Genome Wide Conditional Mouse Knockout Resources.

The International Knockout Mouse Consortium (IKMC) developed high throughput gene trapping and gene targeting pipelines that produced mostly conditional mutations of more than 18,500 genes in C57BL/6N mouse embryonic stem (ES) cells which have been archived and are freely available to the research community as a frozen resource. From this unprecedented resource more than 6,000 mutant mouse strains have been produced by the IKMC and mostly the International Mouse Phenotyping Consortium (IMPC). In addition, a cre-driver resource was established including 250 inducible cre-driver mouse strains in a C57BL/6 background. Complementing the cre-driver resource, a collection of comprising 27 cre-driver rAAVs has also been produced. The resources can be easily accessed at the IKMC/IMPC web portal (www.mousephenotype.org). The IKMC/IMPC resource is a standardized reference library of mouse models with defined genetic backgrounds that enables the analysis of gene-disease associations in mice of different genetic makeup and should therefore have a major impact on biomedical research.

Right Ventricular Epicardial Fibrosis in Mice With Sternal Segment Dislocation.

We report coincident sternal segment dislocation and focally extensive right ventricular epicardial fibrosis observed during routine histopathology evaluation of C57BL/6N mice as part of a high throughput phenotyping screen conducted between 4 and 16 weeks of age. This retrospective case series study was conducted to determine whether cardiac fibrosis was a pathological consequence of sternal segment dislocation. We identified sternal segment dislocation in 51 of the total 1103 mice (4.6%) analyzed at 16 weeks of age. Males were more frequently affected. In all cases but 2, the dislocation occurred at the fourth intersternebral joint. In 42 of the 51 cases (82.4%), the dislocation was encased by regenerative cartilaginous callus that protruded internally into the thoracic cavity (intrathoracic callus) and/or externally to the outer aspect of the sternum (extrathoracic callus). Displacement of dislocated ends of the sternum into the thoracic cavity was present in 19 of 51 cases (36.5%). Coincident minimal or mild right ventricular epicardial and subepicardial fibrosis was observed in 22 of the 51 cases (43%) but was not observed in any of the mice in the absence of sternal segment dislocation. Our data suggest that right ventricular fibrosis was likely caused by direct injury of the right ventricle by the dislocated ends of the sternum and/or by intrathoracic callus that develops post dislocation. Potential pathogenesis for the sternal and cardiac lesions and their implication for the interpretation of phenotypes in mouse models of cardiopulmonary and skeletal disease are discussed.

Arrhythmia and sudden death associated with elevated cardiac chloride channel activity.

The identification and analysis of several cationic ion channels and their associated genes have greatly improved our understanding of the molecular and cellular mechanisms of cardiac arrhythmia. Our objective in this study was to examine the involvement of anionic ion channels in cardiac arrhythmia. We used a transgenic mouse model to overexpress the human cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cAMP-regulated chloride channel. We used RNase protection and in situ hybridization assays to determine the level of CFTR expression, and radiotelemetry and in vivo electrophysiological study in combination with pharmacological intervention to analyse the cardiac function. Cardiac CFTR overexpression leads to stress-related sudden death in this model. In vivo intracardiac electrophysiological studies performed in anaesthetized mice showed no significant differences in baseline conduction parameters including atrial-His bundle (AH) or His bundle-ventricular (HV) conduction intervals, atrioventricular (AV) Wenckebach or 2:1 AV block cycle length and AV nodal functional refractory period. However, following isoproterenol administration, there was marked slowing of conduction parameters, including high-grade AV block in transgenic mice, with non-sustained ventricular tachycardia easily inducible using programmed stimulation or burst pacing. Our sudden death mouse model can be a valuable tool for investigation of the role of chloride channels in arrhythmogenesis and, potentially, for future evaluation of novel anti-arrhythmic therapeutic strategies and pharmacological agents.

Restoration of fertility by orthotopic transplantation of frozen adult mouse ovaries.

