The Mutant Mouse Resource and Research Center (MMRRC): the NIH-supported National Public Repository and Distribution Archive of Mutant Mouse Models in the USA.

The Mutant Mouse Resource and Research Center (MMRRC) Program is the pre-eminent public national mutant mouse repository and distribution archive in the USA, serving as a national resource of mutant mice available to the global scientific community for biomedical research. Established more than two decades ago with grants from the National Institutes of Health (NIH), the MMRRC Program supports a Consortium of regionally distributed and dedicated vivaria, laboratories, and offices (Centers) and an Informatics Coordination and Service Center (ICSC) at three academic teaching and research universities and one non-profit genetic research institution. The MMRRC Program accepts the submission of unique, scientifically rigorous, and experimentally valuable genetically altered and other mouse models donated by academic and commercial scientists and organizations for deposition, maintenance, preservation, and dissemination to scientists upon request. The four Centers maintain an archive of nearly 60,000 mutant alleles as live mice, frozen germplasm, and/or embryonic stem (ES) cells. Since its inception, the Centers have fulfilled 13,184 orders for mutant mouse models from 9591 scientists at 6626 institutions around the globe. Centers also provide numerous services that facilitate using mutant mouse models obtained from the MMRRC, including genetic assays, microbiome analysis, analytical phenotyping and pathology, cryorecovery, mouse husbandry, infectious disease surveillance and diagnosis, and disease modeling. The ICSC coordinates activities between the Centers, manages the website (mmrrc.org) and online catalog, and conducts communication, outreach, and education to the research community. Centers preserve, secure, and protect mutant mouse lines in perpetuity, promote rigor and reproducibility in scientific experiments using mice, provide experiential training and consultation in the responsible use of mice in research, and pursue cutting edge technologies to advance biomedical studies using mice to improve human health. Researchers benefit from an expansive list of well-defined mouse models of disease that meet the highest standards of rigor and reproducibility, while donating investigators benefit by having their mouse lines preserved, protected, and distributed in compliance with NIH policies.

Regression of solid tumors by induction of MazF, a bacterial mRNA endoribonuclease.

MazF from Escherichia coli is an endoribonuclease that specifically cleaves mRNAs at ACA sequences. Its induction in mammalian cells has been shown to cause programmed cell death. Here we explored if a bacterial MazF-MazE toxin-antitoxin system can be used for gene therapy. For this, we first constructed a tetracycline-inducible MazF expression system in human embryonic kidney cells (T-Rex 293-mazF). Solid tumors were formed by injecting T-Rex 293-mazF cells into nude mice. All 8 mice injected with the cells developed solid tumors, which regressed upon induction of MazF. In 4 mice, tumors completely regressed, while in the remaining 4 mice, tumors reappeared after apparent significant regression, which was found to be due to the lack of presence of functional MazF. Notably, the MazF-mediated regression of the tumors was counteracted by the expression of its cognate antitoxin MazE. These results indicate that a bacterial MazF-MazE toxin-antitoxin system may have potential to be used as a therapeutic tool.

Preclinical studies for induced pluripotent stem cell-based therapeutics.

Induced pluripotent stem cells (iPSCs) and their differentiated derivatives can potentially be applied to cell-based therapy for human diseases. The properties of iPSCs are being studied intensively both to understand the basic biology of pluripotency and cellular differentiation and to solve problems associated with therapeutic applications. Examples of specific preclinical applications summarized briefly in this minireview include the use of iPSCs to treat diseases of the liver, nervous system, eye, and heart and metabolic conditions such as diabetes. Early stage studies illustrate the potential of iPSC-derived cells and have identified several challenges that must be addressed before moving to clinical trials. These include rigorous quality control and efficient production of required cell populations, improvement of cell survival and engraftment, and development of technologies to monitor transplanted cell behavior for extended periods of time. Problems related to immune rejection, genetic instability, and tumorigenicity must be solved. Testing the efficacy of iPSC-based therapies requires further improvement of animal models precisely recapitulating human disease conditions.

Large animal models for stem cell therapy.

