Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when homozygous, and proposed model for moderate glutathione deficiency when heterozygous

The biosynthesis of reduced glutathione (GSH) is carried out by the enzymes gamma-glutamylcysteine synthetase (GCL) and GSH synthetase. GCL is the rate-limiting step and represents a heterodimeric enzyme comprised of a catalytic subunit (GCLC) and a ("regulatory"), or modifier, subunit (GCLM). The nonhomologous Gclc and Gclm genes are located on mouse chromosomes 9 and 3, respectively. GCLC owns the catalytic activity, whereas GCLM enhances the enzyme activity by lowering the K(m) for glutamate and increasing the K(i) to GSH inhibition. Humans have been identified with one or two defective GCLC alleles and show low GSH levels. As an initial first step toward understanding the role of GSH in cellular redox homeostasis, we have targeted a disruption of the mouse Gclc gene. The Gclc(-/-) homozygous knockout animal dies before gestational day 13, whereas the Gclc(+/-) heterozygote is viable and fertile. The Gclc(+/-) mouse exhibits a gene-dose decrease in the GCLC protein and GCL activity, but only about a 20% diminution in GSH levels and a compensatory increase of approximately 30% in ascorbate-as compared with that in Gclc(+/+) wild-type littermates. These data show a reciprocal action between falling GSH concentrations and rising ascorbate levels. Therefore, the Gclc(+/-) mouse may be a useful genetic model for mild endogenous oxidative stress.     

Kidney disease models:tools to identify mechanisms and potential therapeutic targets

Acute kidney injury(AKI) and chronic kidney disease(CKD) are worldwide public health problems affecting millions of people and have rapidly increased in prevalence in recent years. Due to the multiple causes of renal failure, many animal models have been developed to advance our understanding of human nephropathy. Among these experimental models, rodents have been extensively used to enable mechanistic understanding of kidney disease induction and progression, as well as to identify potential targets for therapy. In this review, we discuss AKI models induced by surgical operation and drugs or toxins, as well as a variety of CKD models(mainly genetically modified mouse models).Results from recent and ongoing clinical trials and conceptual advances derived from animal models are also explored.     

Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer

Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF) is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mice does not adequately replicate spontaneous bacterial infections observed in the human CF lung. Hence, several laboratories are pursuing alternative animal models of CF in larger species such as the pig, sheep, rabbits, and ferrets. Our laboratory has focused on developing the ferret as a CF animal model. Over the past few years, we have investigated several experimental parameters required for gene targeting and nuclear transfer (NT) cloning in the ferret using somatic cells. In this review, we will discuss our progress and the hurdles to NT cloning and gene-targeting that accompany efforts to generate animal models of genetic diseases in species such as the ferret.     

Animal models of enterovirus 71 infection: applications and limitations

Human enterovirus 71 (EV71) has emerged as a neuroinvasive virus that is responsible for several outbreaks in the Asia-Pacific region over the past 15 years. Appropriate animal models are needed to understand EV71 neuropathogenesis better and to facilitate the development of effective vaccines and drugs. Non-human primate models have been used to characterize and evaluate the neurovirulence of EV71 after the early outbreaks in late 1990s. However, these models were not suitable for assessing the neurovirulence level of the virus and were associated with ethical and economic difficulties in terms of broad application. Several strategies have been applied to develop mouse models of EV71 infection, including strategies that employ virus adaption and immunodeficient hosts. Although these mouse models do not closely mimic human disease, they have been applied to determine the pathogenesis of and treatment and prevention of the disease. EV71 receptor-transgenic mouse models have recently been developed and have significantly advanced our understanding of the biological features of the virus and the host-parasite interactions. Overall, each of these models has advantages and disadvantages, and these models are differentially suited for studies of EV71 pathogenesis and/or the pre-clinical testing of antiviral drugs and vaccines. In this paper, we review the characteristics, applications and limitation of these EV71 animal models, including non-human primate and mouse models.     

Disease model: human aging

Very little is known about the molecular mechanisms of human aging. This, at least in part, derives from a paucity of appropriate animal models of aging. Until recently, the senescence-accelerated mouse was the only mammalian model of aging. However, novel mouse models that exhibit multiple aging phenotypes have been developed in the past few years by disruption of the klotho gene, the telomerase gene and the genes involved in premature aging syndromes. These mouse models are expected to be important tools for aging research.     

