Vaccination with recombinant adenoviruses expressing Ebola virus glycoprotein elicits protection in the interferon alpha/beta receptor knock-out mouse.

PubMed

O'Brien, Lyn M; Stokes, Margaret G; Lonsdale, Stephen G; Maslowski, David R; Smither, Sophie J; Lever, Mark S; Laws, Thomas R; Perkins, Stuart D

2014-03-01

The resistance of adult immunocompetent mice to infection with ebolaviruses has led to the development of alternative small animal models that utilise immunodeficient mice, for example the interferon α/β receptor knock-out mouse (IFNR(-/-)). IFNR(-/-) mice have been shown to be susceptible to infection with ebolaviruses by multiple routes but it is not known if this murine model is suitable for testing therapeutics that rely on the generation of an immune response for efficacy. We have tested recombinant adenovirus vectors for their ability to protect IFNR(-/-) mice from challenge with Ebola virus and have analysed the humoral response generated after immunisation. The recombinant vaccines elicited good levels of protection in the knock-out mouse and the antibody response in IFNR(-/-) mice was similar to that observed in vaccinated wild-type mice. These results indicate that the IFNR(-/-) mouse is a relevant small animal model for studying ebolavirus-specific therapeutics. Copyright © 2014. Published by Elsevier Inc.

Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing.

PubMed

Liu, Zhen; Lu, Zongyang; Yang, Guang; Huang, Shisheng; Li, Guanglei; Feng, Songjie; Liu, Yajing; Li, Jianan; Yu, Wenxia; Zhang, Yu; Chen, Jia; Sun, Qiang; Huang, Xingxu

2018-06-14

A recently developed adenine base editor (ABE) efficiently converts A to G and is potentially useful for clinical applications. However, its precision and efficiency in vivo remains to be addressed. Here we achieve A-to-G conversion in vivo at frequencies up to 100% by microinjection of ABE mRNA together with sgRNAs. We then generate mouse models harboring clinically relevant mutations at Ar and Hoxd13, which recapitulates respective clinical defects. Furthermore, we achieve both C-to-T and A-to-G base editing by using a combination of ABE and SaBE3, thus creating mouse model harboring multiple mutations. We also demonstrate the specificity of ABE by deep sequencing and whole-genome sequencing (WGS). Taken together, ABE is highly efficient and precise in vivo, making it feasible to model and potentially cure relevant genetic diseases.

The untapped potential of genetically-engineered mouse models in chemoprevention research: Opportunities and challenges

PubMed Central

Abate-Shen, Cory; Brown, Powel H.; Colburn, Nancy H.; Gerner, Eugene W.; Green, Jeffery E.; Lipkin, Martin; Nelson, William G.; Threadgill, David

2009-01-01

Summary The past decade has witnessed the unveiling of a powerful new generation of genetically-engineered mouse (GEM) models of human cancer, which are proving to be highly effective for elucidating cancer mechanisms and interrogating novel experimental therapeutics. This new generation of GEM models are well-suited for chemoprevention research, particularly for investigating progressive stages of carcinogenesis, identifying biomarkers for early detection and intervention, and pre-clinical assessment of novel agents or combinations of agents. Here we discuss opportunities and challenges for the application of GEM models in prevention research, as well as strategies to maximize their relevance for human cancer. PMID:19138951

Generation of a mouse model for studying the role of upregulated RTEL1 activity in tumorigenesis.

PubMed

Wu, Xiaoli; Sandhu, Sumit; Nabi, Zinnatun; Ding, Hao

2012-10-01

Regulator of telomere length 1 (RTEL1) is a DNA helicase protein that has been demonstrated to be required for the maintenance of telomere length and genomic stability. It has also been found to be essential for DNA homologous recombination during DNA repairing. Human RTEL1 genomic locus (20q13.3) is frequently amplified in multiple types of human cancers, including hepatocellular carcinoma and gastrointestinal tract tumors, indicating that upregulated RTEL1 activity could be important for tumorigenesis. In this study, we have developed a conditional transgenic mouse model that overexpress mouse Rtel1 in a Cre-excision manner. By crossing with a ubiquitous Cre mouse line, we further demonstrated that these established Rtel1 conditional transgenic mice allow to efficiently and highly express a functional Rtel1 that is able to rescue the embryonic defects of Rtel1 null mouse allele. Furthermore, we demonstrated that more than 70% transgenic mice that widely overexpress Rtel1 developed liver tumors that recapitulate many malignant features of human hepatocellular carcinoma (HCC). Our work not only generated a valuable mouse model for determining the role of RTEL1 in the development of cancers, but also provided the first genetic evidence to support that amplification of RTEL1, as observed in several types of human cancers, is tumorigenic.

Behavioural phenotyping assays for mouse models of autism

PubMed Central

Silverman, Jill L.; Yang, Mu; Lord, Catherine; Crawley, Jacqueline N.

2011-01-01

Autism is a heterogeneous neurodevelopmental disorder of unknown aetiology that affects 1 in 100–150 individuals. Diagnosis is based on three categories of behavioural criteria: abnormal social interactions, communication deficits and repetitive behaviours. Strong evidence for a genetic basis has prompted the development of mouse models with targeted mutations in candidate genes for autism. As the diagnostic criteria for autism are behavioural, phenotyping these mouse models requires behavioural assays with high relevance to each category of the diagnostic symptoms. Behavioural neuroscientists are generating a comprehensive set of assays for social interaction, communication and repetitive behaviours to test hypotheses about the causes of austism. Robust phenotypes in mouse models hold great promise as translational tools for discovering effective treatments for components of autism spectrum disorders. PMID:20559336

Generation and testing anti-influenza human monoclonal antibodies in a new humanized mouse model (DRAGA: HLA-A2. HLA-DR4. Rag1 KO. IL-2Rγc KO. NOD).

PubMed

Mendoza, Mirian; Ballesteros, Angela; Qiu, Qi; Pow Sang, Luis; Shashikumar, Soumya; Casares, Sofia; Brumeanu, Teodor-D

2018-02-01

Pandemic outbreaks of influenza type A viruses have resulted in numerous fatalities around the globe. Since the conventional influenza vaccines (CIV) provide less than 20% protection for individuals with weak immune system, it has been considered that broadly cross-neutralizing antibodies may provide a better protection. Herein, we showed that a recently generated humanized mouse (DRAGA mouse; HLA-A2. HLA-DR4. Rag1KO. IL-2Rgc KO. NOD) that lacks the murine immune system and expresses a functional human immune system can be used to generate cross-reactive, human anti-influenza monoclonal antibodies (hu-mAb). DRAGA mouse was also found to be suitable for influenza virus infection, as it can clear a sub-lethal infection and sustain a lethal infection with PR8/A/34 influenza virus. The hu-mAbs were designed for targeting a human B-cell epitope ( 180 WGIHHPPNSKEQ QNLY 195 ) of hemagglutinin (HA) envelope protein of PR8/A/34 (H1N1) virus with high homology among seven influenza type A viruses. A single administration of HA 180-195 specific hu-mAb in PR8-infected DRAGA mice significantly delayed the lethality by reducing the lung damage. The results demonstrated that DRAGA mouse is a suitable tool to (i) generate heterotype cross-reactive, anti-influenza human monoclonal antibodies, (ii) serve as a humanized mouse model for influenza infection, and (iii) assess the efficacy of anti-influenza antibody-based therapeutics for human use.

Mouse Models for Down Syndrome-Associated Developmental Cognitive Disabilities

PubMed Central

Liu, Chunhong; Belichenko, Pavel V.; Zhang, Li; Fu, Dawei; Kleschevnikov, Alexander M.; Baldini, Antonio; Antonarakis, Stylianos E.; Mobley, William C.; Yu, Y. Eugene

2011-01-01

Down syndrome (DS) is mainly caused by the presence of an extra copy of human chromosome 21 (Hsa21) and is a leading genetic cause for developmental cognitive disabilities in humans. The mouse is a premier model organism for DS because the regions on Hsa21 are syntenically conserved with three regions in the mouse genome, which are located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. With the advance of chromosomal manipulation technologies, new mouse mutants have been generated to mimic DS at both the genotypic and phenotypic levels. Further mouse-based molecular genetic studies in the future may lead to the unraveling of the mechanisms underlying DS-associated developmental cognitive disabilities, which would lay the groundwork for developing effective treatments for this phenotypic manifestation. In this review, we will discuss recent progress and future challenges in modeling DS-associated developmental cognitive disability in mice with an emphasis on hippocampus-related phenotypes. PMID:21865664

Harnessing the Power of Light to See and Treat Breast Cancer

DTIC Science & Technology

2011-10-01

generate sarcomas include LSL- KrasG12D/+;Trp53Flox/Flox, BrafCa/+;Trp53 Flox/Flox and BrafCa/Ca;Trp53Flox/Flox.7,8 Soft tissue sarcomas were generated...temporally restricted mouse model of soft tissue sarcoma , Nat Med, 2007. 13(8): p. 992-7. 8. Dankort, D., et al., A new mouse model to explore the...resolution anatomical images of heterogeneous tissue. To do so we are employing the use of two ex vivo test beds: 1) murine sarcoma margins and 2

Generation of a pancreatic cancer model using a Pdx1-Flp recombinase knock-in allele

PubMed Central

Wu, Jinghai; Liu, Xin; Nayak, Sunayana G.; Pitarresi, Jason R.; Cuitiño, Maria C.; Yu, Lianbo; Hildreth, Blake E.; Thies, Katie A.; Schilling, Daniel J.; Fernandez, Soledad A.; Leone, Gustavo

2017-01-01

The contribution of the tumor microenvironment to the development of pancreatic adenocarcinoma (PDAC) is unclear. The LSL-KrasG12D/+;LSL-p53R172H/+;Pdx-1-Cre (KPC) tumor model, which is widely utilized to faithfully recapitulate human pancreatic cancer, depends on Cre-mediated recombination in the epithelial lineage to drive tumorigenesis. Therefore, specific Cre-loxP recombination in stromal cells cannot be applied in this model, limiting the in vivo investigation of stromal genetics in tumor initiation and progression. To address this issue, we generated a new Pdx1FlpO knock-in mouse line, which represents the first mouse model to physiologically express FlpO recombinase in pancreatic epithelial cells. This mouse specifically recombines Frt loci in pancreatic epithelial cells, including acinar, ductal, and islet cells. When combined with the Frt-STOP-Frt KrasG12D and p53Frt mouse lines, simultaneous Pdx1FlpO activation of mutant Kras and deletion of p53 results in the spectrum of pathologic changes seen in PDAC, including PanIN lesions and ductal carcinoma. Combination of this KPF mouse model with any stroma-specific Cre can be used to conditionally modify target genes of interest. This will provide an excellent in vivo tool to study the roles of genes in different cell types and multiple cell compartments within the pancreatic tumor microenvironment. PMID:28934293

Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors.

PubMed

Miura, Hiromi; Quadros, Rolen M; Gurumurthy, Channabasavaiah B; Ohtsuka, Masato

2018-01-01

CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1-10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts-CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30-60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.

Generation of an immortalized mouse embryonic palatal mesenchyme cell line

PubMed Central

Soriano, Philippe

2017-01-01

Palatogenesis is a complex morphogenetic process, disruptions in which result in highly prevalent birth defects in humans. In recent decades, the use of model systems such as genetically-modified mice, mouse palatal organ cultures and primary mouse embryonic palatal mesenchyme (MEPM) cultures has provided significant insight into the molecular and cellular defects underlying cleft palate. However, drawbacks in each of these systems have prevented high-throughput, large-scale studies of palatogenesis in vitro. Here, we report the generation of an immortalized MEPM cell line that maintains the morphology, migration ability, transcript expression and responsiveness to exogenous growth factors of primary MEPM cells, with increased proliferative potential over primary cultures. The immortalization method described in this study will facilitate the generation of palatal mesenchyme cells with an unlimited capacity for expansion from a single genetically-modified mouse embryo and enable mechanistic studies of palatogenesis that have not been possible using primary culture. PMID:28582446

Massively Parallel Sequencing Reveals the Complex Structure of an Irradiated Human Chromosome on a Mouse Background in the Tc1 Model of Down Syndrome

PubMed Central

Clayton, Stephen; Prigmore, Elena; Langley, Elizabeth; Yang, Fengtang; Maguire, Sean; Fu, Beiyuan; Rajan, Diana; Sheppard, Olivia; Scott, Carol; Hauser, Heidi; Stephens, Philip J.; Stebbings, Lucy A.; Ng, Bee Ling; Fitzgerald, Tomas; Quail, Michael A.; Banerjee, Ruby; Rothkamm, Kai; Tybulewicz, Victor L. J.; Fisher, Elizabeth M. C.; Carter, Nigel P.

2013-01-01

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype – phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439. PMID:23596509

Human androgen deficiency: insights gained from androgen receptor knockout mouse models

PubMed Central

Rana, Kesha; Davey, Rachel A; Zajac, Jeffrey D

2014-01-01

The mechanism of androgen action is complex. Recently, significant advances have been made into our understanding of how androgens act via the androgen receptor (AR) through the use of genetically modified mouse models. A number of global and tissue-specific AR knockout (ARKO) models have been generated using the Cre-loxP system which allows tissue- and/or cell-specific deletion. These ARKO models have examined a number of sites of androgen action including the cardiovascular system, the immune and hemopoetic system, bone, muscle, adipose tissue, the prostate and the brain. This review focuses on the insights that have been gained into human androgen deficiency through the use of ARKO mouse models at each of these sites of action, and highlights the strengths and limitations of these Cre-loxP mouse models that should be considered to ensure accurate interpretation of the phenotype. PMID:24480924

Ghrelin and eating behavior: evidence and insights from genetically-modified mouse models

PubMed Central

Uchida, Aki; Zigman, Jeffrey M.; Perelló, Mario

2013-01-01

Ghrelin is an octanoylated peptide hormone, produced by endocrine cells of the stomach, which acts in the brain to increase food intake and body weight. Our understanding of the mechanisms underlying ghrelin's effects on eating behaviors has been greatly improved by the generation and study of several genetically manipulated mouse models. These models include mice overexpressing ghrelin and also mice with genetic deletion of ghrelin, the ghrelin receptor [the growth hormone secretagogue receptor (GHSR)] or the enzyme that post-translationally modifies ghrelin [ghrelin O-acyltransferase (GOAT)]. In addition, a GHSR-null mouse model in which GHSR transcription is globally blocked but can be cell-specifically reactivated in a Cre recombinase-mediated fashion has been generated. Here, we summarize findings obtained with these genetically manipulated mice, with the aim to highlight the significance of the ghrelin system in the regulation of both homeostatic and hedonic eating, including that occurring in the setting of chronic psychosocial stress. PMID:23882175

Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research.

PubMed

Tetteh, Paul W; Kretzschmar, Kai; Begthel, Harry; van den Born, Maaike; Korving, Jeroen; Morsink, Folkert; Farin, Henner; van Es, Johan H; Offerhaus, G Johan A; Clevers, Hans

2016-10-18

Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1 CreER knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1 CreER KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4 Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1 CreER KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.

Angiogenic Signaling in Living Breast Tumor Models

DTIC Science & Technology

2010-06-01

harmonic generation imaging of the diseased state osteogenesis imperfecta : experiment and simulation,” Biophys. J. 94(11), 4504–4514 (2008). 3. O...biopsies, mouse models of breast cancer, and dermis from mouse models of Osteogenesis Imperfecta (OIM) [1–5,7]. The F/B ratio revealed the length scale of...interest in discriminating skin with Osteogenesis Imperfecta [2] from normal dermis [2] and SHG F/B ratio measurements have been used to help determine

Mouse phenotyping.

PubMed

Fuchs, Helmut; Gailus-Durner, Valérie; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Calzada-Wack, Julia; Da Silva-Buttkus, Patricia; Neff, Frauke; Götz, Alexander; Hans, Wolfgang; Hölter, Sabine M; Horsch, Marion; Kastenmüller, Gabi; Kemter, Elisabeth; Lengger, Christoph; Maier, Holger; Matloka, Mikolaj; Möller, Gabriele; Naton, Beatrix; Prehn, Cornelia; Puk, Oliver; Rácz, Ildikó; Rathkolb, Birgit; Römisch-Margl, Werner; Rozman, Jan; Wang-Sattler, Rui; Schrewe, Anja; Stöger, Claudia; Tost, Monica; Adamski, Jerzy; Aigner, Bernhard; Beckers, Johannes; Behrendt, Heidrun; Busch, Dirk H; Esposito, Irene; Graw, Jochen; Illig, Thomas; Ivandic, Boris; Klingenspor, Martin; Klopstock, Thomas; Kremmer, Elisabeth; Mempel, Martin; Neschen, Susanne; Ollert, Markus; Schulz, Holger; Suhre, Karsten; Wolf, Eckhard; Wurst, Wolfgang; Zimmer, Andreas; Hrabě de Angelis, Martin

2011-02-01

Model organisms like the mouse are important tools to learn more about gene function in man. Within the last 20 years many mutant mouse lines have been generated by different methods such as ENU mutagenesis, constitutive and conditional knock-out approaches, knock-down, introduction of human genes, and knock-in techniques, thus creating models which mimic human conditions. Due to pleiotropic effects, one gene may have different functions in different organ systems or time points during development. Therefore mutant mouse lines have to be phenotyped comprehensively in a highly standardized manner to enable the detection of phenotypes which might otherwise remain hidden. The German Mouse Clinic (GMC) has been established at the Helmholtz Zentrum München as a phenotyping platform with open access to the scientific community (www.mousclinic.de; [1]). The GMC is a member of the EUMODIC consortium which created the European standard workflow EMPReSSslim for the systemic phenotyping of mouse models (http://www.eumodic.org/[2]). Copyright © 2010 Elsevier Inc. All rights reserved.