The field of regenerative medicine is approaching translation to clinical practice, and significant safety concerns and knowledge gaps have become clear as clinical practitioners are considering the potential risks and benefits of cell-based therapy. It is necessary to understand the full spectrum of stem cell actions and preclinical evidence for safety and therapeutic efficacy. The role of animal models for gaining this information has increased substantially. There is an urgent need for novel animal models to expand the range of current studies, most of which have been conducted in rodents. Extant models are providing important information but have limitations for a variety of disease categories and can have different size and physiology relative to humans. These differences can preclude the ability to reproduce the results of animal-based preclinical studies in human trials. Larger animal species, such as rabbits, dogs, pigs, sheep, goats, and non-human primates, are better predictors of responses in humans than are rodents, but in each case it will be necessary to choose the best model for a specific application. There is a wide spectrum of potential stem cell-based products that can be used for regenerative medicine, including embryonic and induced pluripotent stem cells, somatic stem cells, and differentiated cellular progeny. The state of knowledge and availability of these cells from large animals vary among species. In most cases, significant effort is required for establishing and characterizing cell lines, comparing behavior to human analogs, and testing potential applications. Stem cell-based therapies present significant safety challenges, which cannot be addressed by traditional procedures and require the development of new protocols and test systems, for which the rigorous use of larger animal species more closely resembling human behavior will be required. In this article, we discuss the current status and challenges of and several major directions for the future development of large animal models to facilitate advances in stem cell-based regenerative medicine.

The glutathione peroxidase 1-protein tyrosine phosphatase 1B-protein phosphatase 2A axis. A key determinant of airway inflammation and alveolar destruction.

Protein phosphatase-2A (PP2A) is a primary serine-threonine phosphatase that modulates inflammatory responses in asthma and chronic obstructive pulmonary disease (COPD). Despite its importance, the mechanisms that regulate lung PP2A activity remain to be determined. The redox-sensitive enzyme protein tyrosine phosphatase-1B (PTP1B) activates PP2A by dephosphorylating the catalytic subunit of the protein at tyrosine 307. This study aimed to identify how the interaction between the intracellular antioxidant glutathione peroxidase-1 (GPx-1) and PTP1B affected lung PP2A activity and airway inflammation. Experiments using gene silencing techniques in mouse lung or human small airway epithelial cells determined that knocking down PTP1B expression blocked GPx-1's activation of PP2A and negated the anti-inflammatory effects of GPx-1 protein in the lung. Similarly, the expression of human GPx-1 in transgenic mice significantly increased PP2A and PTP1B activities and prevented chronic cigarette smoke-induced airway inflammation and alveolar destruction. GPx-1 knockout mice, however, exhibited an exaggerated emphysema phenotype, correlating with a nonresponsive PP2A pathway. Importantly, GPx-1-PTP1B-PP2A signaling becomes inactivated in advanced lung disease. Indeed, PTP1B protein was oxidized in the lungs of subjects with advanced emphysema, and cigarette smoke did not increase GPx-1 or PTP1B activity within epithelial cells isolated from subjects with COPD, unlike samples of healthy lung epithelial cells. In conclusion, these findings establish that the GPx-1-PTP1B-PP2A axis plays a critical role in countering the inflammatory and proteolytic responses that result in lung-tissue destruction in response to cigarette smoke exposure.

Strategies for improving animal models for regenerative medicine.

The field of regenerative medicine is moving toward translation to clinical practice. However, there are still knowledge gaps and safety concerns regarding stem cell-based therapies. Improving large animal models and methods for transplantation, engraftment, and imaging should help address these issues, facilitating eventual use of stem cells in the clinic.

Animal model of autism using GSTM1 knockout mice and early post-natal sodium valproate treatment.

Autism is a heterogeneous, behaviorally defined developmental disorder with unknown etiology but thought to be the result of environmental insult acting upon the developing brain of a genetically susceptible individual. Approximately 30% of individuals with autism have normal development up to the age of about 30 months after which they experience behavioral regression and lose previously acquired motor, cognitive and social skills. Early post-natal toxicant administration to mice has been used to model autistic regression. To test the hypothesis that genetically altered mice might be more sensitive to toxicant exposure early in life, mice with a deletion of glutathione-S-transferaseM1 (GSTM1; a gene associated with increased risk of autism that codes for an enzyme involved in the management of toxicant-induced oxidative stress) and wild-type controls were exposed to valproic acid (VPA; a toxicant known to cause autism-like behavioral deficits that, in part, are mediated through oxidative stress) on post-natal day 14. VPA treatment caused significant increases in apoptosis in granule cells of the hippocampus and cerebellum. There was a genotype by treatment by sex interaction with wild-type females exhibiting significantly fewer apoptotic cells in these regions compared to all other groups. VPA treatment also resulted in long-lasting deficits in social behaviors and significant alterations in brain chemistry. VPA-treated GSTM1 knockout animals performed significantly fewer crawl-under behaviors compared to saline-treated knockout animals as well as wild-type controls receiving either treatment. Collectively, these studies indicate that VPA-treatment causes cerebellar and hippocampal apoptosis and that having the wild-type GSTM1 genotype may confer protection against VPA-induced neuronal death in female mice.

