.

Successful Derivation of an Induced Pluripotent Stem Cell Line from a Genetically Nonpermissive Enhanced Green Fluorescent Protein-Transgenic FVB/N Mouse Strain.

The embryonic stem cell line derivation from nonpermissive mouse strains is a challenging and highly inefficient process. The cellular reprogramming strategy provides an alternative route for generating pluripotent stem cell (PSC) lines from such strains. In this study, we successfully derived an enhanced green fluorescent protein (EGFP)-transgenic "N9" induced pluripotent stem cell (iPS cell, iPSC) line from the FVB/N strain-derived mouse embryonic fibroblasts (MEFs). The exposure of MEFs to human OCT4, SOX2, KLF4, and c-MYC (OSKM) transgenes via lentiviral transduction resulted in complete reprogramming. The N9 iPS cell line demonstrated all the criteria of a typical mouse PSC line, including normal colony morphology and karyotype (40,XY), high replication and propagation efficiencies, expression of the pluripotency-associated genes, spontaneous differentiation to three germ lineage-derived cell types, and robust potential of chimeric blastocyst formation. Taken together, using human OSKM genes for transduction, we report, for the first time, the successful derivation of an EGFP-expressing iPS cell line from a genetically nonpermissive transgenic FVB/N mouse. This cell line could provide opportunities for designing protocols for efficient derivation of PSC lines from other nonpermissive strains and developing mouse models of human diseases.

Mouse models of human disease: Identification of animal models for translational research

Mouse models of human disease: Identification of animal models for translational research