Genetic characterization and improved genotyping of the dysferlin-deficient mouse strain Dysf (tm1Kcam).

PubMed

Wiktorowicz, Tatiana; Kinter, Jochen; Kobuke, Kazuhiro; Campbell, Kevin P; Sinnreich, Michael

2015-01-01

Mouse models of dysferlinopathies are valuable tools with which to investigate the pathomechanisms underlying these diseases and to test novel therapeutic strategies. One such mouse model is the Dysf (tm1Kcam) strain, which was generated using a targeting vector to replace a 12-kb region of the dysferlin gene and which features a progressive muscular dystrophy. A prerequisite for successful animal studies using genetic mouse models is an accurate genotyping protocol. Unfortunately, the lack of robustness of currently available genotyping protocols for the Dysf (tm1Kcam) mouse has prevented efficient colony management. Initial attempts to improve the genotyping protocol based on the published genomic structure failed. These difficulties led us to analyze the targeted locus of the dysferlin gene of the Dysf (tm1Kcam) mouse in greater detail. In this study we resequenced and analyzed the targeted locus of the Dysf (tm1Kcam) mouse and developed a novel PCR protocol for genotyping. We found that instead of a deletion, the dysferlin locus in the Dysf (tm1Kcam) mouse carries a targeted insertion. This genetic characterization enabled us to establish a reliable method for genotyping of the Dysf (tm1Kcam) mouse, and thus has made efficient colony management possible. Our work will make the Dysf (tm1Kcam) mouse model more attractive for animal studies of dysferlinopathies.

Generation of human acute lymphoblastic leukemia xenografts for use in oncology drug discovery

PubMed Central

Holmfeldt, Linda

2015-01-01

The establishment of reproducible mouse models of acute lymphoblastic leukemia (ALL) is necessary to provide in vivo therapeutic models that recapitulate human ALL, and for amplification of limiting amounts of primary tumor material. A frequently used model is the primary xenograft model that utilizes immunocompromised mice and involves injection of primary patient tumor specimens into mice, and subsequent serial passaging of the tumors by retransplants of cells harvested from the mouse bone marrow and spleen. The tumors generated can then be used for genomic profiling, ex vivo compound testing, mechanistic studies and retransplantation. This unit describes detailed procedures for the establishment and maintenance of primary ALL xenograft panels for potential use in basic research or translational studies. PMID:25737157

Autism Spectrum Disorders: Translating human deficits into mouse behavior.

PubMed

Pasciuto, E; Borrie, S C; Kanellopoulos, A K; Santos, A R; Cappuyns, E; D'Andrea, L; Pacini, L; Bagni, C

2015-10-01

Autism Spectrum Disorders are a heterogeneous group of neurodevelopmental disorders, with rising incidence but little effective therapeutic intervention available. Currently two main clinical features are described to diagnose ASDs: impaired social interaction and communication, and repetitive behaviors. Much work has focused on understanding underlying causes of ASD by generating animal models of the disease, in the hope of discovering signaling pathways and cellular targets for drug intervention. Here we review how ASD behavioral phenotypes can be modeled in the mouse, the most common animal model currently in use in this field, and discuss examples of genetic mouse models of ASD with behavioral features that recapitulate various symptoms of ASD. Copyright © 2015 Elsevier Inc. All rights reserved.

Generation and Characterization of a Transgenic Mouse Carrying a Functional Human β-Globin Gene with the IVSI-6 Thalassemia Mutation

PubMed Central

Mancini, Irene; Lampronti, Ilaria; Salvatori, Francesca; Fabbri, Enrica; Zuccato, Cristina; Cosenza, Lucia C.; Montagner, Giulia; Borgatti, Monica; Altruda, Fiorella; Fagoonee, Sharmila; Carandina, Gianni; Aiello, Vincenzo; Breda, Laura; Rivella, Stefano; Gambari, Roberto

2015-01-01

Mouse models that carry mutations causing thalassemia represent a suitable tool to test in vivo new mutation-specific therapeutic approaches. Transgenic mice carrying the β-globin IVSI-6 mutation (the most frequent in Middle-Eastern regions and recurrent in Italy and Greece) are, at present, not available. We report the production and characterization of a transgenic mouse line (TG-β-IVSI-6) carrying the IVSI-6 thalassemia point mutation within the human β-globin gene. In the TG-β-IVSI-6 mouse (a) the transgenic integration region is located in mouse chromosome 7; (b) the expression of the transgene is tissue specific; (c) as expected, normally spliced human β-globin mRNA is produced, giving rise to β-globin production and formation of a human-mouse tetrameric chimeric hemoglobin mu α-globin2/hu β-globin2 and, more importantly, (d) the aberrant β-globin-IVSI-6 RNAs are present in blood cells. The TG-β-IVSI-6 mouse reproduces the molecular features of IVSI-6 β-thalassemia and might be used as an in vivo model to characterize the effects of antisense oligodeoxynucleotides targeting the cryptic sites responsible for the generation of aberrantly spliced β-globin RNA sequences, caused by the IVSI-6 mutation. These experiments are expected to be crucial for the development of a personalized therapy for β-thalassemia. PMID:26097845

An extended Kalman filter for mouse tracking.

PubMed

Choi, Hongjun; Kim, Mingi; Lee, Onseok

2018-05-19

Animal tracking is an important tool for observing behavior, which is useful in various research areas. Animal specimens can be tracked using dynamic models and observation models that require several types of data. Tracking mouse has several barriers due to the physical characteristics of the mouse, their unpredictable movement, and cluttered environments. Therefore, we propose a reliable method that uses a detection stage and a tracking stage to successfully track mouse. The detection stage detects the surface area of the mouse skin, and the tracking stage implements an extended Kalman filter to estimate the state variables of a nonlinear model. The changes in the overall shape of the mouse are tracked using an oval-shaped tracking model to estimate the parameters for the ellipse. An experiment is conducted to demonstrate the performance of the proposed tracking algorithm using six video images showing various types of movement, and the ground truth values for synthetic images are compared to the values generated by the tracking algorithm. A conventional manual tracking method is also applied to compare across eight experimenters. Furthermore, the effectiveness of the proposed tracking method is also demonstrated by applying the tracking algorithm with actual images of mouse. Graphical abstract.

Mutsβ generates both expansions and contractions in a mouse model of the Fragile X-associated disorders

PubMed Central

Zhao, Xiao-Nan; Kumari, Daman; Gupta, Shikha; Wu, Di; Evanitsky, Maya; Yang, Wei; Usdin, Karen

2015-01-01

Fragile X-associated disorders are Repeat Expansion Diseases that result from expansion of a CGG/CCG-repeat in the FMR1 gene. Contractions of the repeat tract also occur, albeit at lower frequency. However, these contractions can potentially modulate disease symptoms or generate an allele with repeat numbers in the normal range. Little is known about the expansion mechanism and even less about contractions. We have previously demonstrated that the mismatch repair (MMR) protein MSH2 is required for expansions in a mouse model of these disorders. Here, we show that MSH3, the MSH2-binding partner in the MutSβ complex, is required for 98% of germ line expansions and all somatic expansions in this model. In addition, we provide evidence for two different contraction mechanisms that operate in the mouse model, a MutSβ-independent one that generates small contractions and a MutSβ-dependent one that generates larger ones. We also show that MutSβ complexes formed with the repeats have altered kinetics of ATP hydrolysis relative to complexes with bona fide MMR substrates and that MutSβ increases the stability of the CCG-hairpins at physiological temperatures. These data may have important implications for our understanding of the mechanism(s) of repeat instability and for the role of MMR proteins in this process. PMID:26420841

A Rapid Embryonic Stem Cell-Based Mouse Model for B-cell Lymphomas Driven by Epstein-Barr Virus Protein LMP1.

PubMed

Ba, Zhaoqing; Meng, Fei-Long; Gostissa, Monica; Huang, Pei-Yi; Ke, Qiang; Wang, Zhe; Dao, Mai N; Fujiwara, Yuko; Rajewsky, Klaus; Zhang, Baochun; Alt, Frederick W

2015-06-01

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of α/β and γ/δ T cells ("CLT" mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, although introduction of additional activating or knockout mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive, and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT embryonic stem (ES) cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which, like germline CLT mice, harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation ("RDBC") approach die rapidly in association with B-cell lymphoproliferation and lymphoma. Because CLT lymphomas routinely express the activation-induced cytidine deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models. ©2015 American Association for Cancer Research.

A Rapid Embryonic Stem Cell-Based Mouse Model for B-cell Lymphomas Driven by Epstein-Barr Virus Protein LMP1

PubMed Central

Ba, Zhaoqing; Meng, Fei-Long; Gostissa, Monica; Huang, Pei-Yi; Ke, Qiang; Wang, Zhe; Dao, Mai N.; Fujiwara, Yuko; Rajewsky, Klaus; Baochun, Zhang; Alt, Frederick W.

2015-01-01

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of α/β and γ/δ T cells (“CLT” mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, while introduction of additional activating or knock-out mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT ES cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which like germline CLT mice harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation (“RDBC”) approach die rapidly in association with B-cell lymphoproliferation and lymphoma. As CLT lymphomas routinely express the Activation-Induced Cytidine Deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models. PMID:25934172

The Snowballing Literature on Imiquimod-Induced Skin Inflammation in Mice: A Critical Appraisal

PubMed Central

Hawkes, Jason E.; Gudjonsson, Johann E.; Ward, Nicole L.

2016-01-01

Since 2009, the imiquimod- or Aldara-induced (3M Pharmaceuticals, St. Paul, MN) model of acute skin inflammation has become the most widely used mouse model in preclinical psoriasis studies. Although this model offers researchers numerous benefits, there are important limitations and possible confounding variables to consider. The imiquimod model requires careful consideration and warrants scrutiny of the data generated by its use. In this perspective, we provide an overview of the advantages and disadvantages of this mouse model and offer suggestions for its use in psoriasis research. PMID:27955901

Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer

DTIC Science & Technology

2016-11-01

available to the research community. Similarly, any cell lines generated in our studies will also be shared. The EGFR transgenic mouse models used in...Lines and Transgenic Mice Active Completed – May 31, 2015 NIH/NCI R01CA121210 Overcoming Acquired Resistance to EGFR Inhibitors in Lung Cancer...Active Active Labrecque Foundation Not Applicable A Translational Pilot Study on Serum Biomarkers of Lung Cancer Using Transgenic Mouse Models of

The Mouse Genome Database (MGD): facilitating mouse as a model for human biology and disease.

PubMed

Eppig, Janan T; Blake, Judith A; Bult, Carol J; Kadin, James A; Richardson, Joel E

2015-01-01

The Mouse Genome Database (MGD, http://www.informatics.jax.org) serves the international biomedical research community as the central resource for integrated genomic, genetic and biological data on the laboratory mouse. To facilitate use of mouse as a model in translational studies, MGD maintains a core of high-quality curated data and integrates experimentally and computationally generated data sets. MGD maintains a unified catalog of genes and genome features, including functional RNAs, QTL and phenotypic loci. MGD curates and provides functional and phenotype annotations for mouse genes using the Gene Ontology and Mammalian Phenotype Ontology. MGD integrates phenotype data and associates mouse genotypes to human diseases, providing critical mouse-human relationships and access to repositories holding mouse models. MGD is the authoritative source of nomenclature for genes, genome features, alleles and strains following guidelines of the International Committee on Standardized Genetic Nomenclature for Mice. A new addition to MGD, the Human-Mouse: Disease Connection, allows users to explore gene-phenotype-disease relationships between human and mouse. MGD has also updated search paradigms for phenotypic allele attributes, incorporated incidental mutation data, added a module for display and exploration of genes and microRNA interactions and adopted the JBrowse genome browser. MGD resources are freely available to the scientific community. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Bat-mouse bone marrow chimera: a novel animal model for dissecting the uniqueness of the bat immune system.

PubMed

Yong, Kylie Su Mei; Ng, Justin Han Jia; Her, Zhisheng; Hey, Ying Ying; Tan, Sue Yee; Tan, Wilson Wei Sheng; Irac, Sergio Erdal; Liu, Min; Chan, Xue Ying; Gunawan, Merry; Foo, Randy Jee Hiang; Low, Dolyce Hong Wen; Mendenhall, Ian Hewitt; Chionh, Yok Teng; Dutertre, Charles-Antoine; Chen, Qingfeng; Wang, Lin-Fa

2018-03-16

Bats are an important animal model with long lifespans, low incidences of tumorigenesis and an ability to asymptomatically harbour pathogens. Currently, in vivo studies of bats are hampered due to their low reproduction rates. To overcome this, we transplanted bat cells from bone marrow (BM) and spleen into an immunodeficient mouse strain NOD-scid IL-2R -/- (NSG), and have successfully established stable, long-term reconstitution of bat immune cells in mice (bat-mice). Immune functionality of our bat-mouse model was demonstrated through generation of antigen-specific antibody response by bat cells following immunization. Post-engraftment of total bat BM cells and splenocytes, bat immune cells survived, expanded and repopulated the mouse without any observable clinical abnormalities. Utilizing bat's remarkable immunological functions, this novel model has a potential to be transformed into a powerful platform for basic and translational research.

Instability of the insertional mutation in CftrTgH(neoim)Hgu cystic fibrosis mouse model

PubMed Central

Charizopoulou, Nikoletta; Jansen, Silke; Dorsch, Martina; Stanke, Frauke; Dorin, Julia R; Hedrich, Hans-Jürgen; Tümmler, Burkhard

2004-01-01

Background A major boost to the cystic fibrosis disease research was given by the generation of various mouse models using gene targeting in embryonal stem cells. Moreover, the introduction of the same mutation on different inbred strains generating congenic strains facilitated the search for modifier genes. From the original CftrTgH(neoim)Hgu CF mouse model we have generated using strict brother × sister mating two inbred CftrTgH(neoim)Hgu mouse lines (CF/1 and CF/3). Thereafter, the insertional mutation was introgressed from CF/3 into three inbred backgrounds (C57BL/6, BALB/c, DBA/2J) generating congenic animals. In every backcross cycle germline transmission of the insertional mutation was monitored by direct probing the insertion via Southern RFLP. In order to bypass this time consuming procedure we devised an alternative PCR based protocol whereby mouse strains are differentiated at the Cftr locus by Cftr intragenic microsatellite genotypes that are tightly linked to the disrupted locus. Results Using this method we were able to identify animals carrying the insertional mutation based upon the differential haplotypic backgrounds of the three inbred strains and the mutant CftrTgH(neoim)Hgu at the Cftr locus. Moreover, this method facilitated the identification of the precise vector excision from the disrupted Cftr locus in two out of 57 typed animals. This reversion to wild type status took place without any loss of sequence revealing the instability of insertional mutations during the production of congenic animals. Conclusions We present intragenic microsatellite markers as a tool for fast and efficient identification of the introgressed locus of interest in the recipient strain during congenic animal breeding. Moreover, the same genotyping method allowed the identification of a vector excision event, posing questions on the stability of insertional mutations in mice. PMID:15102331

Human gastric cancer modelling using organoids.

PubMed

Seidlitz, Therese; Merker, Sebastian R; Rothe, Alexander; Zakrzewski, Falk; von Neubeck, Cläre; Grützmann, Konrad; Sommer, Ulrich; Schweitzer, Christine; Schölch, Sebastian; Uhlemann, Heike; Gaebler, Anne-Marlene; Werner, Kristin; Krause, Mechthild; Baretton, Gustavo B; Welsch, Thilo; Koo, Bon-Kyoung; Aust, Daniela E; Klink, Barbara; Weitz, Jürgen; Stange, Daniel E

2018-04-27

Gastric cancer is the second leading cause of cancer-related deaths and the fifth most common malignancy worldwide. In this study, human and mouse gastric cancer organoids were generated to model the disease and perform drug testing to delineate treatment strategies. Human gastric cancer organoid cultures were established, samples classified according to their molecular profile and their response to conventional chemotherapeutics tested. Targeted treatment was performed according to specific druggable mutations. Mouse gastric cancer organoid cultures were generated carrying molecular subtype-specific alterations. Twenty human gastric cancer organoid cultures were established and four selected for a comprehensive in-depth analysis. Organoids demonstrated divergent growth characteristics and morphologies. Immunohistochemistry showed similar characteristics to the corresponding primary tissue. A divergent response to 5-fluoruracil, oxaliplatin, irinotecan, epirubicin and docetaxel treatment was observed. Whole genome sequencing revealed a mutational spectrum that corresponded to the previously identified microsatellite instable, genomic stable and chromosomal instable subtypes of gastric cancer. The mutational landscape allowed targeted therapy with trastuzumab for ERBB2 alterations and palbociclib for CDKN2A loss. Mouse cancer organoids carrying Kras and Tp53 or Apc and Cdh1 mutations were characterised and serve as model system to study the signalling of induced pathways. We generated human and mouse gastric cancer organoids modelling typical characteristics and altered pathways of human gastric cancer. Successful interference with activated pathways demonstrates their potential usefulness as living biomarkers for therapy response testing. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

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

PubMed Central

Li, Ziyi; Engelhardt, John F

2003-01-01

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. PMID:14613541

Characterisation of a C1qtnf5 Ser163Arg Knock-In Mouse Model of Late-Onset Retinal Macular Degeneration

PubMed Central

Shu, Xinhua; Luhmann, Ulrich F. O.; Aleman, Tomas S.; Barker, Susan E.; Lennon, Alan; Tulloch, Brian; Chen, Mei; Xu, Heping; Jacobson, Samuel G.; Ali, Robin; Wright, Alan F.