Noncollagenous region of the streptococcal collagen-like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains.

Proper folding of the (Gly-Xaa-Yaa)(n) sequence of animal collagens requires adjacent N- or C-terminal noncollagenous trimerization domains which often contain coiled-coil or beta sheet structure. Collagen-like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen-like protein from Streptococcus pyogenes has an N-terminal globular domain, designated V(sp), adjacent to its triple-helix domain. The V(sp) domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V(sp) domain alone is shown to form trimers with a significant alpha-helix content and to have a thermal stability of T(m) = 45 degrees C. Examination of a new construct shows that the V(sp) domain facilitates efficient in vitro refolding only when it is located N-terminal to the triple-helix domain but not when C-terminal to the triple-helix domain. Fusion of the V(sp) domain N-terminal to a heterologous (Gly-Xaa-Yaa)(n) sequence from Clostridium perfringens led to correct folding and refolding of this triple-helix, which was unable to fold into a triple-helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly-Xaa-Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple-helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.

A Streptococcus pyogenes derived collagen-like protein as a non-cytotoxic and non-immunogenic cross-linkable biomaterial.

A range of bacteria have been shown to contain collagen-like sequences that form triple-helical structures. Some of these proteins have been shown to form triple-helical motifs that are stable around body temperature without the inclusion of hydroxyproline or other secondary modifications to the protein sequence. This makes these collagen-like proteins particularly suitable for recombinant production as only a single gene product and no additional enzyme needs to be expressed. In the present study, we have examined the cytotoxicity and immunogenicity of the collagen-like domain from Streptococcus pyogenes Scl2 protein. These data show that the purified, recombinant collagen-like protein is not cytotoxic to fibroblasts and does not elicit an immune response in SJL/J and Arc mice. The freeze dried protein can be stabilised by glutaraldehyde cross-linking giving a material that is stable at >37 degrees C and which supports cell attachment while not causing loss of viability. These data suggest that bacterial collagen-like proteins, which can be modified to include specific functional domains, could be a useful material for medical applications and as a scaffold for tissue engineering.

Expanding the family of collagen proteins: recombinant bacterial collagens of varying composition form triple-helices of similar stability.

The presence of the (Gly-Xaa-Yaa)(n) open reading frames in different bacteria predicts the existence of an expanded family of collagen-like proteins. To further explore the triple-helix motif and stabilization mechanisms in the absence of hydroxyproline (Hyp), predicted novel collagen-like proteins from Gram-positive and -negative bacteria were expressed in Escherichia coli and characterized. Soluble proteins capable of successful folding and in vitro refolding were observed for collagen proteins from Methylobacterium sp 4-46, Rhodopseudomonas palustris and Solibacter usitatus . In contrast, all protein constructs from Clostridium perfringens were found predominantly in inclusion bodies. However, attachment of a heterologous N-terminal or C-terminal noncollagenous folding domain induced the Clostridium perfringens collagen domain to fold and become soluble. The soluble constructs from different bacteria had typical collagen triple-helical features and showed surprisingly similar thermal stabilities despite diverse amino acid compositions. These collagen-like proteins provide a resource for the development of biomaterials with new properties.

Ischemia-reperfusion-inducible protein modulates cell sensitivity to anticancer drugs by regulating activity of efflux transporter.

Human ischemia-reperfusion-inducible protein (hIRIP) or hYrdC belongs to the SUA5/YrdC/YciO protein family and affects activity of a variety of cellular transporters. We observed that overexpression of wild-type or dominant-negative mutant of hIRIP protein affects the cellular sensitivity to anticancer drugs with different mechanisms of toxicity. Here we investigated in detail the effect of hIRIP on cell sensitivity to doxorubicin and show that hIRIP inhibits the drug efflux. Multidrug-resistant P-glycoprotein was identified as one of the target transporters. IRIP does not influence P-glycoprotein biosynthesis but affects its processing and promotes degradation. We also show that P-glycoprotein is associated with COP-alpha, one of the proteins of the COPI complex. This interaction is sensitive to the level of hIRIP expression. These findings suggest that hIRIP expression can regulate cargo assembly and function of efflux transporters, including P-glycoprotein, which mediates one of the most common mechanisms of the multidrug resistance.

Prolonged toxicokinetics and toxicodynamics of paraquat in mouse brain.