BACKGROUND: Successful thawing and orthotopic transplantation of ovarian tissue has produced live offspring in mice, but until now has only been successful for very young ovary donors. METHODS: Whole and half ovaries from adult C3H/HeNCrlBR (C3H) and whole ovaries from B6129SF1/J were frozen-thawed and then grafted orthotopically into B6C3F1/CrlBR (B6C3F1) and B6129SF1/J recipients, respectively. In bilateral transplant groups (bilateral), recipients underwent a bilateral ovariectomy, followed by orthotopic grafting. In unilateral groups recipients either underwent bilateral ovariectomy followed by unilateral grafting (unilateral(ovx)) or had only one ovary removed and replaced with a graft (unilateral) along with complete transection of the remaining oviduct. RESULTS: Ovary size and number of follicles decreased dramatically in grafted compared with control groups, but the loss in the unilateral(ovx) group was significantly less than in the unilateral group. Similar numbers of litters and litter size were obtained in bilateral and unilateral grafts of fresh ovary. However, a much lower number of litters and litter size were derived from unilateral grafts than from unilateral(ovx) grafts of frozen ovary. CONCLUSIONS: Normal fertility can be restored by orthotopic grafting of fresh or frozen adult mouse ovaries and no significant difference between fresh and frozen ovaries was found. Grafting of half ovaries does not alter the overall fertility rate. Unilateral(ovx) grafting is an efficient procedure to produce live pups and removes the negative effect of recipient native ovaries on post-grafting fertility.

Mouse-based phenogenomics for modelling human disease.

The powerful and wide-ranging genetic tools available in the laboratory mouse make it the major experimental model for studying mammalian gene function in vivo and modelling human disease traits. Large-scale random mutagenesis approaches, either gene-driven or phenotype-driven, promise to identify new clinically relevant phenotypes and their associated genes. Development of appropriate tools for assessing clinical phenotypes in mice is a crucial component of these endeavours, as is the establishment of the infrastructure for archiving and distribution of the growing mutant resource to the community. Integrated, multidisciplinary programs will be needed to fully exploit the power of the mouse in molecular medicine.

Enhanced susceptibility to pulmonary infection with Burkholderia cepacia in Cftr(-/-) mice.

Progressive pulmonary infection is the dominant clinical feature of cystic fibrosis (CF), but the molecular basis for this susceptibility remains incompletely understood. To study this problem, we developed a model of chronic pneumonia by repeated instillation of a clinical isolate of Burkholderia cepacia (genomovar III, ET12 strain), an opportunistic gram-negative bacterium, from a case of CF into the lungs of Cftr (m1unc-/-) (Cftr(-/-)) and congenic Cftr(+/+) controls. Nine days after the last instillation, the CF transmembrane regulator knockout mice showed persistence of viable bacteria with chronic severe bronchopneumonia while wild-type mice remained healthy. The histopathological changes in the lungs of the susceptible Cftr(-/-) mice were characterized by infiltration of a mixed inflammatory-cell population into the peribronchiolar and perivascular spaces, Clara cell hyperplasia, mucus hypersecretion in airways, and exudation into alveolar airspaces by a mixed population of macrophages and neutrophils. An increased proportion of neutrophils was observed in bronchoalveolar lavage fluid from the Cftr(-/-) mice, which, despite an increased bacterial load, demonstrated minimal evidence of activation. Alveolar macrophages from Cftr(-/-) mice also demonstrated suboptimal activation. These observations suggest that the pulmonary host defenses are compromised in lungs from animals with CF, as manifested by increased susceptibility to bacterial infection and lung injury. This murine model of chronic pneumonia thus reflects, in part, the situation in human patients and may help elucidate the mechanisms leading to defective host defense in CF.

T cells of multiple sclerosis patients target a common environmental peptide that causes encephalitis in mice.

Multiple sclerosis (MS) is a chronic autoimmune disease triggered by unknown environmental factors in genetically susceptible hosts. MS risk was linked to high rates of cow milk protein (CMP) consumption, reminiscent of a similar association in autoimmune diabetes. A recent rodent study showed that immune responses to the CMP, butyrophilin, can lead to encephalitis through antigenic mimicry with myelin oligodendrocyte glycoprotein. In this study, we show abnormal T cell immunity to several other CMPs in MS patients comparable to that in diabetics. Limited epitope mapping with the milk protein BSA identified one specific epitope, BSA(193), which was targeted by most MS but not diabetes patients. BSA(193) was encephalitogenic in SJL/J mice subjected to a standard protocol for the induction of experimental autoimmune encephalitis. These data extend the possible, immunological basis for the association of MS risk, CMP, and CNS autoimmunity. To pinpoint the same peptide, BSA(193), in encephalitis-prone humans and rodents may imply a common endogenous ligand, targeted through antigenic mimicry.

A model of atypical absence seizures: EEG, pharmacology, and developmental characterization.