2011-01-01

A single founder mutation resulting in a Ser163Arg substitution in the C1QTNF5 gene product causes autosomal dominant late-onset retinal macular degeneration (L-ORMD) in humans, which has clinical and pathological features resembling age-related macular degeneration. We generated and characterised a mouse “knock-in” model carrying the Ser163Arg mutation in the orthologous murine C1qtnf5 gene by site-directed mutagenesis and homologous recombination into mouse embryonic stem cells. Biochemical, immunological, electron microscopic, fundus autofluorescence, electroretinography and laser photocoagulation analyses were used to characterise the mouse model. Heterozygous and homozygous knock-in mice showed no significant abnormality in any of the above measures at time points up to 2 years. This result contrasts with another C1qtnf5 Ser163Arg knock-in mouse which showed most of the features of L-ORMD but differed in genetic background and targeting construct. PMID:22110650

Scattered Dose Calculations and Measurements in a Life-Like Mouse Phantom

PubMed Central

Welch, David; Turner, Leah; Speiser, Michael; Randers-Pehrson, Gerhard; Brenner, David J.

2017-01-01

Anatomically accurate phantoms are useful tools for radiation dosimetry studies. In this work, we demonstrate the construction of a new generation of life-like mouse phantoms in which the methods have been generalized to be applicable to the fabrication of any small animal. The mouse phantoms, with built-in density inhomogeneity, exhibit different scattering behavior dependent on where the radiation is delivered. Computer models of the mouse phantoms and a small animal irradiation platform were devised in Monte Carlo N-Particle code (MCNP). A baseline test replicating the irradiation system in a computational model shows minimal differences from experimental results from 50 Gy down to 0.1 Gy. We observe excellent agreement between scattered dose measurements and simulation results from X-ray irradiations focused at either the lung or the abdomen within our phantoms. This study demonstrates the utility of our mouse phantoms as measurement tools with the goal of using our phantoms to verify complex computational models. PMID:28140787

Treatment of d-galactose induced mouse aging with Lycium barbarum polysaccharides and its mechanism study.

PubMed

Tang, Tao; He, Bixiu

2013-01-01

We evaluated the effects of Lycium barbarum polysaccharides LBP) on D-galactose aging model mouse, and explored its possible mechanism. Kunming mice were randomly divided into the control group, the model group, the high-dose LBP group, and the low-dose LBP group. Except the control group, D-galactose was used for modelling. The drug was administrated when modelling. Mouse behavioural, learning and memory changes were observed, and the contents of lipid peroxidation (LPO), lipofuscin (LF) and monoamine oxidase B (MAO-B) in mouse brain tissue and the weight of immune organs were measured after 6 weeks. Compared with the control group, mouse weight gain in the model group reduced significantly. Compared with model group, after mice drank LBP, the times of electric shock was less than aging mice (in which, the high-dose LBP group, P<0.05), and electric shock incubation period was longer (P<0.01). On Day 45 after modelling and drug administration, the contents of LPO, LF and MAO-B in mouse brain tissue in the model group increased significantly, while those in the drug administration groups decreased significantly. The thymus index in the aging model group decreased significantly; the thymus index and the spleen index in the high-dose LBP group and the low-dose LBP group rebounded significantly (P<0.01). We concluded that LBP has an anti-aging effect on D-galactose induced aging model mouse, and its mechanism may be related with the alleviation of glucose metabolism disorder and the resistance of the generation of lipid peroxide and other substances, which damage cell membrane lipid.

Humanized Mouse Models for the Study of Human Malaria Parasite Biology, Pathogenesis, and Immunity.

PubMed

Minkah, Nana K; Schafer, Carola; Kappe, Stefan H I

2018-01-01

Malaria parasite infection continues to inflict extensive morbidity and mortality in resource-poor countries. The insufficiently understood parasite biology, continuously evolving drug resistance and the lack of an effective vaccine necessitate intensive research on human malaria parasites that can inform the development of new intervention tools. Humanized mouse models have been greatly improved over the last decade and enable the direct study of human malaria parasites in vivo in the laboratory. Nevertheless, no small animal model developed so far is capable of maintaining the complete life cycle of Plasmodium parasites that infect humans. The ultimate goal is to develop humanized mouse systems in which a Plasmodium infection closely reproduces all stages of a parasite infection in humans, including pre-erythrocytic infection, blood stage infection and its associated pathology, transmission as well as the human immune response to infection. Here, we discuss current humanized mouse models and the future directions that should be taken to develop next-generation models for human malaria parasite research.

New generation humanized mice for virus research: Comparative aspects and future prospects

PubMed Central

Akkina, Ramesh

2014-01-01

Work with human specific viruses will greatly benefit from the use of an in vivo system that provides human target cells and tissues in a physiological setting. In this regard humanized mice (hu-Mice) have played an important role in our understanding of viral pathogenesis and testing of therapeutic strategies. Limitations with earlier versions of hu-Mice that lacked a functioning human immune system are currently being overcome. The new generation hu-Mouse models are capable of multilineage human hematopoiesis and generate T cells, B cells, macrophages and dendritic cells required for an adaptive human immune response. Now any human specific pathogen that can infect humanized mice can be studied in the context of ongoing infection and immune responses. Two leading humanized mouse models are currently employed: the hu-HSC model is created by transplantation of human hematopoietic stem cells (HSC), whereas the BLT mouse model is prepared by transplantation of human fetal liver, thymus and HSC. A number of human specific viruses such as HIV-1, dengue, EBV and HCV are being studied intensively in these systems. Both models permit infection by mucosal routes with viruses such as HIV-1 thus allowing transmission prevention studies. Cellular and humoral immune responses are seen in both the models. While there is efficient antigen specific IgM production, IgG responses are suboptimal due to inefficient immunoglobulin class switching. With the maturation of T cells occurring in the autologous human thymus, BLT mice permit human HLA restricted T cell responses in contrast to hu-HSC mice. However, the strength of the immune responses needs further improvement in both models to reach the levels seen in humans. The scope of hu-Mice use is further broadened by transplantation of additional tissues like human liver thus permitting immunopathogenesis studies on hepatotropic viruses such as HCV. Numerous studies that encompass antivirals, gene therapy, viral evolution, and the generation of human monoclonal antibodies have been conducted with promising results in these mice. For further improvement of the new hu-Mouse models, ongoing work is focused on generating new strains of immunodeficient mice transgenic for human HLA molecules to strengthen immune responses and human cytokines and growth factors to improve human cell reconstitution and their homeostatic maintenance. PMID:23217612

An Immunocompetent Mouse Model of Zika Virus Infection.

PubMed

Gorman, Matthew J; Caine, Elizabeth A; Zaitsev, Konstantin; Begley, Matthew C; Weger-Lucarelli, James; Uccellini, Melissa B; Tripathi, Shashank; Morrison, Juliet; Yount, Boyd L; Dinnon, Kenneth H; Rückert, Claudia; Young, Michael C; Zhu, Zhe; Robertson, Shelly J; McNally, Kristin L; Ye, Jing; Cao, Bin; Mysorekar, Indira U; Ebel, Gregory D; Baric, Ralph S; Best, Sonja M; Artyomov, Maxim N; Garcia-Sastre, Adolfo; Diamond, Michael S

2018-05-09

Progress toward understanding Zika virus (ZIKV) pathogenesis is hindered by lack of immunocompetent small animal models, in part because ZIKV fails to effectively antagonize Stat2-dependent interferon (IFN) responses in mice. To address this limitation, we first passaged an African ZIKV strain (ZIKV-Dak-41525) through Rag1 -/- mice to obtain a mouse-adapted virus (ZIKV-Dak-MA) that was more virulent than ZIKV-Dak-41525 in mice treated with an anti-Ifnar1 antibody. A G18R substitution in NS4B was the genetic basis for the increased replication, and resulted in decreased IFN-β production, diminished IFN-stimulated gene expression, and the greater brain infection observed with ZIKV-Dak-MA. To generate a fully immunocompetent mouse model of ZIKV infection, human STAT2 was introduced into the mouse Stat2 locus (hSTAT2 KI). Subcutaneous inoculation of pregnant hSTAT2 KI mice with ZIKV-Dak-MA resulted in spread to the placenta and fetal brain. An immunocompetent mouse model of ZIKV infection may prove valuable for evaluating countermeasures to limit disease. Copyright © 2018 Elsevier Inc. All rights reserved.

[Generation and comparison of two genetically engineered mouse models of ErbB2/Neu positive-PTEN deficient breast cancer].

PubMed

Wang, Qing-fei; Ding, Hui; Liu, Bao-rui; Zhang, Kui

2014-07-01

To generate two genetically engineered mouse models of ErbB2/Neu positive-PTEN deficient breast cancer and to compare their biological properties. The genetically engineered mice previously developed with mouse mammary tumor virus (MMTV) promoter driven expression of activated ErbB2/Neu and recombinant Cre (FVB/N-MMTV-NIC) were interbred with Flox-PTEN mice; and FVB/N-ErbB2KI mice, harboring endogenous promoter driven activated ErbB2/Neu expression, FVB/N-MMTV-Cre mice and the flox-PTEN mice were interbred. Neu, Cre and PTEN genes were amplified by PCR for genotyping of the offsprings. ErbB2/Neu and PTEN expression in mammary tumors were detected by immunohistochemistry. Tumor formation time, tumor number, histopathology and lung metastasis were compared between two models, Ki-67 expression was detected by immunohistochemistry, and TUNEL staining of tumor tissues was performed. Two genetically engineered mouse models of ErbB2/Neu positive-PTEN homozygous deficient breast cancer were generated. The models were confirmed by genotyping and immunohistochemistry. One model with exogenous MMTV promoter driven expression of activated ErbB2/Neu and Cre coupling PTEN disruption was designated as NIC/PTEN(-/-) mice, and the other with MMTV-Cre induced endogenous promoter driven expression of activated ErbB2/Neu with PTEN disruption was designated as ErbB2KI/PTEN(-/-) mice. The tumor formation time in NIC/PTEN(-/-) mice was significantly shorter than that of ErbB2KI/PTEN(-/-) mice (30 vs 368 d, P<0.01); the number of tumor and incidence of lung metastasis was also significantly higher in NIC/PTEN(-/-) mice (10 vs 1-2 and 75.0% vs 37.5%, respectively, Ps<0.01). The Two models displayed distinct histopathological morphology. NIC/PTEN(-/-) tumor showed more Ki-67 positive cells than ErbB2KI/PTEN(-/-) tumor did (86.9%±2.8% vs 37.4%±7.2%, P<0.01), while the amount of cell apoptosis in tumors was not significantly different between two models. Two genetically engineered mouse models of ErbB2/Neu positive-PTEN homozygous deficient breast cancer with different phenotypes have been successfully generated, which may provide useful resource for further investigation of the initiation and progression of HER2/ErbB2 breast cancer, as well as for the development of novel prevention and treatment regimens of this malignance.

Establishing a laboratory animal model from a transgenic animal: RasH2 mice as a model for carcinogenicity studies in regulatory science.

PubMed

Urano, K; Tamaoki, N; Nomura, T

2012-01-01

Transgenic animal models have been used in small numbers in gene function studies in vivo for a period of time, but more recently, the use of a single transgenic animal model has been approved as a second species, 6-month alternative (to the routine 2-year, 2-animal model) used in short-term carcinogenicity studies for generating regulatory application data of new drugs. This article addresses many of the issues associated with the creation and use of one of these transgenic models, the rasH2 mouse, for regulatory science. The discussion includes strategies for mass producing mice with the same stable phenotype, including constructing the transgene, choosing a founder mouse, and controlling both the transgene and background genes; strategies for developing the model for regulatory science, including measurements of carcinogen susceptibility, stability of a large-scale production system, and monitoring for uniform carcinogenicity responses; and finally, efficient use of the transgenic animal model on study. Approximately 20% of mouse carcinogenicity studies for new drug applications in the United States currently use transgenic models, typically the rasH2 mouse. The rasH2 mouse could contribute to animal welfare by reducing the numbers of animals used as well as reducing the cost of carcinogenicity studies. A better understanding of the advantages and disadvantages of the transgenic rasH2 mouse will result in greater and more efficient use of this animal model in the future.

Mice with Reconstituted Human Immune System Components as a Tool to Study Immune Cell Interactions in EBV Infection.

PubMed

Heuts, Frank; Nagy, Noemi

2017-01-01

Recent developments in mouse models that harbor part of a human immune system have proved extremely valuable to study the in vivo immune response to human specific pathogens such as Epstein-Barr virus. Over the last decades, advances in immunodeficient mouse strains that can be used as recipients for human immune cells have greatly enhanced the use of these models. Here, we describe the generation of mice with reconstituted human immune system (HIS mice) using immunocompromised mice transplanted with human CD34 + hematopoietic stem cells. We will also describe how such mice, in which human immune cells are generated de novo, can be used to study EBV infection.

The mouse age phenome knowledgebase and disease-specific inter-species age mapping.

PubMed

Geifman, Nophar; Rubin, Eitan

2013-01-01

Similarities between mice and humans lead to generation of many mouse models of human disease. However, differences between the species often result in mice being unreliable as preclinical models for human disease. One difference that might play a role in lowering the predictivity of mice models to human diseases is age. Despite the important role age plays in medicine, it is too often considered only casually when considering mouse models. We developed the mouse-Age Phenotype Knowledgebase, which holds knowledge about age-related phenotypic patterns in mice. The knowledgebase was extensively populated with literature-derived data using text mining techniques. We then mapped between ages in humans and mice by comparing the age distribution pattern for 887 diseases in both species. The knowledgebase was populated with over 9800 instances generated by a text-mining pipeline. The quality of the data was manually evaluated, and was found to be of high accuracy (estimated precision >86%). Furthermore, grouping together diseases that share similar age patterns in mice resulted in clusters that mirror actual biomedical knowledge. Using these data, we matched age distribution patterns in mice and in humans, allowing for age differences by shifting either of the patterns. High correlation (r(2)>0.5) was found for 223 diseases. The results clearly indicate a difference in the age mapping between different diseases: age 30 years in human is mapped to 120 days in mice for Leukemia, but to 295 days for Anemia. Based on these results we generated a mice-to-human age map which is publicly available. We present here the development of the mouse-APK, its population with literature-derived data and its use to map ages in mice and human for 223 diseases. These results present a further step made to bridging the gap between humans and mice in biomedical research.

Gene Suppression of Mouse Testis In Vivo Using Small Interfering RNA Derived from Plasmid Vectors

PubMed Central

Takizawa, Takami; Ishikawa, Tomoko; Kosuge, Takuji; Mizuguchi, Yoshiaki; Sato, Yoko; Koji, Takehiko; Araki, Yoshihiko; Takizawa, Toshihiro

2012-01-01

We evaluated whether inhibiting gene expression by small interfering RNA (siRNA) can be used for an in vivo model using a germ cell-specific gene (Tex101) as a model target in mouse testis. We generated plasmid-based expression vectors of siRNA targeting the Tex101 gene and transfected them into postnatal day 10 mouse testes by in vivo electroporation. After optimizing the electroporation conditions using a vector transfected into the mouse testis, a combination of high- and low-voltage pulses showed excellent transfection efficiency for the vectors with minimal tissue damage, but gene suppression was transient. Gene suppression by in vivo electroporation may be helpful as an alternative approach when designing experiments to unravel the basic role of testicular molecules. PMID:22489107

Preservation of Long-Term Memory and Synaptic Plasticity Despite Short-Term Impairments in the Tc1 Mouse Model of Down Syndrome

ERIC Educational Resources Information Center

Morice, Elise; Andreae, Laura C.; Cooke, Sam F.; Vanes, Lesley; Fisher, Elizabeth M. C.; Tybulewicz, Victor L. J.; Bliss, Timothy V. P.

2008-01-01

Down syndrome (DS) is a genetic disorder arising from the presence of a third copy of the human chromosome 21 (Hsa21). Recently, O'Doherty and colleagues in an earlier study generated a new genetic mouse model of DS (Tc1) that carries an almost complete Hsa21. Since DS is the most common genetic cause of mental retardation, we have undertaken a…

3D engineered cardiac tissue models of human heart disease: learning more from our mice.

PubMed

Ralphe, J Carter; de Lange, Willem J

2013-02-01

Mouse engineered cardiac tissue constructs (mECTs) are a new tool available to study human forms of genetic heart disease within the laboratory. The cultured strips of cardiac cells generate physiologic calcium transients and twitch force, and respond to electrical pacing and adrenergic stimulation. The mECT can be made using cells from existing mouse models of cardiac disease, providing a robust readout of contractile performance and allowing a rapid assessment of genotype-phenotype correlations and responses to therapies. mECT represents an efficient and economical extension to the existing tools for studying cardiac physiology. Human ECTs generated from iPSCMs represent the next logical step for this technology and offer significant promise of an integrated, fully human, cardiac tissue model. Copyright © 2013. Published by Elsevier Inc.