BACKGROUND: Paraquat (PQ) has been implicated as a risk factor for the Parkinson disease phenotype (PDP) in humans and mice using epidemiologic or experimental approaches. The toxicokinetics (TK) and toxicodynamics (TD) of PQ in the brain are not well understood. OBJECTIVES: The TK and TD of PQ in brain were measured after single or repeated doses. METHODS: Brain regions were analyzed for PQ levels, amount of lipid peroxidation, and functional activity of the 20S proteasome. RESULTS: Paraquat (10 mg/kg, ip) was found to be persistent in mouse ventral midbrain (VM) with an apparent half-life of approximately 28 days and was cumulative with a linear pattern between one and five doses. PQ was also absorbed orally with a concentration in brain rising linearly after single doses between 10 and 50 mg/kg. The level of tissue lipid peroxides (LPO) was differentially elevated in three regions, being highest in VM, lower in striatum (STR), and least in frontal cortex (FCtx), with the earliest significant elevation detected at 1 day. An elevated level of LPO was still present in VM after 28 days. Despite the cumulative tissue levels of PQ after one, three, and five doses, the level of LPO was not further increased. The activity of the 20S proteasome in the striatum was altered after a single dose and reduced after five doses. CONCLUSIONS: These data have implications for PQ as a risk factor in humans and in rodent models of the PDP.

DNA immobilization and SAW response in ZnO nanotips grown on LiNbO3 substrates.

DNA immobilization enhancement is demonstrated in a structure consisting of ZnO nanotips on 128 degrees Y-cut LiNbO3. The ZnO nanotips are grown by metalorganic chemical vapor deposition (MOCVD) on the top of a SiO2 layer that is deposited and patterned on the LiNbO3 SAW delay path. The effects of ZnO nanotips on the SAW response are investigated. X-ray diffraction and scanning electron microscopy are used to analyze the ZnO nanotips, which are of single crystalline quality, and they are uniformly aligned with their c-axis perpendicular to the substrate surface. The photoluminescence (PL) spectrum of the ZnO nanotips shows strong near bandedge transition with insignificant deep level emission, confirming their good optical property. DNA immobilization enhancement is experimentally validated by radioactive labeling tests and SAW response changes. The ZnO nanotips enhance the DNA immobilization by a factor of 200 compared to ZnO film with flat surface. DNA hybridization with complementary and noncomplementary second strand DNA oligonucleotides is used to study the selective binding of the structure. This device structure possesses the advantages of both traditional SAW sensors and ZnO nanostructures.

The expression profile of microRNAs in mouse embryos.

MicroRNAs (miRNAs), which are non-coding RNAs 18-25 nt in length, regulate a variety of biological processes, including vertebrate development. To identify new species of miRNA and to simultaneously obtain a comprehensive quantitative profile of small RNA expression in mouse embryos, we used the massively parallel signature sequencing technology that potentially identifies virtually all of the small RNAs in a sample. This approach allowed us to detect a total of 390 miRNAs, including 195 known miRNAs covering approximately 80% of previously registered mouse miRNAs as well as 195 new miRNAs, which are so far unknown in mouse. Some of these miRNAs showed temporal expression profiles during prenatal development (E9.5, E10.5 and E11.5). Several miRNAs were positioned in polycistron clusters, including one particular large transcription unit consisting of 16 known and 23 new miRNAs. Our results indicate existence of a significant number of new miRNAs expressed at specific stages of mammalian embryonic development and which were not detected by earlier methods.

Superoxide dismutase expression attenuates cigarette smoke- or elastase-generated emphysema in mice.

RATIONALE: Oxidants are believed to play a major role in the development of emphysema. OBJECTIVES: This study aimed to determine if the expression of human copper-zinc superoxide dismutase (CuZnSOD) within the lungs of mice protects against the development of emphysema. METHODS: Transgenic CuZnSOD and littermate mice were exposed to cigarette smoke (6 h/d, 5 d/wk, for 1 yr) and compared with nonexposed mice. A second group was treated with intratracheal elastase to induce emphysema. MEASUREMENTS: Lung inflammation was measured by cell counts and myeloperoxidase levels. Oxidative damage was assessed by immunofluorescence for 3-nitrotyrosine and 8-hydroxydeoxyguanosine and lipid peroxidation levels. The development of emphysema was determined by measuring the mean linear intercept (Lm). MAIN RESULTS: Smoke exposure caused a fourfold increase in neutrophilic inflammation and doubled lung myeloperoxidase activity. This inflammatory response did not occur in the smoke-exposed CuZnSOD mice. Similarly, CuZnSOD expression prevented the 58% increase in lung lipid peroxidation products that occurred after smoke exposure. Most important, CuZnSOD prevented the onset of emphysema in both the smoke-induced model (Lm, 68 exposed control vs. 58 exposed transgenic; p < 0.04) and elastase-generated model (Lm, 80 exposed control vs. 63 exposed transgenic; p < 0.03). These results demonstrate for the first time that antioxidants can prevent smoke-induced inflammation and can counteract the proteolytic cascade that leads to emphysema formation in two separate animal models of the disease. CONCLUSIONS: These findings indicate that strategies aimed at enhancing or supplementing lung antioxidants could be effective for the prevention and treatment of this disease.