OBJECTIVE AND BACKGROUND: Atypical absence seizures differ markedly from typical absence seizures in EEG findings, ictal behavior, and neurodevelopmental outcome. The object of these experiments was to provide electrical, behavioral, pharmacologic, and developmental characterization of a putative animal model of atypical absence seizures. METHODS: Atypical absence seizures were induced in Long Evans hooded rats by treatment with a cholesterol biosynthesis inhibitor, AY-9944 (AY), during development. Prolonged video EEG recordings were made from chronically implanted depth electrodes in the waking and sleep states in adult and developing animals during and after AY treatment. Also, the response of AY-induced atypical absence seizures to drugs known to exacerbate and block typical absence seizures was ascertained. RESULTS: AY treatment resulted in spontaneous, bilaterally synchronous, slow spike-and-wave discharges (SWD), which were frequent, recurrent, prolonged, and lifelong. SWD began as early as postnatal day 21, occurred throughout all stages of sleep, and were associated with myoclonic jerks during sleep. The SWD were significantly prolonged by carbamazepine, gamma-hydroxybutyrate, and the gamma-aminobutyrate type B (GABA(B)) receptor (GABA(B)R) agonist baclofen. AY-induced seizures were abolished by diazepam, ethosuximide, and the GABA(B)R antagonist CGP 35348 but returned as the drugs were eliminated. Atypical features of absence seizures in this model are slow spike-wave, emanation of SWD from hippocampus, gradual onset and offset of ictal behavior, and the ability of the animals to move during the spike-and-wave bursts. CONCLUSION: The AY-treated rat represents a predictable, reproducible, and clinically relevant animal model of atypical absence seizures that may be used to investigate the pathogenesis and treatment of this malignant disorder.

Type I diabetes and multiple sclerosis patients target islet plus central nervous system autoantigens; nonimmunized nonobese diabetic mice can develop autoimmune encephalitis.

Type I diabetes and multiple sclerosis (MS) are distinct autoimmune diseases where T cells target either islet or CNS self-proteins. Unexpectedly, we found that autoreactive T cells in diabetic patients, relatives with high diabetes risk, nonobese diabetic (NOD) mice, and MS patients routinely target classical islet as well as CNS autoantigens. The pathogenic potential of CNS autoreactivity was testable in NOD mice. Pertussis holotoxin, without additional Ags or adjuvants, allowed development of an NOD mouse-specific, autoimmune encephalitis with variable primary-progressive, monophasic, and relapsing-remitting courses. T cells from diabetic donors transferred CNS disease to pertussis toxin-pretreated NOD.scid mice, with accumulation of CD3/IFN-gamma transcripts in the brain. Diabetes and MS appear more closely related than previously perceived. NOD mouse-specific, autoimmune encephalitis provides a new MS model to identify factors that determine alternative disease outcomes in hosts with similar autoreactive T cell repertoires.

Lung recruitment and lung volume maintenance: a strategy for improving oxygenation and preventing lung injury during both conventional mechanical ventilation and high-frequency oscillation.

OBJECTIVE: To determine whether using a small tidal volume (5 ml/kg) ventilation following sustained inflation with positive endexpiratory pressure (PEEP) set above the critical closing pressure (CCP) allows oxygenation equally well and induces as little lung damage as high-frequency oscillation following sustained inflation with a continuous distending pressure (CDP) slightly above the CCP of the lung. MATERIAL AND METHODS: Twelve surfactant-depleted adult New Zealand rabbits were ventilated for 4 h after being randomly assigned to one of two groups: group 1, conventional mechanical ventilation, tidal volume 5 ml/kg, sustained inflation followed by PEEP > CCP; group 2, high-frequency oscillation, sustained inflation followed by CDP > CCP. RESULTS: In both groups oxygenation improved substantially after sustained inflation (P < 0.05) and remained stable over 4 h of ventilation without any differences between the groups. Histologically, both groups showed only little airway injury to bronchioles, alveolar ducts, and alveolar airspace, with no difference between the two groups. Myeloperoxidase content in homogenized lung tissue, as a marker of leukocyte infiltration, was equivalent in the two groups. CONCLUSIONS: We conclude that a volume recruitment strategy during small tidal volume ventilation and maintaining lung volumes above lung closing is as protective as that of high-frequency oscillation at similar lung volumes in this model of lung injury

Distribution of pulmonary blood flow in the perfluorocarbon-filled lung.