Unexpected effects of different genetic backgrounds on identification of genomic rearrangements via whole-genome next generation sequencing.

PubMed

Chen, Zhangguo; Gowan, Katherine; Leach, Sonia M; Viboolsittiseri, Sawanee S; Mishra, Ameet K; Kadoishi, Tanya; Diener, Katrina; Gao, Bifeng; Jones, Kenneth; Wang, Jing H

2016-10-21

Whole genome next generation sequencing (NGS) is increasingly employed to detect genomic rearrangements in cancer genomes, especially in lymphoid malignancies. We recently established a unique mouse model by specifically deleting a key non-homologous end-joining DNA repair gene, Xrcc4, and a cell cycle checkpoint gene, Trp53, in germinal center B cells. This mouse model spontaneously develops mature B cell lymphomas (termed G1XP lymphomas). Here, we attempt to employ whole genome NGS to identify novel structural rearrangements, in particular inter-chromosomal translocations (CTXs), in these G1XP lymphomas. We sequenced six lymphoma samples, aligned our NGS data with mouse reference genome (in C57BL/6J (B6) background) and identified CTXs using CREST algorithm. Surprisingly, we detected widespread CTXs in both lymphomas and wildtype control samples, majority of which were false positive and attributable to different genetic backgrounds. In addition, we validated our NGS pipeline by sequencing multiple control samples from distinct tissues of different genetic backgrounds of mouse (B6 vs non-B6). Lastly, our studies showed that widespread false positive CTXs can be generated by simply aligning sequences from different genetic backgrounds of mouse. We conclude that mapping and alignment with reference genome might not be a preferred method for analyzing whole-genome NGS data obtained from a genetic background different from reference genome. Given the complex genetic background of different mouse strains or the heterogeneity of cancer genomes in human patients, in order to minimize such systematic artifacts and uncover novel CTXs, a preferred method might be de novo assembly of personalized normal control genome and cancer cell genome, instead of mapping and aligning NGS data to mouse or human reference genome. Thus, our studies have critical impact on the manner of data analysis for cancer genomics.

Additive interactions between 1-methyl-1,2,3,4-tetrahydroisoquinoline and clobazam in the mouse maximal electroshock-induced tonic seizure model--an isobolographic analysis for parallel dose-response relationship curves.

PubMed

Andres-Mach, Marta; Haratym-Maj, Agnieszka; Zagaja, Mirosław; Luszczki, Jarogniew J

2014-01-01

The aim of this study was to characterize the anticonvulsant effect of 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTHIQ) in combination with clobazam (CLB) in the mouse maximal electroshock-induced seizure (MES) model. The anticonvulsant interaction profile between 1-MeTHIQ and CLB in the mouse MES model was determined using an isobolographic analysis for parallel dose-response relationship curves. Electroconvulsions were produced in albino Swiss mice by a current (sine wave, 25 mA, 500 V, 50 Hz, 0.2-second stimulus duration) delivered via auricular electrodes by a Hugo Sachs generator. There was an additive effect of the combination of 1-MeTHIQ with CLB (at the fixed ratios of 1:3, 1:1 and 3:1) in the mouse MES-induced tonic seizure model. The additive interaction of the combination of 1-MeTHIQ with CLB (at fixed-ratios of 1:3, 1:1 and 3:1) in the mouse MES model seems to be pharmacodynamic in nature and worth of considering in further clinical practice. © 2014 S. Karger AG, Basel.

The NOTCH3 score: a pre-clinical CADASIL biomarker in a novel human genomic NOTCH3 transgenic mouse model with early progressive vascular NOTCH3 accumulation.

PubMed

Rutten, Julie W; Klever, Roselin R; Hegeman, Ingrid M; Poole, Dana S; Dauwerse, Hans G; Broos, Ludo A M; Breukel, Cor; Aartsma-Rus, Annemieke M; Verbeek, J Sjef; van der Weerd, Louise; van Duinen, Sjoerd G; van den Maagdenberg, Arn M J M; Lesnik Oberstein, Saskia A J

2015-12-29

CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is a hereditary small vessel disease caused by mutations in the NOTCH3 gene, leading to toxic NOTCH3 protein accumulation in the small- to medium sized arterioles. The accumulation is systemic but most pronounced in the brain vasculature where it leads to clinical symptoms of recurrent stroke and dementia. There is no therapy for CADASIL, and therapeutic development is hampered by a lack of feasible clinical outcome measures and biomarkers, both in mouse models and in CADASIL patients. To facilitate pre-clinical therapeutic interventions for CADASIL, we aimed to develop a novel, translational CADASIL mouse model. We generated transgenic mice in which we overexpressed the full length human NOTCH3 gene from a genomic construct with the archetypal c.544C > T, p.Arg182Cys mutation. The four mutant strains we generated have respective human NOTCH3 RNA expression levels of 100, 150, 200 and 350 % relative to endogenous mouse Notch3 RNA expression. Immunohistochemistry on brain sections shows characteristic vascular human NOTCH3 accumulation in all four mutant strains, with human NOTCH3 RNA expression levels correlating with age at onset and progression of NOTCH3 accumulation. This finding was the basis for developing the 'NOTCH3 score', a quantitative measure for the NOTCH3 accumulation load. This score proved to be a robust and sensitive method to assess the progression of NOTCH3 accumulation, and a feasible biomarker for pre-clinical therapeutic testing. This novel, translational CADASIL mouse model is a suitable model for pre-clinical testing of therapeutic strategies aimed at delaying or reversing NOTCH3 accumulation, using the NOTCH3 score as a biomarker.

Biology and therapy of inherited retinal degenerative disease: insights from mouse models

PubMed Central

Veleri, Shobi; Lazar, Csilla H.; Chang, Bo; Sieving, Paul A.; Banin, Eyal; Swaroop, Anand

2015-01-01

Retinal neurodegeneration associated with the dysfunction or death of photoreceptors is a major cause of incurable vision loss. Tremendous progress has been made over the last two decades in discovering genes and genetic defects that lead to retinal diseases. The primary focus has now shifted to uncovering disease mechanisms and designing treatment strategies, especially inspired by the successful application of gene therapy in some forms of congenital blindness in humans. Both spontaneous and laboratory-generated mouse mutants have been valuable for providing fundamental insights into normal retinal development and for deciphering disease pathology. Here, we provide a review of mouse models of human retinal degeneration, with a primary focus on diseases affecting photoreceptor function. We also describe models associated with retinal pigment epithelium dysfunction or synaptic abnormalities. Furthermore, we highlight the crucial role of mouse models in elucidating retinal and photoreceptor biology in health and disease, and in the assessment of novel therapeutic modalities, including gene- and stem-cell-based therapies, for retinal degenerative diseases. PMID:25650393

The Dwarf Phenotype in GH240B Mice, Haploinsufficient for the Autism Candidate Gene Neurobeachin, Is Caused by Ectopic Expression of Recombinant Human Growth Hormone

PubMed Central

Fu, Quili; Stijnen, Pieter; Pruniau, Vincent; Meulemans, Sandra; Vankelecom, Hugo; Creemers, John W. M.

2014-01-01

Two knockout mouse models for the autism candidate gene Neurobeachin (Nbea) have been generated independently. Although both models have similar phenotypes, one striking difference is the dwarf phenotype observed in the heterozygous configuration of the GH240B model that is generated by the serendipitous insertion of a promoterless human growth hormone (hGH) genomic fragment in the Nbea gene. In order to elucidate this discrepancy, the dwarfism present in this Nbea mouse model was investigated in detail. The growth deficiency in Nbea +/− mice coincided with an increased percentage of fat mass and a decrease in bone mineral density. Low but detectable levels of hGH were detected in the pituitary and hypothalamus of Nbea +/− mice but not in liver, hippocampus nor in serum. As a consequence, several members of the mouse growth hormone (mGH) signaling cascade showed altered mRNA levels, including a reduction in growth hormone-releasing hormone mRNA in the hypothalamus. Moreover, somatotrope cells were less numerous in the pituitary of Nbea +/− mice and both contained and secreted significantly less mGH resulting in reduced levels of circulating insulin-like growth factor 1. These findings demonstrate that the random integration of the hGH transgene in this mouse model has not only inactivated Nbea but has also resulted in the tissue-specific expression of hGH causing a negative feedback loop, mGH hyposecretion and dwarfism. PMID:25333629

The dwarf phenotype in GH240B mice, haploinsufficient for the autism candidate gene Neurobeachin, is caused by ectopic expression of recombinant human growth hormone.

PubMed

Nuytens, Kim; Tuand, Krizia; Fu, Quili; Stijnen, Pieter; Pruniau, Vincent; Meulemans, Sandra; Vankelecom, Hugo; Creemers, John W M

2014-01-01

Two knockout mouse models for the autism candidate gene Neurobeachin (Nbea) have been generated independently. Although both models have similar phenotypes, one striking difference is the dwarf phenotype observed in the heterozygous configuration of the GH240B model that is generated by the serendipitous insertion of a promoterless human growth hormone (hGH) genomic fragment in the Nbea gene. In order to elucidate this discrepancy, the dwarfism present in this Nbea mouse model was investigated in detail. The growth deficiency in Nbea+/- mice coincided with an increased percentage of fat mass and a decrease in bone mineral density. Low but detectable levels of hGH were detected in the pituitary and hypothalamus of Nbea+/- mice but not in liver, hippocampus nor in serum. As a consequence, several members of the mouse growth hormone (mGH) signaling cascade showed altered mRNA levels, including a reduction in growth hormone-releasing hormone mRNA in the hypothalamus. Moreover, somatotrope cells were less numerous in the pituitary of Nbea+/- mice and both contained and secreted significantly less mGH resulting in reduced levels of circulating insulin-like growth factor 1. These findings demonstrate that the random integration of the hGH transgene in this mouse model has not only inactivated Nbea but has also resulted in the tissue-specific expression of hGH causing a negative feedback loop, mGH hyposecretion and dwarfism.

Generation of gene-targeted mice using embryonic stem cells derived from a transgenic mouse model of Alzheimer's disease.

PubMed

Yamamoto, Satoshi; Ooshima, Yuki; Nakata, Mitsugu; Yano, Takashi; Matsuoka, Kunio; Watanabe, Sayuri; Maeda, Ryouta; Takahashi, Hideki; Takeyama, Michiyasu; Matsumoto, Yoshio; Hashimoto, Tadatoshi

2013-06-01

Gene-targeting technology using mouse embryonic stem (ES) cells has become the "gold standard" for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer's disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409-421, 2003) harboring 3 mutated genes (APPswe, TauP301L, and PS1M146V) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F1 mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background.

Phenotyping male infertility in the mouse: how to get the most out of a 'non-performer'.

PubMed

Borg, Claire L; Wolski, Katja M; Gibbs, Gerard M; O'Bryan, Moira K

2010-01-01

Functional male gametes are produced through complex processes that take place within the testis, epididymis and female reproductive tract. A breakdown at any of these phases can result in male infertility. The production of mutant mouse models often yields an unexpected male infertility phenotype. It is with this in mind that the current review has been written. The review aims to act as a guide to the 'non-reproductive biologist' to facilitate a systematic analysis of sterile or subfertile mice and to assist in extracting the maximum amount of information from each model. This is a review of the original literature on defects in the processes that take a mouse spermatogonial stem cell through to a fully functional spermatozoon, which result in male infertility. Based on literature searches and personal experience, we have outlined a step-by-step strategy for the analysis of an infertile male mouse line. A wide range of methods can be used to define the phenotype of an infertile male mouse. These methods range from histological methods such as electron microscopy and immunohistochemistry, to hormone analyses and methods to assess sperm maturation status and functional competence. With the increased rate of genetically modified mouse production, the generation of mouse models with unexpected male infertility is increasing. This manuscript will help to ensure that the maximum amount of information is obtained from each mouse model and, by extension, will facilitate the knowledge of both normal fertility processes and the causes of human infertility.

Machine Learning Model Analysis and Data Visualization with Small Molecules Tested in a Mouse Model of Mycobacterium tuberculosis Infection (2014–2015)

PubMed Central

2016-01-01

The renewed urgency to develop new treatments for Mycobacterium tuberculosis (Mtb) infection has resulted in large-scale phenotypic screening and thousands of new active compounds in vitro. The next challenge is to identify candidates to pursue in a mouse in vivo efficacy model as a step to predicting clinical efficacy. We previously analyzed over 70 years of this mouse in vivo efficacy data, which we used to generate and validate machine learning models. Curation of 60 additional small molecules with in vivo data published in 2014 and 2015 was undertaken to further test these models. This represents a much larger test set than for the previous models. Several computational approaches have now been applied to analyze these molecules and compare their molecular properties beyond those attempted previously. Our previous machine learning models have been updated, and a novel aspect has been added in the form of mouse liver microsomal half-life (MLM t1/2) and in vitro-based Mtb models incorporating cytotoxicity data that were used to predict in vivo activity for comparison. Our best Mtbin vivo models possess fivefold ROC values > 0.7, sensitivity > 80%, and concordance > 60%, while the best specificity value is >40%. Use of an MLM t1/2 Bayesian model affords comparable results for scoring the 60 compounds tested. Combining MLM stability and in vitroMtb models in a novel consensus workflow in the best cases has a positive predicted value (hit rate) > 77%. Our results indicate that Bayesian models constructed with literature in vivoMtb data generated by different laboratories in various mouse models can have predictive value and may be used alongside MLM t1/2 and in vitro-based Mtb models to assist in selecting antitubercular compounds with desirable in vivo efficacy. We demonstrate for the first time that consensus models of any kind can be used to predict in vivo activity for Mtb. In addition, we describe a new clustering method for data visualization and apply this to the in vivo training and test data, ultimately making the method accessible in a mobile app. PMID:27335215

A Dynamic Simulation of Musculoskeletal Function in the Mouse Hindlimb During Trotting Locomotion

PubMed Central

Charles, James P.; Cappellari, Ornella; Hutchinson, John R.

2018-01-01

Mice are often used as animal models of various human neuromuscular diseases, and analysis of these models often requires detailed gait analysis. However, little is known of the dynamics of the mouse musculoskeletal system during locomotion. In this study, we used computer optimization procedures to create a simulation of trotting in a mouse, using a previously developed mouse hindlimb musculoskeletal model in conjunction with new experimental data, allowing muscle forces, activation patterns, and levels of mechanical work to be estimated. Analyzing musculotendon unit (MTU) mechanical work throughout the stride allowed a deeper understanding of their respective functions, with the rectus femoris MTU dominating the generation of positive and negative mechanical work during the swing and stance phases. This analysis also tested previous functional inferences of the mouse hindlimb made from anatomical data alone, such as the existence of a proximo-distal gradient of muscle function, thought to reflect adaptations for energy-efficient locomotion. The results do not strongly support the presence of this gradient within the mouse musculoskeletal system, particularly given relatively high negative net work output from the ankle plantarflexor MTUs, although more detailed simulations could test this further. This modeling analysis lays a foundation for future studies of the control of vertebrate movement through the development of neuromechanical simulations. PMID:29868576

Anti-Aß immunotherapy in Alzheimer's disease; relevance of transgenic mouse studies to clinical trials

PubMed Central

Wilcock, Donna M.; Colton, Carol A.

2009-01-01

Therapeutic approaches to the treatment of Alzheimer's disease are focused primarily on the Aß peptide which aggregates to form amyloid deposits in the brain. The amyloid hypothesis states that amyloid is the precipitating factor that results in the other pathologies of Alzheimer's, namely neurofibrillary tangles and neurodegeneration, as well as the clinical dementia. One such therapy that has attracted significant attention is anti-Aß immunotherapy. First described in 1999, immunotherapy uses anti-Aß antibodies to lower brain amyloid levels. Active immunization, in which Aß is combined with an adjuvant to stimulate an immune response producing antibodies and passive immunization, in which antibodies are directly injected, were shown to lower brain amyloid levels and improve cognition in multiple transgenic mouse models. Mechanisms of action were studied in these mice and revealed a complex set of mechanisms that depended on the type of antibody used. When active immunization advanced to clinical trials a subset of patients developed meningoencephalitis; an event not predicted in mouse studies. However, it was suspected that a T-cell response due to the type of adjuvant used was the cause of the meningoencephalitis and studies in mice indicated alternative methods of vaccination. Passive immunization has also advanced to phase III clinical trials on the basis of successful transgenic mouse studies. Reports from the active immunization clinical trial indicated that, indeed, amyloid levels in brain were reduced. While APP transgenic mouse models are useful in studying amyloid pathology these mice do not generate significant tau pathology or neuron loss. Continued development of new mouse models that do generate all of these pathologies will be critical in more accurately testing therapeutics and predicting the clinical outcome of such therapeutics. PMID:19096156

Dysregulation of mitochondrial calcium signaling and superoxide flashes cause mitochondrial genomic DNA damage in Huntington disease.