Animal models of CRH excess and CRH receptor deficiency display altered adaptations to stress

This review highlights new information gained from studies using recently developed animal models that harbor specific alterations in corticotropin-releasing hormone (CRH) pathways. We discuss features of a transgenic mouse model of chronic CRH overexpression and two mouse models that lack either CRH receptor type 1 (CRH-R1) or type 2 (CRH-R2). Together these models provide new insights into the role of CRH pathways in promoting stability through adaptive changes, a process known as allostasis.     

Mouse model of surgically-induced endometriosis by auto-transplantation of uterine tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat and later transferred to the mouse. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system, hormones and environmental factors affect endometriosis as well as the effects of endometriosis on fertility and pain.     

Genetically engineered mice and their use in aging research

Genetically engineered animal models have been and will continue to be invaluable for exploring the basic mechanisms involved in the aging process as well as in extending our understanding of diseases found to be more prevalent in the older human population. Continued development of such in vivo systems will allow scientists to further dissect the role genetic and environmental factors play in aging and in age-related disease states and to enhance our understanding of these processes. In this article we discuss techniques involved in the development of such models and review some examples of laboratory mouse strains that have been used to study either normal aging or select diseases associated with aging.     

Now and future of mouse mutagenesis for human disease models

One of the major objectives of the Human Genome Project is to understand the biological function of the gene and genome as well as to develop clinical applications for human diseases. For this purpose, the experimental validations and preclinical trails by using animal models are indispensable. The mouse (Mus musculus) is one of the best animal models because genetics is well established in the mouse and embryonic manipulation technologies are also well developed. Large-scale mouse mutagenesis projects have been conducted to de-velop various mouse models since 1997. Originally, the phenotype-driven mutagenesis with N-ethyl-N-nitrosourea (ENU) has been the major efforts internationally then knockout/conditional mouse projects and gene-driven mutagenesis have been following. At the beginning, simple monogenic traits in the experimental condition have been elucidated. Then, more complex traits with variety of environmental interactions and gene-to-gene interactions (epistasis) have been challenged with mutant mice. In addition, chromosomal substitution swains and collaborative cross strains are also available to elucidate the complex Waits in the mouse. Altogether, mouse models with mutagenesis and various laboratory strains will accelerate the studies of functional genomics in the mouse as well as in human.     

Variable regulation of glutamate cysteine ligase subunit proteins affects glutathione biosynthesis in response to oxidative stress

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.     

Human pathways in animal models: possibilities and limitations

Animal models are crucial for advancing our knowledge about the molecular pathways involved in human diseases. However, it remains unclear to what extent tissue expression of pathways in healthy individuals is conserved between species. In addition, organism-specific information on pathways in animal models is often lacking. Within these limitations, we explore the possibilities that arise from publicly available data for the animal models mouse, rat, and pig. We approximate the animal pathways activity by integrating the human counterparts of curated pathways with tissue expression data from the models. Specifically, we compare whether the animal orthologs of the human genes are expressed in the same tissue. This is complicated by the lower coverage and worse quality of data in rat and pig as compared to mouse. Despite that, from 203 human KEGG pathways and the seven tissues with best experimental coverage, we identify 95 distinct pathways, for which the tissue expression in one animal model agrees better with human than the others. Our systematic pathway-tissue comparison between human and three animal modes points to specific similarities with human and to distinct differences among the animal models, thereby suggesting the most suitable organism for modeling a human pathway or tissue.     

Choosing The Right Animal Model for Renal Cancer Research

The increase in the life expectancy of patients with renal cell carcinoma (RCC) in the last decade is due to changes that have occurred in the area of preclinical studies. Understanding cancer pathophysiology and the emergence of new therapeutic options, including immunotherapy, would not be possible without proper research. Before new approaches to disease treatment are developed and introduced into clinical practice they must be preceded by preclinical tests, in which animal studies play a significant role. This review describes the progress in animal model development in kidney cancer research starting from the oldest syngeneic or chemically-induced models, through genetically modified mice, finally to xenograft, especially patient-derived, avatar and humanized mouse models. As there are a number of subtypes of RCC, our aim is to help to choose the right animal model for a particular kidney cancer subtype. The data on genetic backgrounds, biochemical parameters, histology, different stages of carcinogenesis and metastasis in various animal models of RCC as well as their translational relevance are summarized. Moreover, we shed some light on imaging methods, which can help define tumor microstructure, assist in the analysis of its metabolic changes and track metastasis development.     