OBJECTIVE: Partial liquid ventilation (PLV) improves gas exchange in animal studies of lung injury. Perfluorocarbons (PFCs) are heavy liquids and are therefore preferentially delivered to the most dependent areas of lung. We hypothesised that improved oxygenation during PLV might be the consequence of a redistribution of pulmonary blood flow away from poorly ventilated, dependent alveoli, leading to improved ventilation/perfusion (V/Q) matching. This study investigated whether partially filling the lung with PFC would result in a redistribution of pulmonary blood flow. DESIGN: Prospective experimental study. SETTING: Hospital research institute laboratory. PARTICIPANTS: Six anaesthetised pigs without lung injury. INTERVENTIONS: Animals were anaesthetised and ventilated (gas tidal volume 12 ml/kg, PEEP 5, FIO2 1.0, rate 16). Whilst the pigs were maintained in the supine position, regional pulmonary blood flow was measured during conventional gas ventilation and repeated during PLV. Flow to regions of lung was determined by injection of radioactive microspheres (Co(57), Sn(113), Sc(46)). Measurements were performed with ventilation held at end-expiratory pressure and, in two PLV animals only, repeated with ventilation held at peak inspiratory pressure. RESULTS: During conventional gas ventilation, blood flow followed a linear distribution with the highest flow to the most dependent lung. In the lung partially filled with PFC a diversion of blood flow away from the most dependent lung was seen (p = 0.007), resulting in a more uniform distribution of flow down the lung (p = 0.006). Linear regression analysis (r2 = 0.75) also confirmed a difference in distribution pattern. On applying an inspiratory hold to the liquid-containing lung, blood flow was redistributed back towards the dependent lung. CONCLUSIONS: Partially filling the lung with PFC results in a redistribution of pulmonary blood flow away from the dependent region of the lung. During PLV a different blood flow distribution may be seen between inspiration and expiration. The clinical significance of these findings has yet to be determined.

Characterization of graft-versus-host disease in SCID mice and prevention by physicochemical stressors.

BACKGROUND: Graft versus host disease (GVHD) prevents potentially curative allogeneic stem cell transplantation from being offered to cancer patients who lack a suitably matched donor. New methods to prevent GVHD are required to allow successful transplants across major histocompatibility complex barriers. METHODS: A model of GVHD in C.B-17 SCID mice was developed to allow the study of allo-activated donor T cells without confounding effects of host lymphocytes. The abilities of cyclosporin-A, anticytokine antibodies, and oxidative stress to prevent GVHD in this model was studied. RESULTS: T cells from major histocompatibility-mismatched donor mice caused severe GVHD in sublethally irradiated SCID hosts that could be ameliorated by coadministration of donor bone marrow but not by cyclosporine-A or anticytokine antibodies. In contrast, three-log more T cells could be injected without clinical consequences if they had been pretreated with a combination of heat, ultraviolet light, and oxygenation. The effect was not the trivial result of donor T cell destruction because T cell reconstitution, although delayed, recovered to normal levels within 2 weeks. Protection from GVHD required oxygenation and was associated with normalization of the CD4/CD8 donor T cell ratio, recovery of host hematopoiesis, and decreased inflammatory cytokine production. CONCLUSION: Pretreatment of donor T cells with a combination of physicochemical stressors effectively prevents GVHD caused by major major histocompatibility disparities and may facilitate the safe transplantation of patients without HLA-identical donors.

Glypican-3-deficient mice exhibit developmental overgrowth and some of the abnormalities typical of Simpson-Golabi-Behmel syndrome.

Glypicans are a family of heparan sulfate proteoglycans that are linked to the cell surface through a glycosyl-phosphatidylinositol anchor. One member of this family, glypican-3 (Gpc3), is mutated in patients with the Simpson-Golabi-Behmel syndrome (SGBS). These patients display pre- and postnatal overgrowth, and a varying range of dysmorphisms. The clinical features of SGBS are very similar to the more extensively studied Beckwith-Wiedemann syndrome (BWS). Since BWS has been associated with biallelic expression of insulin-like growth factor II (IGF-II), it has been proposed that GPC3 is a negative regulator of IGF-II. However, there is still no biochemical evidence indicating that GPC3 plays such a role.Here, we report that GPC3-deficient mice exhibit several of the clinical features observed in SGBS patients, including developmental overgrowth, perinatal death, cystic and dyplastic kidneys, and abnormal lung development. A proportion of the mutant mice also display mandibular hypoplasia and an imperforate vagina. In the particular case of the kidney, we demonstrate that there is an early and persistent developmental abnormality of the ureteric bud/collecting system due to increased proliferation of cells in this tissue element. The degree of developmental overgrowth of the GPC3-deficient mice is similar to that of mice deficient in IGF receptor type 2 (IGF2R), a well characterized negative regulator of IGF-II. Unlike the IGF2R-deficient mice, however, the levels of IGF-II in GPC3 knockouts are similar to those of the normal littermates.