PubMed

Wang, Jiu-Qiang; Chen, Qian; Wang, Xianhua; Wang, Qiao-Chu; Wang, Yun; Cheng, He-Ping; Guo, Caixia; Sun, Qinmiao; Chen, Quan; Tang, Tie-Shan

2013-02-01

Huntington disease (HD) is an inherited, fatal neurodegenerative disorder characterized by the progressive loss of striatal medium spiny neurons. Indications of oxidative stress are apparent in brain tissues from both HD patients and HD mouse models; however, the origin of this oxidant stress remains a mystery. Here, we used a yeast artificial chromosome transgenic mouse model of HD (YAC128) to investigate the potential connections between dysregulation of cytosolic Ca(2+) signaling and mitochondrial oxidative damage in HD cells. We found that YAC128 mouse embryonic fibroblasts exhibit a strikingly higher level of mitochondrial matrix Ca(2+) loading and elevated superoxide generation compared with WT cells, indicating that both mitochondrial Ca(2+) signaling and superoxide generation are dysregulated in HD cells. The excessive mitochondrial oxidant stress is critically dependent on mitochondrial Ca(2+) loading in HD cells, because blocking mitochondrial Ca(2+) uptake abolished elevated superoxide generation. Similar results were obtained using neurons from HD model mice and fibroblast cells from HD patients. More importantly, mitochondrial Ca(2+) loading in HD cells caused a 2-fold higher level of mitochondrial genomic DNA (mtDNA) damage due to the excessive oxidant generation. This study provides strong evidence to support a new causal link between dysregulated mitochondrial Ca(2+) signaling, elevated mitochondrial oxidant stress, and mtDNA damage in HD. Our results also indicate that reducing mitochondrial Ca(2+) uptake could be a therapeutic strategy for HD.

Novel mouse models of oculopharyngeal muscular dystrophy (OPMD) reveal early onset mitochondrial defects and suggest loss of PABPN1 may contribute to pathology.

PubMed

Vest, Katherine E; Phillips, Brittany L; Banerjee, Ayan; Apponi, Luciano H; Dammer, Eric B; Xu, Weiting; Zheng, Dinghai; Yu, Julia; Tian, Bin; Pavlath, Grace K; Corbett, Anita H

2017-09-01

Oculopharyngeal muscular dystrophy (OPMD) is a late onset disease caused by polyalanine expansion in the poly(A) binding protein nuclear 1 (PABPN1). Several mouse models have been generated to study OPMD; however, most of these models have employed transgenic overexpression of alanine-expanded PABPN1. These models do not recapitulate the OPMD patient genotype and PABPN1 overexpression could confound molecular phenotypes. We have developed a knock-in mouse model of OPMD (Pabpn1+/A17) that contains one alanine-expanded Pabpn1 allele under the control of the native promoter and one wild-type Pabpn1 allele. This mouse is the closest available genocopy of OPMD patients. We show that Pabpn1+/A17 mice have a mild myopathic phenotype in adult and aged animals. We examined early molecular and biochemical phenotypes associated with expressing native levels of A17-PABPN1 and detected shorter poly(A) tails, modest changes in poly(A) signal (PAS) usage, and evidence of mitochondrial damage in these mice. Recent studies have suggested that a loss of PABPN1 function could contribute to muscle pathology in OPMD. To investigate a loss of function model of pathology, we generated a heterozygous Pabpn1 knock-out mouse model (Pabpn1+/Δ). Like the Pabpn1+/A17 mice, Pabpn1+/Δ mice have mild histologic defects, shorter poly(A) tails, and evidence of mitochondrial damage. However, the phenotypes detected in Pabpn1+/Δ mice only partially overlap with those detected in Pabpn1+/A17 mice. These results suggest that loss of PABPN1 function could contribute to but may not completely explain the pathology detected in Pabpn1+/A17 mice. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Expression, function and regulation of mouse cytochrome P450 enzymes: comparison with human P450 enzymes.

PubMed

Hrycay, E G; Bandiera, S M

2009-12-01

The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

Mouse-based genetic modeling and analysis of Down syndrome

PubMed Central

Xing, Zhuo; Li, Yichen; Pao, Annie; Bennett, Abigail S.; Tycko, Benjamin; Mobley, William C.; Yu, Y. Eugene

2016-01-01

Introduction Down syndrome (DS), caused by human trisomy 21 (Ts21), can be considered as a prototypical model for understanding the effects of chromosomal aneuploidies in other diseases. Human chromosome 21 (Hsa21) is syntenically conserved with three regions in the mouse genome. Sources of data A review of recent advances in genetic modeling and analysis of DS. Using Cre/loxP-mediated chromosome engineering, a substantial number of new mouse models of DS have recently been generated, which facilitates better understanding of disease mechanisms in DS. Areas of agreement Based on evolutionary conservation, Ts21 can be modeled by engineered triplication of Hsa21 syntenic regions in mice. The validity of the models is supported by the exhibition of DS-related phenotypes. Areas of controversy Although substantial progress has been made, it remains a challenge to unravel the relative importance of specific candidate genes and molecular mechanisms underlying the various clinical phenotypes. Growing points Further understanding of mechanisms based on data from mouse models, in parallel with human studies, may lead to novel therapies for clinical manifestations of Ts21 and insights to the roles of aneuploidies in other developmental disorders and cancers. PMID:27789459

Molecular dissection of Norrie disease.

PubMed

Berger, W

1998-01-01

Norrie disease (ND) is a severe form of congenital blindness accompanied by mental retardation and/or deafness in at least one third of the patients. This article summarizes advances in the molecular genetic analysis of this disease during the last 13 years, including mapping and cloning of the human gene and the generation and characterization of a mouse model. Genetic linkage studies and physical mapping strategies have assigned the ND locus to the proximal short arm of the human X chromosome. The identification of chromosomal rearrangements in several patients, such as microdeletions, enabled the isolation of the ND gene by a positional cloning approach. Numerous point mutations in this gene have been identified in three distinct clinical entities: (1) ND, (2) familial and sporadic exudative vitreoretinopathy, and (3) retinopathy of prematurity. The gene encodes a relatively small protein, consisting of 133 amino acids. The function of the gene product is yet unknown, although homologies with known proteins and molecular modelling data suggest a role in the regulation of cell interaction or differentiation processes. A mouse model has been generated to shed more light on early pathogenic events involved in ND and allelic disorders. The mouse homologous protein is highly identical (94%) to the human polypeptide. The gene is expressed in the neuronal layers of the mouse retina, the cerebellum and olfactory epithelium. Mutant mice show snowflake-like opacities within the vitreous, dysgenesis of the ganglion cell layer and occasionally degeneration of photoreceptor cells. The mouse phenotype does not include phthisis bulbi and, overall, resembles a mild form of ND. Electrophysiological studies revealed a severely altered electroretinogram b-wave. These results suggest a primary defect in the inner neuronal layers of the retina. Defects in the vitreous and photoreceptor cell layer are most likely secondary effects. Further histological, functional and molecular studies of the mouse model are needed to provide additional information on disease associated pathways.

Translational animal models of autism and neurodevelopmental disorders.

PubMed

Crawley, Jacqueline N

2012-09-01

Autism is a neurodevelopmental disorder whose diagnosis is based on three behavioral criteria: unusual reciprocal social interactions, deficits in communication, and stereotyped repetitive behaviors with restricted interests. A large number of de novo single gene mutations and chromosomal deletions are associated with autism spectrum disorders. Based on the strong genetic evidence, mice with targeted mutations in homologous genes have been generated as translational research tools. Mouse models of autism have revealed behavioral and biological outcomes of mutations in risk genes. The field is now poised to employ the most robust phenotypes in the most replicable mouse models for preclinical screening of novel therapeutics.

Conditional Allele Mouse Planner (CAMP): software to facilitate the planning and design of breeding strategies involving mice with conditional alleles.

PubMed

Hoffert, Jason D; Pisitkun, Trairak; Miller, R Lance

2012-06-01

Transgenic and conditional knockout mouse models play an important role in biomedical research and their use has grown exponentially in the last 5-10 years. Generating conditional knockouts often requires breeding multiple alleles onto the background of a single mouse or group of mice. Breeding these mice depends on parental genotype, litter size, transmission frequency, and the number of breeding rounds. Therefore, a well planned breeding strategy is critical for keeping costs to a minimum. However, designing a viable breeding strategy can be challenging. With so many different variables this would be an ideal task for a computer program. To facilitate this process, we created a Java-based program called Conditional Allele Mouse Planner (CAMP). CAMP is designed to provide an estimate of the number of breeders, amount of time, and costs associated with generating mice of a particular genotype. We provide a description of CAMP, how to use it, and offer it freely as an application.

Mice Expressing RHAG and RHD Human Blood Group Genes

PubMed Central

Goossens, Dominique; da Silva, Nelly; Metral, Sylvain; Cortes, Ulrich; Callebaut, Isabelle; Picot, Julien; Mouro-Chanteloup, Isabelle; Cartron, Jean-Pierre

2013-01-01

Anti-RhD prophylaxis of haemolytic disease of the fetus and newborn (HDFN) is highly effective, but as the suppressive mechanism remains uncertain, a mouse model would be of interest. Here we have generated transgenic mice expressing human RhAG and RhD erythrocyte membrane proteins in the presence and, for human RhAG, in the absence, of mouse Rhag. Human RhAG associates with mouse Rh but not mouse Rhag on red blood cells. In Rhag knockout mice transgenic for human RHAG, the mouse Rh protein is “rescued” (re-expressed), and co-immunoprecipitates with human RhAG, indicating the presence of hetero-complexes which associate mouse and human proteins. RhD antigen was expressed from a human RHD gene on a BAC or from RHD cDNA under control of β-globin regulatory elements. RhD was never observed alone, strongly indicative that its expression absolutely depends on the presence of transgenic human RhAG. This first expression of RhD in mice is an important step in the creation of a mouse model of RhD allo-immunisation and HDFN, in conjunction with the Rh-Rhag knockout mice we have developed previously. PMID:24260394

Maternal Supply of Cas9 to Zygotes Facilitates the Efficient Generation of Site-Specific Mutant Mouse Models

PubMed Central

Cebrian-Serrano, Alberto; Zha, Shijun; Hanssen, Lars; Biggs, Daniel; Preece, Christopher

2017-01-01

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply. PMID:28081254

The Prx1 limb enhancers: targeted gene expression in developing zebrafish pectoral fins.

PubMed

Hernández-Vega, Amayra; Minguillón, Carolina

2011-08-01

Limbs represent an excellent model to study the induction, growth, and patterning of several organs. A breakthrough to study gene function in various tissues has been the characterization of regulatory elements that allow tissue-specific interference of gene function. The mouse Prx1 promoter has been used to generate limb-specific mutants and overexpress genes in tetrapod limbs. Although zebrafish possess advantages that favor their use to study limb morphogenesis, there is no driver described suitable for specifically interfering with gene function in developing fins. We report the generation of zebrafish lines that express enhanced green fluorescent protein (EGFP) driven by the mouse Prx1 enhancer in developing pectoral fins. We also describe the expression pattern of the zebrafish prrx1 genes and identify three conserved non-coding elements (CNEs) that we use to generate fin-specific EGFP reporter lines. Finally, we show that the mouse and zebrafish regulatory elements may be used to modify gene function in pectoral fins. Copyright © 2011 Wiley-Liss, Inc.

Astonishing advances in mouse genetic tools for biomedical research.

PubMed

Kaczmarczyk, Lech; Jackson, Walker S

2015-01-01

The humble house mouse has long been a workhorse model system in biomedical research. The technology for introducing site-specific genome modifications led to Nobel Prizes for its pioneers and opened a new era of mouse genetics. However, this technology was very time-consuming and technically demanding. As a result, many investigators continued to employ easier genome manipulation methods, though resulting models can suffer from overlooked or underestimated consequences. Another breakthrough, invaluable for the molecular dissection of disease mechanisms, was the invention of high-throughput methods to measure the expression of a plethora of genes in parallel. However, the use of samples containing material from multiple cell types could obfuscate data, and thus interpretations. In this review we highlight some important issues in experimental approaches using mouse models for biomedical research. We then discuss recent technological advances in mouse genetics that are revolutionising human disease research. Mouse genomes are now easily manipulated at precise locations thanks to guided endonucleases, such as transcription activator-like effector nucleases (TALENs) or the CRISPR/Cas9 system, both also having the potential to turn the dream of human gene therapy into reality. Newly developed methods of cell type-specific isolation of transcriptomes from crude tissue homogenates, followed by detection with next generation sequencing (NGS), are vastly improving gene regulation studies. Taken together, these amazing tools simplify the creation of much more accurate mouse models of human disease, and enable the extraction of hitherto unobtainable data.

Dissecting Alzheimer disease in Down syndrome using mouse models

PubMed Central

Choong, Xun Yu; Tosh, Justin L.; Pulford, Laura J.; Fisher, Elizabeth M. C.

2015-01-01

Down syndrome (DS) is a common genetic condition caused by the presence of three copies of chromosome 21 (trisomy 21). This greatly increases the risk of Alzheimer disease (AD), but although virtually all people with DS have AD neuropathology by 40 years of age, not all develop dementia. To dissect the genetic contribution of trisomy 21 to DS phenotypes including those relevant to AD, a range of DS mouse models has been generated which are trisomic for chromosome segments syntenic to human chromosome 21. Here, we consider key characteristics of human AD in DS (AD-DS), and our current state of knowledge on related phenotypes in AD and DS mouse models. We go on to review important features needed in future models of AD-DS, to understand this type of dementia and so highlight pathogenic mechanisms relevant to all populations at risk of AD. PMID:26528151

Dissecting Alzheimer disease in Down syndrome using mouse models.

PubMed

Choong, Xun Yu; Tosh, Justin L; Pulford, Laura J; Fisher, Elizabeth M C

2015-01-01

Down syndrome (DS) is a common genetic condition caused by the presence of three copies of chromosome 21 (trisomy 21). This greatly increases the risk of Alzheimer disease (AD), but although virtually all people with DS have AD neuropathology by 40 years of age, not all develop dementia. To dissect the genetic contribution of trisomy 21 to DS phenotypes including those relevant to AD, a range of DS mouse models has been generated which are trisomic for chromosome segments syntenic to human chromosome 21. Here, we consider key characteristics of human AD in DS (AD-DS), and our current state of knowledge on related phenotypes in AD and DS mouse models. We go on to review important features needed in future models of AD-DS, to understand this type of dementia and so highlight pathogenic mechanisms relevant to all populations at risk of AD.

Behavioral Phenotyping Assays for Genetic Mouse Models of Neurodevelopmental, Neurodegenerative, and Psychiatric Disorders.

PubMed

Sukoff Rizzo, Stacey J; Crawley, Jacqueline N

2017-02-08

Animal models offer heuristic research tools to understand the causes of human diseases and to identify potential treatments. With rapidly evolving genetic engineering technologies, mutations identified in a human disorder can be generated in the mouse genome. Phenotypic outcomes of the mutation are then explicated to confirm hypotheses about causes and to discover effective therapeutics. Most neurodevelopmental, neurodegenerative, and psychiatric disorders are diagnosed primarily by their prominent behavioral symptoms. Mouse behavioral assays analogous to the human symptoms have been developed to analyze the consequences of mutations and to evaluate proposed therapeutics preclinically. Here we describe the range of mouse behavioral tests available in the established behavioral neuroscience literature, along with examples of their translational applications. Concepts presented have been successfully used in other species, including flies, worms, fish, rats, pigs, and nonhuman primates. Identical strategies can be employed to test hypotheses about environmental causes and gene × environment interactions.

Hyperpolarized 13C pyruvate mouse brain metabolism with absorptive-mode EPSI at 1 T

NASA Astrophysics Data System (ADS)

Miloushev, Vesselin Z.; Di Gialleonardo, Valentina; Salamanca-Cardona, Lucia; Correa, Fabian; Granlund, Kristin L.; Keshari, Kayvan R.

2017-02-01

The expected signal in echo-planar spectroscopic imaging experiments was explicitly modeled jointly in spatial and spectral dimensions. Using this as a basis, absorptive-mode type detection can be achieved by appropriate choice of spectral delays and post-processing techniques. We discuss the effects of gradient imperfections and demonstrate the implementation of this sequence at low field (1.05 T), with application to hyperpolarized [1-13C] pyruvate imaging of the mouse brain. The sequence achieves sufficient signal-to-noise to monitor the conversion of hyperpolarized [1-13C] pyruvate to lactate in the mouse brain. Hyperpolarized pyruvate imaging of mouse brain metabolism using an absorptive-mode EPSI sequence can be applied to more sophisticated murine disease and treatment models. The simple modifications presented in this work, which permit absorptive-mode detection, are directly translatable to human clinical imaging and generate improved absorptive-mode spectra without the need for refocusing pulses.

The intramammary efficacy of first generation cephalosporins against Staphylococcus aureus mastitis in mice.

PubMed

Demon, Dieter; Ludwig, Carolin; Breyne, Koen; Guédé, David; Dörner, Julia-Charlotte; Froyman, Robrecht; Meyer, Evelyne

2012-11-09

Staphylococcus aureus-induced mastitis in cattle causes important financial losses in the dairy industry due to lower yield and bad milk quality. Although S. aureus is susceptible to many antimicrobials in vitro, treatment often fails to cure the infected udder. Hence, comprehensive evaluation of antimicrobials against S. aureus mastitis is desirable to direct treatment strategies. The mouse mastitis model is an elegant tool to evaluate antimicrobials in vivo while circumventing the high costs associated with bovine experiments. An evaluation of the antimicrobial efficacy of the intramammary (imam) applied first generation cephalosporins cefalexin, cefalonium, cefapirin and cefazolin, was performed using the S. aureus mouse mastitis model. In vivo determination of the effective dose 2log(10) (ED(2log10)), ED(4log10), protective dose 50 (PD(50)) and PD(100) in mouse mastitis studies, support that in vitro MIC data of the cephalosporins did not fully concur with the in vivo clinical outcome. Cefazolin was shown to be the most efficacious first generation cephalosporin to treat S. aureus mastitis whereas the MIC data indicate that cefalonium and cefapirin were more active in vitro. Changing the excipient for imam application from mineral oil to miglyol 812 further improved the antimicrobial efficacy of cefazolin, confirming that the excipient can influence the in vivo efficacy. Additionally, statistical analysis of the variation of S. aureus-infected, excipient-treated mice from fourteen studies emphasizes the strength of the mouse mastitis model as a fast, cost-effective and highly reproducible screening tool to assess the efficacy of antimicrobial compounds against intramammary S. aureus infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Mouse Models for Investigating the Developmental Bases of Human Birth Defects

PubMed Central

MOON, ANNE M.