Applicability of bioengineered human skin: from preclinical skin humanized mouse models to clinical regenerative therapies

Ongoing progress in the field of regenerative medicine, in combination with the development of tissue-engineered skin products, has opened new possibilities for the treatment of certain diseases in which current treatments are aimed at alleviating symptoms but are not able to get a permanent cure. Our laboratory has developed a fibrin-based bioengineered human skin that has been successfully used for permanent regenerative therapies in different situations in the clinic. Moreover, we have been able to stably regenerate human skin by orthotopic grafting of this skin equivalent onto the back of immunodeficient mice. The so-called skin-humanized mouse model system has permitted us to model several monogenic skin diseases, when keratinocytes and fibroblasts harboring the genetic defect were used. In most cases different gene therapy approaches for ex vivo correction of cells have proved effective in reverting the phenotype using this model. More importantly, the feasibility of the system has allowed us to generate a skin humanized mouse model for psoriasis, a common chronic inflammatory disease where the immune component has a pivotal role in the pathogenesis. Establishing reliable humanized animal models for skin diseases is necessary to gain a deeper knowledge of the pathogenesis and to develop novel therapeutic strategies. In this sense, the skin humanized mouse model developed in our laboratory meets the needs of this field of research.     

Determination of glutathione in lymphocytes and possible association of redox state and proliferative capacity of lymphocytes.

The glutathione (GSH) content of mouse T- and B-cells was determined and compared with the GSH content of human peripheral blood lymphocytes and human erythrocytes. Owing to the difficulty of obtaining large numbers of purified lymphocytes, a technique was developed to measure picomolar quantities of GSH. By this technique, mouse T- and B-cells, as well as mouse peripheral-blood lymphocytes, were found to contain approx. 30% of the GSH found in human peripheral-blood lymphocytes. The concanavalin A response of human peripheral-blood lymphocytes and human spleen cells was insensitive to 2-mercaptoethanol as well as to culture in 17% O2, whereas mouse lymphocyte responses were altered by 2-mercaptoethanol and inhibited by 17% O2. The capacity of human peripheral-blood lymphocytes, human erythrocytes, mouse T-cells and mouse B-cells to regenerate GSH stores after chemical oxidation by diamide was tested, and it was found that mouse cells were less capable of regenerating GSH than human erythrocytes or human peripheral-blood lymphocytes. In addition, the latter lymphocytes were less sensitive to oxidation of GSH and to inhibition of proliferation by diamide.     

The LEGSKO Mouse: A Mouse Model of Age-Related Nuclear Cataract Based on Genetic Suppression of Lens Glutathione Synthesis

Age-related nuclear cataracts are associated with progressive post-synthetic modifications of crystallins from various physical chemical and metabolic insults, of which oxidative stress is a major factor. The latter is normally suppressed by high concentrations of glutathione (GSH), which however are very low in the nucleus of the old lens. Here we generated a mouse model of oxidant stress by knocking out glutathione synthesis in the mouse in the hope of recapitulating some of the changes observed in human age-related nuclear cataract (ARNC). A floxed Gclc mouse was generated and crossed with a transgenic mouse expressing Cre in the lens to generate the LEGSKO mouse in which de novo GSH synthesis was completely abolished in the lens. Lens GSH levels were reduced up to 60% in homozygous LEGSKO mice, and a decreasing GSH gradient was noticed from cortical to nuclear region at 4 months of age. Oxidation of crystallin methionine and sulfhydryls into sulfoxides was dramatically increased, but methylglyoxal hydroimidazolones levels that are GSH/glyoxalase dependent were surprisingly normal. Homozygous LEGSKO mice developed nuclear opacities starting at 4 months that progressed into severe nuclear cataract by 9 months. We conclude that the LEGSKO mouse lens mimics several features of human ARNC and is thus expected to be a useful model for the development of anti-cataract agents.     