Overexpression of glutathione peroxidase protects immature murine neurons from oxidative stress.

Neuronal enzyme systems involved in free radical detoxification are developmentally regulated such that intracellular glutathione peroxidase (GPx-1) activity is low in the newborn mouse brain. We hypothesized that neurons expressing a higher level of GPx-1 will be more resistant to hydrogen peroxide (H(2)O(2)) exposure. We show a dose-dependent protection against H(2)O(2) in primary neuronal cultures from fetuses overexpressing human GPx-1 compared to wild types of the same genetic background. Exogenous antioxidants completely protected neurons, even at extremely high H(2)O(2 )concentrations and regardless of the genotype. Specific depletion of glutathione with buthionine sulfoximine increased cell death in transgenic cultures exposed to 200 microM H(2)O(2), reducing protection afforded by increased GPx-1 activity. Increased GPx-1 expression in immature cortical neurons confers protection from oxidative stress, but availability of reducing equivalents determines susceptibility to oxidative cell death.

IRIP, a new ischemia/reperfusion-inducible protein that participates in the regulation of transporter activity.

We report the identification and characterization of a new ischemia/reperfusion-inducible protein (IRIP), which belongs to the SUA5/YrdC/YciO protein family. IRIP cDNA was isolated in a differential display analysis of an ischemia/reperfusion-treated kidney RNA sample. Mouse IRIP mRNA was expressed in all tissues tested, the highest level being in the testis, secretory, and endocrine organs. Besides ischemia/reperfusion, endotoxemia also activated the expression of IRIP in the liver, lung, and spleen. The transporter regulator RS1 was identified as an IRIP-interacting protein in yeast two-hybrid screening. The interaction between IRIP and RS1 was further confirmed in coimmunoprecipitation assays. A possible role of IRIP in regulating transporter activity was subsequently investigated. IRIP overexpression inhibited endogenous 1-methyl-4-phenylpyridinium (MPP+) uptake activity in HeLa cells. The activities of exogenous organic cation transporters (OCT2 and OCT3), organic anion transporter (OAT1), and monoamine transporters were also inhibited by IRIP. Conversely, inhibition of IRIP expression by small interfering RNA or antisense RNA increased MPP+ uptake. We measured transport kinetics of OCT2-mediated uptake and demonstrated that IRIP overexpression significantly decreased V(max) but did not affect K(m). On the basis of these results, we propose that IRIP regulates the activity of a variety of transporters under normal and pathological conditions.

[WITHDRAWN] Overexpression of superoxide dismutase or glutathione peroxidase protects against the paraquat + maneb-induced Parkinson disease phenotype.

Oxidative stress has been implicated in the pathogenesis of Parkinson disease based on its role in the cascade of biochemical changes that lead to dopaminergic neuronal death. This study analyzed the role of oxidative stress as a mechanism of the dopaminergic neurotoxicity produced by the combined paraquat and maneb model of the Parkinson disease phenotype. Transgenic mice overexpressing either Cu,Zn superoxide dismutase or intracellular glutathione peroxidase and non-transgenic mice were exposed to saline, paraquat, or the combination of paraquat + maneb twice a week for 9 weeks. Non-transgenic mice chronically exposed to paraquat + maneb exhibited significant reductions in locomotor activity, levels of striatal dopamine and metabolites, and dopaminergic neurons in the substantia nigra pars compacta. In contrast, no corresponding effects were observed in either Cu,Zn superoxide dismutase or glutathione peroxidase transgenic mice. Similarly, the increase in levels of lipid hydroperoxides in the midbrain and striatum of paraquat + maneb-treated non-transgenic mice was not detected in either Cu,Zn superoxide dismutase or glutathione peroxidase transgenic mice. To begin to determine critical pathways of paraquat + maneb neurotoxicity, the functions of cell death-inducing and protective mechanisms were analyzed. Even a single injection of paraquat + maneb in the non-transgenic treated group modulated several key pro- and anti-apoptotic proteins, including Bax, Bad, Bcl-xL, and upstream stress-induced cascade. Collectively, these findings support the assertion that protective mechanisms against paraquat + maneb-induced neurodegeneration could involve modulation of the level of reactive oxygen species and alterations of the functions of specific signaling cascades.