2006-01-01

Clinicians and basic scientists share an interest in discovering how genetic or environmental factors interact to perturb normal development and cause birth defects and human disease. Given the complexity of such interactions, it is not surprising that 4% of human infants are born with a congenital malformation, and cardiovascular defects occur in nearly 1%. Our research is based on the fundamental hypothesis that an understanding of normal and abnormal development will permit us to generate effective strategies for both prevention and treatment of human birth defects. Animal models are invaluable in these efforts because they allow one to interrogate the genetic, molecular and cellular events that distinguish normal from abnormal development. Several features of the mouse make it a particularly powerful experimental model: it is a mammalian system with similar embryology, anatomy and physiology to humans; genes, proteins and regulatory programs are largely conserved between human and mouse; and finally, gene targeting in murine embryonic stem cells has made the mouse genome amenable to sophisticated genetic manipulation currently unavailable in any other model organism. PMID:16641221

eIF4E/Fmr1 double mutant mice display cognitive impairment in addition to ASD-like behaviors.

PubMed

Huynh, Thu N; Shah, Manan; Koo, So Yeon; Faraud, Kirsten S; Santini, Emanuela; Klann, Eric

2015-11-01

Autism spectrum disorder (ASD) is a group of heritable disorders with complex and unclear etiology. Classic ASD symptoms include social interaction and communication deficits as well as restricted, repetitive behaviors. In addition, ASD is often comorbid with intellectual disability. Fragile X syndrome (FXS) is the leading genetic cause of ASD, and is the most commonly inherited form of intellectual disability. Several mouse models of ASD and FXS exist, however the intellectual disability observed in ASD patients is not well modeled in mice. Using the Fmr1 knockout mouse and the eIF4E transgenic mouse, two previously characterized mouse models of fragile X syndrome and ASD, respectively, we generated the eIF4E/Fmr1 double mutant mouse. Our study shows that the eIF4E/Fmr1 double mutant mice display classic ASD behaviors, as well as cognitive dysfunction. Importantly, the learning impairments displayed by the double mutant mice spanned multiple cognitive tasks. Moreover, the eIF4E/Fmr1 double mutant mice display increased levels of basal protein synthesis. The results of our study suggest that the eIF4E/Fmr1 double mutant mouse may be a reliable model to study cognitive dysfunction in the context of ASD. Copyright © 2015 Elsevier Inc. All rights reserved.

Coherence in the Visual Imagination.

PubMed

Vertolli, Michael O; Kelly, Matthew A; Davies, Jim

2018-04-01

An incoherent visualization is when aspects of different senses of a word (e.g., the biological "mouse" vs. the computer "mouse") are present in the same visualization (e.g., a visualization of a biological mouse in the same image with a computer tower). We describe and implement a new model of creating contextual coherence in the visual imagination called Coherencer, based on the SOILIE model of imagination. We show that Coherencer is able to generate scene descriptions that are more coherent than SOILIE's original approach as well as a parallel connectionist algorithm that is considered competitive in the literature on general coherence. We also show that co-occurrence probabilities are a better association representation than holographic vectors and that better models of coherence improve the resulting output independent of the association type that is used. Theoretically, we show that Coherencer is consistent with other models of cognitive generation. In particular, Coherencer is a similar, but more cognitively plausible model than the C 3 model of concept combination created by Costello and Keane (2000). We show that Coherencer is also consistent with both the modal schematic indices of perceptual symbol systems theory (Barsalou, 1999) and the amodal contextual constraints of Thagard's (2002) theory of coherence. Finally, we describe how Coherencer is consistent with contemporary research on the hippocampus, and we show evidence that the process of making a visualization coherent is serial. Copyright © 2017 Cognitive Science Society, Inc.

Monitoring blood-flow in the mouse cochlea using an endoscopic laser speckle contrast imaging system

PubMed Central

Yu, Sunkon; Jung, Byungjo; Choi, Jin Sil

2018-01-01

Laser speckle contrast imaging (LSCI) enables continuous high-resolution assessment of microcirculation in real-time. We applied an endoscope to LSCI to measure cochlear blood-flow in an ischemia–reperfusion mouse model. We also explored whether using xenon light in combination with LSCI facilitates visualization of anatomical position. Based on a previous preliminary study, the appropriate wavelength for penetrating the thin bony cochlea was 830 nm. A 2.7-mm-diameter endoscope was used, as appropriate for the size of the mouse cochlea. Our endoscopic LSCI system was used to illuminate the right cochlea after dissection of the mouse. We observed changes in the speckle signals when we applied the endoscopic LSCI system to the ischemia-reperfusion mouse model. The anatomical structure of the mouse cochlea and surrounding structures were clearly visible using the xenon light. The speckle signal of the cochlea was scattered, with an intensity that varied between that of the stapes (with the lowest signal), the negative control, and the stapedial artery (with the highest signal), the positive control. In the cochlear ischemia–reperfusion mouse model, the speckle signal of the cochlea decreased during the ischemic phase, and increased during the reperfusion phase, clearly reflecting cochlear blood-flow. The endoscopic LSCI system generates high-resolution images in real-time, allowing visualization of blood-flow and its changes in the mouse cochlea. Anatomical structures were clearly matched using LSCI along with visible light. PMID:29489849

Monitoring blood-flow in the mouse cochlea using an endoscopic laser speckle contrast imaging system.

PubMed

Kong, Tae Hoon; Yu, Sunkon; Jung, Byungjo; Choi, Jin Sil; Seo, Young Joon

2018-01-01

Laser speckle contrast imaging (LSCI) enables continuous high-resolution assessment of microcirculation in real-time. We applied an endoscope to LSCI to measure cochlear blood-flow in an ischemia-reperfusion mouse model. We also explored whether using xenon light in combination with LSCI facilitates visualization of anatomical position. Based on a previous preliminary study, the appropriate wavelength for penetrating the thin bony cochlea was 830 nm. A 2.7-mm-diameter endoscope was used, as appropriate for the size of the mouse cochlea. Our endoscopic LSCI system was used to illuminate the right cochlea after dissection of the mouse. We observed changes in the speckle signals when we applied the endoscopic LSCI system to the ischemia-reperfusion mouse model. The anatomical structure of the mouse cochlea and surrounding structures were clearly visible using the xenon light. The speckle signal of the cochlea was scattered, with an intensity that varied between that of the stapes (with the lowest signal), the negative control, and the stapedial artery (with the highest signal), the positive control. In the cochlear ischemia-reperfusion mouse model, the speckle signal of the cochlea decreased during the ischemic phase, and increased during the reperfusion phase, clearly reflecting cochlear blood-flow. The endoscopic LSCI system generates high-resolution images in real-time, allowing visualization of blood-flow and its changes in the mouse cochlea. Anatomical structures were clearly matched using LSCI along with visible light.

Nucleotide excision repair deficient mouse models and neurological disease

PubMed Central

Niedernhofer, Laura J.

2008-01-01

Nucleotide excision repair (NER) is a highly conserved mechanism to remove helix-distorting DNA base damage. A major substrate for NER is DNA damage caused by environmental genotoxins, most notably ultraviolet radiation. Xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy are three human diseases caused by inherited defects in NER. The symptoms and severity of these diseases vary dramatically, ranging from profound developmental delay to cancer predisposition and accelerated aging. All three syndromes include neurological disease, indicating an important role for NER in protecting against spontaneous DNA damage as well. To study the pathophysiology caused by DNA damage, numerous mouse models of NER deficiency were generated by knocking-out genes required for NER or knocking-in disease-causing human mutations. This review explores the utility of these mouse models to study neurological disease caused by NER deficiency. PMID:18272436

CRISPR/Cas9-Mediated Insertion of loxP Sites in the Mouse Dock7 Gene Provides an Effective Alternative to Use of Targeted Embryonic Stem Cells.

PubMed

Bishop, Kathleen A; Harrington, Anne; Kouranova, Evguenia; Weinstein, Edward J; Rosen, Clifford J; Cui, Xiaoxia; Liaw, Lucy

2016-07-07

Targeted gene mutation in the mouse is a primary strategy to understand gene function and relation to phenotype. The Knockout Mouse Project (KOMP) had an initial goal to develop a public resource of mouse embryonic stem (ES) cell clones that carry null mutations in all genes. Indeed, many useful novel mouse models have been generated from publically accessible targeted mouse ES cell lines. However, there are limitations, including incorrect targeting or cassette structure, and difficulties with germline transmission of the allele from chimeric mice. In our experience, using a small sample of targeted ES cell clones, we were successful ∼50% of the time in generating germline transmission of a correctly targeted allele. With the advent of CRISPR/Cas9 as a mouse genome modification tool, we assessed the efficiency of creating a conditional targeted allele in one gene, dedicator of cytokinesis 7 (Dock7), for which we were unsuccessful in generating a null allele using a KOMP targeted ES cell clone. The strategy was to insert loxP sites to flank either exons 3 and 4, or exons 3 through 7. By coinjecting Cas9 mRNA, validated sgRNAs, and oligonucleotide donors into fertilized eggs from C57BL/6J mice, we obtained a variety of alleles, including mice homozygous for the null alleles mediated by nonhomologous end joining, alleles with one of the two desired loxP sites, and correctly targeted alleles with both loxP sites. We also found frequent mutations in the inserted loxP sequence, which is partly attributable to the heterogeneity in the original oligonucleotide preparation. Copyright © 2016 Bishop et al.

New Rodent Population Models May Inform Human Health Risk Assessment and Identification of Genetic Susceptibility to Environmental Exposures.

PubMed

Harrill, Alison H; McAllister, Kimberly A

2017-08-15

This paper provides an introduction for environmental health scientists to emerging population-based rodent resources. Mouse reference populations provide an opportunity to model environmental exposures and gene-environment interactions in human disease and to inform human health risk assessment. This review will describe several mouse populations for toxicity assessment, including older models such as the Mouse Diversity Panel (MDP), and newer models that include the Collaborative Cross (CC) and Diversity Outbred (DO) models. This review will outline the features of the MDP, CC, and DO mouse models and will discuss published case studies investigating the use of these mouse population resources in each step of the risk assessment paradigm. These unique resources have the potential to be powerful tools for generating hypotheses related to gene-environment interplay in human disease, performing controlled exposure studies to understand the differential responses in humans for susceptibility or resistance to environmental exposures, and identifying gene variants that influence sensitivity to toxicity and disease states. These new resources offer substantial advances to classical toxicity testing paradigms by including genetically sensitive individuals that may inform toxicity risks for sensitive subpopulations. Both in vivo and complementary in vitro resources provide platforms with which to reduce uncertainty by providing population-level data around biological variability. https://doi.org/10.1289/EHP1274.

New Rodent Population Models May Inform Human Health Risk Assessment and Identification of Genetic Susceptibility to Environmental Exposures

PubMed Central

Harrill, Alison H.

2017-01-01

Background: This paper provides an introduction for environmental health scientists to emerging population-based rodent resources. Mouse reference populations provide an opportunity to model environmental exposures and gene–environment interactions in human disease and to inform human health risk assessment. Objectives: This review will describe several mouse populations for toxicity assessment, including older models such as the Mouse Diversity Panel (MDP), and newer models that include the Collaborative Cross (CC) and Diversity Outbred (DO) models. Methods: This review will outline the features of the MDP, CC, and DO mouse models and will discuss published case studies investigating the use of these mouse population resources in each step of the risk assessment paradigm. Discussion: These unique resources have the potential to be powerful tools for generating hypotheses related to gene–environment interplay in human disease, performing controlled exposure studies to understand the differential responses in humans for susceptibility or resistance to environmental exposures, and identifying gene variants that influence sensitivity to toxicity and disease states. Conclusions: These new resources offer substantial advances to classical toxicity testing paradigms by including genetically sensitive individuals that may inform toxicity risks for sensitive subpopulations. Both in vivo and complementary in vitro resources provide platforms with which to reduce uncertainty by providing population-level data around biological variability. https://doi.org/10.1289/EHP1274 PMID:28886592

Generation of the first Autosomal Dominant Osteopetrosis Type II (ADO2) disease models

PubMed Central

Alam, Imranul; Gray, Amie K.; Chu, Kang; Ichikawa, Shoji; Mohammad, Khalid S.; Capannolo, Marta; Capulli, Mattia; Maurizi, Antonio; Muraca, Maurizio; Teti, Anna; Econs, Michael J.; Fattore, Andrea Del

2013-01-01

Autosomal Dominant Osteopetrosis Type II (ADO2) is a heritable osteosclerotic disorder dependent on osteoclast impairment. In most patients it results from heterozygous missense mutations in the chloride channel 7 (CLCN7) gene, encoding for a 2Cl−/1H+ antiporter. By a knock-in strategy inserting a missense mutation in the Clcn7 gene, our two research groups independently generated mouse models of ADO2 on different genetic backgrounds carrying the homolog of the most frequent heterozygous mutation (p.G213R) in the Clcn7 gene found in humans. Our results demonstrate that the heterozygous model holds true presenting with higher bone mass, increased numbers of poorly resorbing osteoclasts and a lethal phenotype in the homozygous state. Considerable variability is observed in the heterozygous mice according with the mouse background, suggesting that modifier genes could influence the penetrance of the disease gene. PMID:24185277

Automated evaluation of liver fibrosis in thioacetamide, carbon tetrachloride, and bile duct ligation rodent models using second-harmonic generation/two-photon excited fluorescence microscopy.

PubMed

Liu, Feng; Chen, Long; Rao, Hui-Ying; Teng, Xiao; Ren, Ya-Yun; Lu, Yan-Qiang; Zhang, Wei; Wu, Nan; Liu, Fang-Fang; Wei, Lai

2017-01-01

Animal models provide a useful platform for developing and testing new drugs to treat liver fibrosis. Accordingly, we developed a novel automated system to evaluate liver fibrosis in rodent models. This system uses second-harmonic generation (SHG)/two-photon excited fluorescence (TPEF) microscopy to assess a total of four mouse and rat models, using chemical treatment with either thioacetamide (TAA) or carbon tetrachloride (CCl 4 ), and a surgical method, bile duct ligation (BDL). The results obtained by the new technique were compared with that using Ishak fibrosis scores and two currently used quantitative methods for determining liver fibrosis: the collagen proportionate area (CPA) and measurement of hydroxyproline (HYP) content. We show that 11 shared morphological parameters faithfully recapitulate Ishak fibrosis scores in the models, with high area under the receiver operating characteristic (ROC) curve (AUC) performance. The AUC values of 11 shared parameters were greater than that of the CPA (TAA: 0.758-0.922 vs 0.752-0.908; BDL: 0.874-0.989 vs 0.678-0.966) in the TAA mice and BDL rat models and similar to that of the CPA in the TAA rat and CCl 4 mouse models. Similarly, based on the trends in these parameters at different time points, 9, 10, 7, and 2 model-specific parameters were selected for the TAA rats, TAA mice, CCl 4 mice, and BDL rats, respectively. These parameters identified differences among the time points in the four models, with high AUC accuracy, and the corresponding AUC values of these parameters were greater compared with those of the CPA in the TAA rat and mouse models (rats: 0.769-0.894 vs 0.64-0.799; mice: 0.87-0.93 vs 0.739-0.836) and similar to those of the CPA in the CCl 4 mouse and BDL rat models. Similarly, the AUC values of 11 shared parameters and model-specific parameters were greater than those of HYP in the TAA rats, TAA mice, and CCl 4 mouse models and were similar to those of HYP in the BDL rat models. The automated evaluation system, combined with 11 shared parameters and model-specific parameters, could specifically, accurately, and quantitatively stage liver fibrosis in animal models.

Generation and Characterization of Transgenic Mice Expressing Mouse Ins1 Promoter for Pancreatic β-Cell-Specific Gene Overexpression and Knockout.

PubMed

Cheng, Yulong; Su, Yutong; Shan, Aijing; Jiang, Xiuli; Ma, Qinyun; Wang, Weiqing; Ning, Guang; Cao, Yanan

2015-07-01

The technologies for pancreatic β-cell-specific gene overexpression or knockout are fundamental for investigations of functional genes in vivo. Here we generated the Ins1-Cre-Dsred and Ins1-rtTA mouse models, which expressed the Cre recombinase or reverse tetracycline regulatable transactivator (rtTA) without hGH minigene under the control of mouse Ins1 promoter. Our data showed that the Cre-mediated recombination and rtTA-mediated activation could be efficiently detected at embryonic day 13.5 when these models were crossed with the reporter mice (ROSA(mT/mG) or tetO-HIST1H2BJ/GFP). The Cre and rtTA expression was restricted to β-cells without leakage in the brain and other tissues. Moreover, both the transgenic lines showed normal glucose tolerance and insulin secretion. These results suggested that the Ins1-Cre-Dsred and Ins1-rtTA mice could be used to knock out or overexpress target genes in embryos and adults to facilitate β-cell researches.