Bone marrow failure in the Fanconi anemia group C mouse model after DNA damage.

Fanconi anemia (FA) is a pleiotropic inherited disease that causes bone marrow failure in children. However, the specific involvement of FA genes in hematopoiesis and their relation to bone marrow (BM) failure is still unclear. The increased sensitivity of FA cells to DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB), including the induction of chromosomal aberrations and delay in the G2 phase of the cell cycle, have suggested a role for the FA genes in DNA repair, cell cycle regulation, and apoptosis. We previously reported the cloning of the FA group C gene (FAC) and the generation of a Fac mouse model. Surprisingly, the Fac -/- mice did not show any of the hematologic defects found in FA patients. To better understand the relationship of FA gene functions to BM failure, we have analyzed the in vivo effect of an FA-specific DNA damaging agent in Fac -/- mice. The mice were found to be highly sensitive to DNA cross-linking agents; acute exposure to MMC produced a marked BM hypoplasia and degeneration of proliferative tissues and caused death within a few days of treatment. However, sequential, nonlethal doses of MMC caused a progressive decrease in all peripheral blood parameters of Fac -/- mice. This treatment targeted specifically the BM compartment, with no effect on other proliferative tissues. The progressive pancytopenia resulted from a reduction in the number of early and committed hematopoietic progenitors. These results indicate that the FA genes are involved in the physiologic response of hematopoietic progenitor cells to DNA damage.

Time-dependent induction of protective anti-influenza immune responses in human peripheral blood lymphocyte/SCID mice.

The human (hu) PBL/SCID mouse model has the potential to provide a powerful tool for the study of human immune function. However, at peak engraftment (4-8 wk postinjection), recovered human T cells are largely unresponsive to foreign Ag and have converted to an activated/memory-type phenotype. Here we show that this conversion is not a prerequisite for engraftment because at early stages (2 wk) a substantial fraction of human T cells detected in SCID peripheral blood retains the unactivated/naive phenotype of donor PBL. This early stage is also associated with a TCR repertoire in both the CD4 and CD8 subsets that is similar to that in the donor. Importantly, we show that strong HLA class I allele- and peptide-specific cytotoxic T lymphocyte as well as humoral responses can be generated in this model when human cells encounter Ag (infection with influenza A) at early, but not late, stages in engraftment. This early human response was also functional, as partial protection against influenza-induced pathology and death in SCIDs was observed. Taken together, these results demonstrate that the huPBL/SCID model can support the generation of potent and specific CTL and humoral responses provided that Ag is introduced early, presumably before the time-dependent generalized xenoactivation of engrafted human cells.

Lung disease in mice with cystic fibrosis.

The leading cause of mortality and morbidity in humans with cystic fibrosis is lung disease. Advances in our understanding of the pathogenesis of the lung disease of cystic fibrosis, as well as development of innovative therapeutic interventions, have been compromised by the lack of a natural animal model. The utility of the CFTR-knockout mouse in studying the pathogenesis of cystic fibrosis has been limited because of their failure, despite the presence of severe intestinal disease, to develop lung disease. Herein, we describe the phenotype of an inbred congenic strain of CFTR-knockout mouse that develops spontaneous and progressive lung disease of early onset. The major features of the lung disease include failure of effective mucociliary transport, postbronchiolar over inflation of alveoli and parenchymal interstitial thickening, with evidence of fibrosis and inflammatory cell recruitment. We speculate that the basis for development of lung disease in the congenic CFTR-knockout mice is their observed lack of a non-CFTR chloride channel normally found in CFTR-knockout mice of mixed genetic background.

Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia.

Fanconi anaemia (FA) is an autosomal recessive disease characterized by bone marrow failure, variable congenital malformations and predisposition to malignancies. Cells derived from FA patients show elevated levels of chromosomal breakage and an increased sensitivity to bifunctional alkylating agents such as mitomycin C (MMC) and diepoxybutane (DEB). Five complementation groups have been identified by somatic cell methods, and we have cloned the gene defective in group C (FAC)(7). To understand the in vivo role of this gene, we have disrupted murine Fac and generated mice homozygous for the targeted allele. The -/- mice did not exhibit developmental abnormalities nor haematologic defects up to 9 months of age. However, their spleen cells had dramatically increased numbers of chromosomal aberrations in response to MMC and DEB. Homozygous male and female mice also had compromised gametogenesis, leading to markedly impaired fertility, a characteristic of FA patients. Thus, inactivation of Fac replicates some of the features of the human disease.