Production of mitochondrial DNA transgenic mice using zygotes

Several animal models of human disease, which have been developed by random or targeted modifications of genomic DNA sequences, have furthered our understanding of pathogenesis and the development of therapeutics. However, these models have not facilitated studies on mitochondrial diseases, since modifications to mitochondrial DNA (mtDNA) sequences are not possible using current recombination techniques. Consequently, information on human mitochondrial diseases is relatively sparse, and issues related to mitochondrial pathogenesis and inheritance remain unresolved. Recently, we reported the development of a new technique to generate mice carrying mutant mtDNA from a mouse cell line. In this report, we describe our techniques in detail, with emphasis on the preparation of donor cytoplasts and the micromanipulative procedures for electrofusion of cytoplasts and recipient zygotes. These steps are critically important for the successful introduction of exogenous mtDNA into embryos, and thereby into animals, so that the mutant mtDNA is efficiently propagated in subsequent generations.     

Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities: RELEVANCE TO MOUSE MODELS OF HUMAN INFLAMMATORY DISEASES*

It is widely accepted that neutrophil serine proteases (NSPs) play a critical role in neutrophil-associated lung inflammatory and tissue-destructive diseases. To investigate NSP pathogenic role(s), various mouse experimental models have been developed that mimic acutely or chronically injured human lungs. We and others are using mouse exposure to cigarette smoke as a model for chronic obstructive pulmonary disease with or without exacerbation. However, the relative contribution of NSPs to lung disease processes as well as their underlying mechanisms remains still poorly understood. And the lack of purified mouse NSPs and their specific substrates have hampered advances in these studies. In this work, we compared mouse and human NSPs and generated three-dimensional models of murine NSPs based on three-dimensional structures of their human homologs. Analyses of these models provided compelling evidence that peptide substrate specificities of human and mouse NSPs are different despite their conserved cleft and close structural resemblance. These studies allowed us to synthesize for the first time novel sensitive fluorescence resonance energy transfer substrates for individual mouse NSPs. Our findings and the newly identified substrates should better our understanding about the role of NSPs in the pathogenesis of cigarette-associated chronic obstructive pulmonary disease as well as other neutrophils-associated inflammatory diseases.     

Th1 and Th2 cells are required for both eosinophil- and neutrophil-associated airway inflammatory responses in mice

Most current animal models focus on eosinophil-mediated asthma, despite compelling evidence that a neutrophil-mediated disease occurs in some asthma patients. Using intranasal challenge of mice sensitized either orally or nasally with whole peanut protein extract in the presence of cholera toxin, we developed mouse models of eosinophil- and neutrophil-mediated asthma, respectively. In this study, mice deficient in Th1 (IL-12 and IFN-gamma) or Th2 (IL-4 and IL-13) pathways were used to characterize the role played by Th1 and Th2 cytokines during the initial priming phase in the two models. Antigen-specific Ab responses were controlled primarily by Th2 cytokines in mice sensitized by the oral route, whereas Th1 cytokines appeared to play a predominant role in mice sensitized by the nasal route. Furthermore, the absence of key Th1 or Th2 cytokines during the initial phase of priming reduced lung reactivity in both mouse models of airway inflammation.     

Invited review: Identifying new mouse models of cardiovascular disease: a review of high-throughput screens of mutagenized and inbred strains.

The mouse is a proven model for studying human disease. Many strains exist that exhibit either natural or engineered genetic variation and thereby enable the elucidation of pathways involved in the development of cardiovascular disease. Although those mouse models have been fundamental to advancing our knowledge base, we are still at an early stage in understanding how genes contribute to complex disorders. There remains a need for new animal models that closely represent human disease. To expedite their development, we have established the Center for New Mouse Models of Heart, Lung, Blood, and Sleep Disorders at The Jackson Laboratory. We are using a phenotype-driven approach to identify mutations leading to atherosclerosis, hypertension, obesity, blood disorders, lung dysfunction, thrombosis, and disordered sleep. Our high-throughput, comprehensive phenotyping draws from two sources for new models: 1) the natural variation among over 40 inbred mouse strains and 2) chemically induced, whole-genome mutagenized mice. Here, we review our cardiovascular screens and present some hypertensive, obese, and cardiovascular models identified with this approach.