Modeling anaplastic thyroid carcinoma in the mouse.

PubMed

Champa, Devora; Di Cristofano, Antonio

2015-02-01

Anaplastic thyroid carcinoma is the least common form of thyroid cancer; however, it accounts for the majority of deaths associated with this family of malignancies. A number of genetically engineered immunocompetent mouse models recapitulating the genetic and histological features of anaplastic thyroid cancer have been very recently generated and represent an invaluable tool to dissect the mechanisms involved in the progression from indolent, well-differentiated tumors to aggressive, undifferentiated carcinomas and to identify novel therapeutic targets. In this review, we focus on the relevant characteristics associated with these models and on what we have learned in terms of anaplastic thyroid cancer biology, genetics, and response to targeted therapy.

Modeling anaplastic thyroid carcinoma in the mouse

PubMed Central

Champa, Devora; Di Cristofano, Antonio

2014-01-01

Anaplastic thyroid carcinoma is the least common form of thyroid cancer; however, it accounts for the majority of deaths associated with this family of malignancies. A number of genetically engineered immunocompetent mouse models recapitulating the genetic and histological features of anaplastic thyroid cancer have been very recently generated and represent an invaluable tool to dissect the mechanisms involved in the progression from indolent, well differentiated tumors to aggressive, undifferentiated carcinomas, and to identify novel therapeutic targets. In this review, we focus on the relevant characteristics associated with these models and on what we have learned in terms of anaplastic thyroid cancer biology, genetics, and response to targeted therapy. PMID:25420535

Translational animal models of autism and neurodevelopmental disorders

PubMed Central

Crawley, Jacqueline N.

2012-01-01

Autism is a neurodevelopmental disorder whose diagnosis is based on three behavioral criteria: unusual reciprocal social interactions, deficits in communication, and stereotyped repetitive behaviors with restricted interests. A large number of de novo single gene mutations and chromosomal deletions are associated with autism spectrum disorders. Based on the strong genetic evidence, mice with targeted mutations in homologous genes have been generated as translational research tools. Mouse models of autism have revealed behavioral and biological outcomes of mutations in risk genes. The field is now poised to employ the most robust phenotypes in the most replicable mouse models for preclinical screening of novel therapeutics. PMID:23226954

Pain, motor and gait assessment of murine osteoarthritis in a cruciate ligament transection model

PubMed Central

Ruan, Merry ZC; Patel, Ronak M; Dawson, Brian C; Jiang, Ming-ming; Lee, Brendan HI

2013-01-01

Objective The major complaint of Osteoarthritis (OA) patients is pain. However, due to the nature of clinical studies and the limitation of animal studies, few studies have linked function impairment and behavioral changes in OA animal models to cartilage loss and histopathology. Our objective was to study surrogate markers of functional impairment in relation to cartilage loss and pathological changes in a post-traumatic mouse model of OA. Method We performed a battery of functional analyses in a mouse model of OA generated by cruciate ligament transection (CLT). The changes in functional analyses were linked to histological changes graded by OARSI standards, histological grading of synovitis, and volumetric changes of the articular cartilage and osteophytes quantified by phase contrast micro-CT. Results OA generated by CLT led to decreased time on rotarod, delayed response on hotplate analysis, and altered gait starting from 4 weeks after surgery. Activity in open field analysis did not change at 4, 8, or 12 weeks after CLT. The magnitude of behavioral changes was directly correlated with higher OARSI histological scores of OA, synovitis in the knee joints, cartilage volume loss, and osteophyte formation. Conclusion Our findings link functional analyses to histological grading, synovitis, comprehensive 3-dimensional assessment of cartilage volume and osteophyte formation. This serves as a reference for a mouse model in predicting outcomes of OA treatment. PMID:23973150

A Humanized Mouse Model Generated Using Surplus Neonatal Tissue.

PubMed

Brown, Matthew E; Zhou, Ying; McIntosh, Brian E; Norman, Ian G; Lou, Hannah E; Biermann, Mitch; Sullivan, Jeremy A; Kamp, Timothy J; Thomson, James A; Anagnostopoulos, Petros V; Burlingham, William J

2018-04-10

Here, we describe the NeoThy humanized mouse model created using non-fetal human tissue sources, cryopreserved neonatal thymus and umbilical cord blood hematopoietic stem cells (HSCs). Conventional humanized mouse models are made by engrafting human fetal thymus and HSCs into immunocompromised mice. These mice harbor functional human T cells that have matured in the presence of human self-peptides and human leukocyte antigen molecules. Neonatal thymus tissue is more abundant and developmentally mature and allows for creation of up to ∼50-fold more mice per donor compared with fetal tissue models. The NeoThy has equivalent frequencies of engrafted human immune cells compared with fetal tissue humanized mice and exhibits T cell function in assays of ex vivo cell proliferation, interferon γ secretion, and in vivo graft infiltration. The NeoThy model may provide significant advantages for induced pluripotent stem cell immunogenicity studies, while bypassing the requirement for fetal tissue. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Zika viral infection and neutralizing human antibody response in a BLT humanized mouse model.

PubMed

Schmitt, Kimberly; Charlins, Paige; Veselinovic, Milena; Kinner-Bibeau, Lauren; Hu, Shuang; Curlin, James; Remling-Mulder, Leila; Olson, Ken E; Aboellail, Tawfik; Akkina, Ramesh

2018-02-01

Many murine and non-human primate animal models have been recently developed to understand Zika viral pathogenesis. However, a major limitation with these models is the inability to directly examine the human-specific immune response. Here, we utilized a BLT humanized mouse model endowed with a transplanted human immune system. Plasma viremia could be detected within 48h after viral challenge and viremia persisted for as long as 220 days in some mice. Neutralizing human antibody was detected in infected mice and mouse sera showed reactivity with the viral envelope and capsid proteins in a radio-immunoprecipitation assay. Human monocytes/macrophages, B cells and hematopoietic stem cells in the bone marrow were found to be virus infected. These data establish that BLT mice are permissive for Zika viral infection and are capable of generating viral-specific human immune responses thus providing a human surrogate model for future testing of vaccine and antiviral therapeutic candidates. Copyright © 2017 Elsevier Inc. All rights reserved.

Whole transcriptome profiling of patient-derived xenograft models as a tool to identify both tumor and stromal specific biomarkers.

PubMed

Bradford, James R; Wappett, Mark; Beran, Garry; Logie, Armelle; Delpuech, Oona; Brown, Henry; Boros, Joanna; Camp, Nicola J; McEwen, Robert; Mazzola, Anne Marie; D'Cruz, Celina; Barry, Simon T

2016-04-12

The tumor microenvironment is emerging as a key regulator of cancer growth and progression, however the exact mechanisms of interaction with the tumor are poorly understood. Whilst the majority of genomic profiling efforts thus far have focused on the tumor, here we investigate RNA-Seq as a hypothesis-free tool to generate independent tumor and stromal biomarkers, and explore tumor-stroma interactions by exploiting the human-murine compartment specificity of patient-derived xenografts (PDX).Across a pan-cancer cohort of 79 PDX models, we determine that mouse stroma can be separated into distinct clusters, each corresponding to a specific stromal cell type. This implies heterogeneous recruitment of mouse stroma to the xenograft independent of tumor type. We then generate cross-species expression networks to recapitulate a known association between tumor epithelial cells and fibroblast activation, and propose a potentially novel relationship between two hypoxia-associated genes, human MIF and mouse Ddx6. Assessment of disease subtype also reveals MMP12 as a putative stromal marker of triple-negative breast cancer. Finally, we establish that our ability to dissect recruited stroma from trans-differentiated tumor cells is crucial to identifying stem-like poor-prognosis signatures in the tumor compartment.In conclusion, RNA-Seq is a powerful, cost-effective solution to global analysis of human tumor and mouse stroma simultaneously, providing new insights into mouse stromal heterogeneity and compartment-specific disease markers that are otherwise overlooked by alternative technologies. The study represents the first comprehensive analysis of its kind across multiple PDX models, and supports adoption of the approach in pre-clinical drug efficacy studies, and compartment-specific biomarker discovery.

Phenotypic screening of hepatocyte nuclear factor (HNF) 4-{gamma} receptor knockout mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gerdin, Anna Karin; Surve, Vikas V.; Joensson, Marie

2006-10-20

Using the mouse as a model organism in pharmaceutical research presents unique advantages as its physiology in many ways resembles the human physiology, it also has a relatively short generation time, low breeding and maintenance costs, and is available in a wide variety of inbred strains. The ability to genetically modify mouse embryonic stem cells to generate mouse models that better mimic human disease is another advantage. In the present study, a comprehensive phenotypic screening protocol is applied to elucidate the phenotype of a novel mouse knockout model of hepatocyte nuclear factor (HNF) 4-{gamma}. HNF4-{gamma} is expressed in the kidneys,more » gut, pancreas, and testis. First level of the screen is aimed at general health, morphologic appearance, normal cage behaviour, and gross neurological functions. The second level of the screen looks at metabolic characteristics and lung function. The third level of the screen investigates behaviour more in-depth and the fourth level consists of a thorough pathological characterisation, blood chemistry, haematology, and bone marrow analysis. When compared with littermate wild-type mice (HNF4-{gamma}{sup +/+}), the HNF4-{gamma} knockout (HNF4-{gamma}{sup -/-}) mice had lowered energy expenditure and locomotor activity during night time that resulted in a higher body weight despite having reduced intake of food and water. HNF4-{gamma}{sup -/-} mice were less inclined to build nest and were found to spend more time in a passive state during the forced swim test.« less

Transgenic mouse model harboring the transcriptional fusion ccl20-luciferase as a novel reporter of pro-inflammatory response.

PubMed

Crispo, Martina; Van Maele, Laurye; Tabareau, Julien; Cayet, Delphine; Errea, Agustina; Ferreira, Ana María; Rumbo, Martin; Sirard, Jean Claude

2013-01-01

The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo.

Transgenic Mouse Model Harboring the Transcriptional Fusion Ccl20-Luciferase as a Novel Reporter of Pro-Inflammatory Response

PubMed Central

Crispo, Martina; Van Maele, Laurye; Tabareau, Julien; Cayet, Delphine; Errea, Agustina; Ferreira, Ana María; Rumbo, Martin; Sirard, Jean Claude

2013-01-01

The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo. PMID:24265691

Design and analysis of sustainable computer mouse using design for disassembly methodology

NASA Astrophysics Data System (ADS)

Roni Sahroni, Taufik; Fitri Sukarman, Ahmad; Agung Mahardini, Karunia

2017-12-01

This paper presents the design and analysis of computer mouse using Design for Disassembly methodology. Basically, the existing computer mouse model consist a number of unnecessary part that cause the assembly and disassembly time in production. The objective of this project is to design a new computer mouse based on Design for Disassembly (DFD) methodology. The main methodology of this paper was proposed from sketch generation, concept selection, and concept scoring. Based on the design screening, design concept B was selected for further analysis. New design of computer mouse is proposed using fastening system. Furthermore, three materials of ABS, Polycarbonate, and PE high density were prepared to determine the environmental impact category. Sustainable analysis was conducted using software SolidWorks. As a result, PE High Density gives the lowers amount in the environmental category with great maximum stress value.

Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.

PubMed

Quadros, Rolen M; Miura, Hiromi; Harms, Donald W; Akatsuka, Hisako; Sato, Takehito; Aida, Tomomi; Redder, Ronald; Richardson, Guy P; Inagaki, Yutaka; Sakai, Daisuke; Buckley, Shannon M; Seshacharyulu, Parthasarathy; Batra, Surinder K; Behlke, Mark A; Zeiner, Sarah A; Jacobi, Ashley M; Izu, Yayoi; Thoreson, Wallace B; Urness, Lisa D; Mansour, Suzanne L; Ohtsuka, Masato; Gurumurthy, Channabasavaiah B

2017-05-17

Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient. Here we describe Easi-CRISPR (Efficient additions with ssDNA inserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi-CRISPR generates correctly targeted conditional and insertion alleles in 8.5-100% of the resulting live offspring. Easi-CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi-CRISPR offers a comprehensive means of building large-scale Cre-LoxP animal resources.

Use of “MGE Enhancers” for Labeling and Selection of Embryonic Stem Cell-Derived Medial Ganglionic Eminence (MGE) Progenitors and Neurons

PubMed Central

Chen, Ying-Jiun J.; Vogt, Daniel; Wang, Yanling; Visel, Axel; Silberberg, Shanni N.; Nicholas, Cory R.; Danjo, Teruko; Pollack, Joshua L.; Pennacchio, Len A.; Anderson, Stewart; Sasai, Yoshiki; Baraban, Scott C.; Kriegstein, Arnold R.; Alvarez-Buylla, Arturo; Rubenstein, John L. R.

2013-01-01

The medial ganglionic eminence (MGE) is an embryonic forebrain structure that generates the majority of cortical interneurons. MGE transplantation into specific regions of the postnatal central nervous system modifies circuit function and improves deficits in mouse models of epilepsy, Parkinson's disease, pain, and phencyclidine-induced cognitive deficits. Herein, we describe approaches to generate MGE-like progenitor cells from mouse embryonic stem (ES) cells. Using a modified embryoid body method, we provided gene expression evidence that mouse ES-derived Lhx6+ cells closely resemble immature interneurons generated from authentic MGE-derived Lhx6+ cells. We hypothesized that enhancers that are active in the mouse MGE would be useful tools in detecting when ES cells differentiate into MGE cells. Here we demonstrate the utility of enhancer elements [422 (DlxI12b), Lhx6, 692, 1056, and 1538] as tools to mark MGE-like cells in ES cell differentiation experiments. We found that enhancers DlxI12b, 692, and 1538 are active in Lhx6-GFP+ cells, while enhancer 1056 is active in Olig2+ cells. These data demonstrate unique techniques to follow and purify MGE-like derivatives from ES cells, including GABAergic cortical interneurons and oligodendrocytes, for use in stem cell-based therapeutic assays and treatments. PMID:23658702

Rett syndrome treatment in mouse models: searching for effective targets and strategies.

PubMed

Ricceri, Laura; De Filippis, Bianca; Laviola, Giovanni

2013-05-01

Rett syndrome (RTT) is a pervasive developmental disorder, primarily affecting girls with a prevalence of 1 in every 10,000 births; it represents the second most common cause of intellectual disability in females. Mutations in the gene encoding methyl-CpG-binding protein 2 (MECP2) have been identified as clear etiological factors in more than 90% of classical RTT cases. Whereas the mechanisms leading to the severe, progressive and specific neurological dysfunctions when this gene is mutated still remain to be elucidated, a series of different mouse models have been generated, bearing different Mecp2 mutation. Neurobehavioural analysis in these mouse lines have been carried out and phenotyping analysis can be now utilised to preclinically evaluate the effects of potential RTT treatments. This review summarizes the different results achieved in this research field taking into account different key targets identified to ameliorate RTT phenotype in mouse models, including those not directly downstream of MeCP2 and those limited to the early phases of postnatal development. This article is part of the Special Issue entitled 'Neurodevelopmental Disorders'. Copyright © 2012 Elsevier Ltd. All rights reserved.

Skeletal Characterization of the Fgfr3 Mouse Model of Achondroplasia Using Micro-CT and MRI Volumetric Imaging.

PubMed

Shazeeb, Mohammed Salman; Cox, Megan K; Gupta, Anurag; Tang, Wen; Singh, Kuldeep; Pryce, Cynthia T; Fogle, Robert; Mu, Ying; Weber, William D; Bangari, Dinesh S; Ying, Xiaoyou; Sabbagh, Yves

2018-01-11

Achondroplasia, the most common form of dwarfism, affects more than a quarter million people worldwide and remains an unmet medical need. Achondroplasia is caused by mutations in the fibroblast growth factor receptor 3 (FGFR3) gene which results in over-activation of the receptor, interfering with normal skeletal development leading to disproportional short stature. Multiple mouse models have been generated to study achondroplasia. The characterization of these preclinical models has been primarily done with 2D measurements. In this study, we explored the transgenic model expressing mouse Fgfr3 containing the achondroplasia mutation G380R under the Col2 promoter (Ach). Survival and growth rate of the Ach mice were reduced compared to wild-type (WT) littermates. Axial skeletal defects and abnormalities of the sternebrae and vertebrae were observed in the Ach mice. Further evaluation of the Ach mouse model was performed by developing 3D parameters from micro-computed tomography (micro-CT) and magnetic resonance imaging (MRI). The 3-week-old mice showed greater differences between the Ach and WT groups compared to the 6-week-old mice for all parameters. Deeper understanding of skeletal abnormalities of this model will help guide future studies for evaluating novel and effective therapeutic approaches for the treatment of achondroplasia.

A Chimeric Humanized Mouse Model by Engrafting the Human Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cell for the Chronic Hepatitis B Virus Infection

PubMed Central

Yuan, Lunzhi; Liu, Xuan; Zhang, Liang; Li, Xiaoling; Zhang, Yali; Wu, Kun; Chen, Yao; Cao, Jiali; Hou, Wangheng; Zhang, Jun; Zhu, Hua; Yuan, Quan; Tang, Qiyi; Cheng, Tong; Xia, Ningshao

2018-01-01

Humanized mouse model generated by grafting primary human hepatocytes (PHHs) to immunodeficient mouse has contributed invaluably to understanding the pathogenesis of hepatitis B virus (HBV). However, the source of PHHs is limited, which necessitates the search for alternatives. Recently, hepatocyte-like cells (HLCs) generated from human induced pluripotent stem cells (hiPSCs) have been used for in vitro HBV infection. Herein, we developed a robust human liver chimeric animal model to study in vivo HBV infection by engrafting the hiPSC-HLCs to Fah-/-Rag2-/-IL-2Rγc-/- SCID (FRGS) mice. After being optimized by a small molecule, XMU-MP-1, the hiPSC-HLCs engrafted FRGS (hHLC-FRGS) mice displayed approximately 40% liver chimerism at week 6 after engraftment and maintained at this level for at least 14 weeks. Viremia and HBV infection markers include antigens, RNA, DNA, and covalently closed circular DNA were detectable in HBV infected hHLC-FRGS mice. Furthermore, hiPSC-HLCs and hHLC-FRGS mice were successfully used to evaluate different antivirals. Therefore, we established a humanized mouse model for not only investigating HBV pathogenesis but also testing the effects of the anti-HBV drugs. Highlights:    (1) The implanted hiPSC-HLCs established a long-term chimerism in FRGS mice liver.    (2) hHLC-FRGS mice are adequate to support chronic HBV infection with a full viral life cycle.    (3) hiPSC-HLCs and hHLC-FRGS mice are useful tools for evaluation of antivirals against HBV infection in vitro and in vivo. Research in Context  To overcome the disadvantages of using primary human hepatocytes, we induced human pluripotent stem cells to hepatocyte-like cells (hiPSC-HLCs) that developed the capability to express important liver functional markers and critical host factors for HBV infection. The hiPSC-HLCs were permissive for the HBV infection and supported a full HBV replication. The hiPSC-HLCs were then engrafted to immunodeficient mouse to establish a chimeric liver mouse model, which was capable of supporting HBV infection in vivo and evaluating the effects of antiviral drugs. Our results shed light into improving the cellular and animal models for studying HBV and other hepatotropic viruses. PMID:29867819

Absence of Prenatal Forebrain Defects in the Dp(16)1Yey/+ Mouse Model of Down Syndrome

PubMed Central

Goodliffe, Joseph W.; Olmos-Serrano, Jose Luis; Aziz, Nadine M.; Pennings, Jeroen L.A.; Guedj, Faycal; Bianchi, Diana W.

2016-01-01

Studies in humans with Down syndrome (DS) show that alterations in fetal brain development are followed by postnatal deficits in neuronal numbers, synaptic plasticity, and cognitive and motor function. This same progression is replicated in several mouse models of DS. Dp(16)1Yey/+ (hereafter called Dp16) is a recently developed mouse model of DS in which the entire region of mouse chromosome 16 that is homologous to human chromosome 21 has been triplicated. As such, Dp16 mice may more closely reproduce neurodevelopmental changes occurring in humans with DS. Here, we present the first comprehensive cellular and behavioral study of the Dp16 forebrain from embryonic to adult stages. Unexpectedly, our results demonstrate that Dp16 mice do not have prenatal brain defects previously reported in human fetal neocortex and in the developing forebrains of other mouse models, including microcephaly, reduced neurogenesis, and abnormal cell proliferation. Nevertheless, we found impairments in postnatal developmental milestones, fewer inhibitory forebrain neurons, and deficits in motor and cognitive performance in Dp16 mice. Therefore, although this new model does not express prenatal morphological phenotypes associated with DS, abnormalities in the postnatal period appear sufficient to produce significant cognitive deficits in Dp16. SIGNIFICANCE STATEMENT Down syndrome (DS) leads to intellectual disability. Several mouse models have increased our understanding of the neuropathology of DS and are currently being used to test therapeutic strategies. A new mouse model that contains an expanded number of DS-related genes, known as Dp(16)1Yey/+ (Dp16), has been generated recently. We sought to determine whether the extended triplication creates a better phenocopy of DS-related brain pathologies. We measured embryonic development, forebrain maturation, and perinatal/adult behavior and revealed an absence of prenatal phenotypes in Dp16 fetal brain, but specific cellular and behavioral deficits after the first 2 postnatal weeks. These results uncover important differences in prenatal phenotype between Dp16 animals and humans with DS and other DS mouse models. PMID:26961948

Direct production of mouse disease models by embryo microinjection of TALENs and oligodeoxynucleotides

PubMed Central

Wefers, Benedikt; Meyer, Melanie; Ortiz, Oskar; Hrabé de Angelis, Martin; Hansen, Jens; Wurst, Wolfgang; Kühn, Ralf

2013-01-01

The study of genetic disease mechanisms relies mostly on targeted mouse mutants that are derived from engineered embryonic stem (ES) cells. Nevertheless, the establishment of mutant ES cells is laborious and time-consuming, restricting the study of the increasing number of human disease mutations discovered by high-throughput genomic analysis. Here, we present an advanced approach for the production of mouse disease models by microinjection of transcription activator-like effector nucleases (TALENs) and synthetic oligodeoxynucleotides into one-cell embryos. Within 2 d of embryo injection, we created and corrected chocolate missense mutations in the small GTPase RAB38; a regulator of intracellular vesicle trafficking and phenotypic model of Hermansky-Pudlak syndrome. Because ES cell cultures and targeting vectors are not required, this technology enables instant germline modifications, making heterozygous mutants available within 18 wk. The key features of direct mutagenesis by TALENs and oligodeoxynucleotides, minimal effort and high speed, catalyze the generation of future in vivo models for the study of human disease mechanisms and interventions. PMID:23426636

New mouse models for metabolic bone diseases generated by genome-wide ENU mutagenesis.

PubMed

Sabrautzki, Sibylle; Rubio-Aliaga, Isabel; Hans, Wolfgang; Fuchs, Helmut; Rathkolb, Birgit; Calzada-Wack, Julia; Cohrs, Christian M; Klaften, Matthias; Seedorf, Hartwig; Eck, Sebastian; Benet-Pagès, Ana; Favor, Jack; Esposito, Irene; Strom, Tim M; Wolf, Eckhard; Lorenz-Depiereux, Bettina; Hrabě de Angelis, Martin

2012-08-01

Metabolic bone disorders arise as primary diseases or may be secondary due to a multitude of organ malfunctions. Animal models are required to understand the molecular mechanisms responsible for the imbalances of bone metabolism in disturbed bone mineralization diseases. Here we present the isolation of mutant mouse models for metabolic bone diseases by phenotyping blood parameters that target bone turnover within the large-scale genome-wide Munich ENU Mutagenesis Project. A screening panel of three clinical parameters, also commonly used as biochemical markers in patients with metabolic bone diseases, was chosen. Total alkaline phosphatase activity and total calcium and inorganic phosphate levels in plasma samples of F1 offspring produced from ENU-mutagenized C3HeB/FeJ male mice were measured. Screening of 9,540 mice led to the identification of 257 phenodeviants of which 190 were tested by genetic confirmation crosses. Seventy-one new dominant mutant lines showing alterations of at least one of the biochemical parameters of interest were confirmed. Fifteen mutations among three genes (Phex, Casr, and Alpl) have been identified by positional-candidate gene approaches and one mutation of the Asgr1 gene, which was identified by next-generation sequencing. All new mutant mouse lines are offered as a resource for the scientific community.

Monoamine oxidases are mediators of endothelial dysfunction in the mouse aorta.

PubMed

Sturza, Adrian; Leisegang, Matthias S; Babelova, Andrea; Schröder, Katrin; Benkhoff, Sebastian; Loot, Annemarieke E; Fleming, Ingrid; Schulz, Rainer; Muntean, Danina M; Brandes, Ralf P

2013-07-01

Monoamine oxidases (MAOs) generate H(2)O(2) as a by-product of their catalytic cycle. Whether MAOs are mediators of endothelial dysfunction is unknown and was determined here in the angiotensin II and lipopolysaccharide-models of vascular dysfunction in mice. Quantitative real-time polymerase chain reaction revealed that mouse aortas contain enzymes involved in catecholamine generation and MAO-A and MAO-B mRNA. MAO-A and -B proteins could be detected by Western blot not only in mouse aortas but also in human umbilical vein endothelial cells. Ex vivo incubation of mouse aorta with recombinant MAO-A increased H(2)O(2) formation and induced endothelial dysfunction that was attenuated by polyethylene glycol-catalase and MAO inhibitors. In vivo lipopolysaccharide (8 mg/kg IP overnight) or angiotensin II (1 mg/kg per day, 2 weeks, minipump) treatment induced vascular MAO-A and -B expressions and resulted in attenuated endothelium-dependent relaxation of the aorta in response to acetylcholine. MAO inhibitors reduced the lipopolysaccharide- and angiotensin II-induced aortic reactive oxygen species formation by 50% (ferrous oxidation xylenol orange assay) and partially normalized endothelium-dependent relaxation. MAO-A and MAO-B inhibitors had an additive effect; combined application completely restored endothelium-dependent relaxation. To determine how MAO-dependent H(2)O(2) formation induces endothelial dysfunction, cyclic GMP was measured. Histamine stimulation of human umbilical vein endothelial cells to activate endothelial NO synthase resulted in an increase in cyclic GMP, which was almost abrogated by MAO-A exposure. MAO inhibition prevented this effect, suggesting that MAO-induced H(2)O(2) formation is sufficient to attenuate endothelial NO release. Thus, MAO-A and MAO-B are both expressed in the mouse aorta, induced by in vivo lipopolysaccharide and angiotensin II treatment and contribute via the generation of H(2)O(2) to endothelial dysfunction in vascular disease models.

Translational Mouse Models of Autism: Advancing Toward Pharmacological Therapeutics

PubMed Central

Kazdoba, Tatiana M.; Leach, Prescott T.; Yang, Mu; Silverman, Jill L.; Solomon, Marjorie

2016-01-01

Animal models provide preclinical tools to investigate the causal role of genetic mutations and environmental factors in the etiology of autism spectrum disorder (ASD). Knockout and humanized knock-in mice, and more recently knockout rats, have been generated for many of the de novo single gene mutations and copy number variants (CNVs) detected in ASD and comorbid neurodevelopmental disorders. Mouse models incorporating genetic and environmental manipulations have been employed for preclinical testing of hypothesis-driven pharmacological targets, to begin to develop treatments for the diagnostic and associated symptoms of autism. In this review, we summarize rodent behavioral assays relevant to the core features of autism, preclinical and clinical evaluations of pharmacological interventions, and strategies to improve the translational value of rodent models of autism. PMID:27305922

Myostatin gene mutated mice induced with tale nucleases.

PubMed

Zhou, Fangfang; Sun, Ruilin; Chen, Hongyan; Fei, Jian; Lu, Daru

2015-01-01

Myostain gene (MSTN) is expressed primarily in skeletal muscle, and negatively regulates skeletal muscle mass; it has been suggested that mice with MSTN inhibition have reduced adiposity and improved insulin sensitivity. Therefore, it is important to establish a fast and effective gene editing method. In this report, we established the myostatin mutated-mouse model by microinjection of Transcription Activator-Like Effector Nucleases (TALENs) mRNA within the mouse fertilized oocytes and achieved high rates of mutagenesis of the mouse MSTN in C57BL/6J. Six of 45 born mice carried target mutations and we appointed one as the parental mating with wild mouse to produce the F1 and backcross to produce the F2 generation. All the mutations of the mice were examined quickly and efficiently by high-resolution melting curve analysis (HRMA) and then verified by direct sequencing. We obtained the homozygous of the F2 generation which transmitted the mutant alleles to the progeny with 100% efficiency. Mutant mice exhibited increases in muscle mass comparable to those observed in wild-type mice. Therefore, combining TALEN-mediated gene targeting with HRMA technology is a superior method of constructing genetically modified mice through microinjection in the mouse fertilized oocytes with high efficiency and short time of selection.

GAA repeat expansion mutation mouse models of Friedreich ataxia exhibit oxidative stress leading to progressive neuronal and cardiac pathology.

PubMed

Al-Mahdawi, Sahar; Pinto, Ricardo Mouro; Varshney, Dhaval; Lawrence, Lorraine; Lowrie, Margaret B; Hughes, Sian; Webster, Zoe; Blake, Julian; Cooper, J Mark; King, Rosalind; Pook, Mark A

2006-11-01

Friedreich ataxia (FRDA) is a neurodegenerative disorder caused by an unstable GAA repeat expansion mutation within intron 1 of the FXN gene. However, the origins of the GAA repeat expansion, its unstable dynamics within different cells and tissues, and its effects on frataxin expression are not yet completely understood. Therefore, we have chosen to generate representative FRDA mouse models by using the human FXN GAA repeat expansion itself as the genetically modified mutation. We have previously reported the establishment of two lines of human FXN YAC transgenic mice that contain unstable GAA repeat expansions within the appropriate genomic context. We now describe the generation of FRDA mouse models by crossbreeding of both lines of human FXN YAC transgenic mice with heterozygous Fxn knockout mice. The resultant FRDA mice that express only human-derived frataxin show comparatively reduced levels of frataxin mRNA and protein expression, decreased aconitase activity, and oxidative stress, leading to progressive neurodegenerative and cardiac pathological phenotypes. Coordination deficits are present, as measured by accelerating rotarod analysis, together with a progressive decrease in locomotor activity and increase in weight. Large vacuoles are detected within neurons of the dorsal root ganglia (DRG), predominantly within the lumbar regions in 6-month-old mice, but spreading to the cervical regions after 1 year of age. Secondary demyelination of large axons is also detected within the lumbar roots of older mice. Lipofuscin deposition is increased in both DRG neurons and cardiomyocytes, and iron deposition is detected in cardiomyocytes after 1 year of age. These mice represent the first GAA repeat expansion-based FRDA mouse models that exhibit progressive FRDA-like pathology and thus will be of use in testing potential therapeutic strategies, particularly GAA repeat-based strategies.

'Mitochondrial energy imbalance and lipid peroxidation cause cell death in Friedreich's ataxia'.

PubMed

Abeti, R; Parkinson, M H; Hargreaves, I P; Angelova, P R; Sandi, C; Pook, M A; Giunti, P; Abramov, A Y

2016-05-26

Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease. The mutation consists of a GAA repeat expansion within the FXN gene, which downregulates frataxin, leading to abnormal mitochondrial iron accumulation, which may in turn cause changes in mitochondrial function. Although, many studies of FRDA patients and mouse models have been conducted in the past two decades, the role of frataxin in mitochondrial pathophysiology remains elusive. Are the mitochondrial abnormalities only a side effect of the increased accumulation of reactive iron, generating oxidative stress? Or does the progressive lack of iron-sulphur clusters (ISCs), induced by reduced frataxin, cause an inhibition of the electron transport chain complexes (CI, II and III) leading to reactive oxygen species escaping from oxidative phosphorylation reactions? To answer these crucial questions, we have characterised the mitochondrial pathophysiology of a group of disease-relevant and readily accessible neurons, cerebellar granule cells, from a validated FRDA mouse model. By using live cell imaging and biochemical techniques we were able to demonstrate that mitochondria are deregulated in neurons from the YG8R FRDA mouse model, causing a decrease in mitochondrial membrane potential (▵Ψm) due to an inhibition of Complex I, which is partially compensated by an overactivation of Complex II. This complex activity imbalance leads to ROS generation in both mitochondrial matrix and cytosol, which results in glutathione depletion and increased lipid peroxidation. Preventing this increase in lipid peroxidation, in neurons, protects against in cell death. This work describes the pathophysiological properties of the mitochondria in neurons from a FRDA mouse model and shows that lipid peroxidation could be an important target for novel therapeutic strategies in FRDA, which still lacks a cure.

Establishment of a patient-derived orthotopic osteosarcoma mouse model.

PubMed

Blattmann, Claudia; Thiemann, Markus; Stenzinger, Albrecht; Roth, Eva K; Dittmar, Anne; Witt, Hendrik; Lehner, Burkhard; Renker, Eva; Jugold, Manfred; Eichwald, Viktoria; Weichert, Wilko; Huber, Peter E; Kulozik, Andreas E

2015-04-30

Osteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue. Fresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin. A primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established. We report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS.

Cloning of ES cells and mice by nuclear transfer.

PubMed

Wakayama, Sayaka; Kishigami, Satoshi; Wakayama, Teruhiko

2009-01-01

We have been able to develop a stable nuclear transfer (NT) method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although the piezo unit is a complex tool, once mastered it is of great help not only in NT experiments, but also in almost all other forms of micromanipulation. Using this technique, embryonic stem (ntES) cell lines established from somatic cell nuclei can be generated relatively easily from a variety of mouse genotypes and cell types. Such ntES cells can be used not only for experimental models of human therapeutic cloning but also as a means of preserving mouse genomes instead of preserving germ cells. Here, we describe our most recent protocols for mouse cloning.

Pain, motor and gait assessment of murine osteoarthritis in a cruciate ligament transection model.

PubMed

Ruan, M Z C; Patel, R M; Dawson, B C; Jiang, M-M; Lee, B H L

2013-09-01

The major complaint of Osteoarthritis (OA) patients is pain. However, due to the nature of clinical studies and the limitation of animal studies, few studies have linked function impairment and behavioral changes in OA animal models to cartilage loss and histopathology. Our objective was to study surrogate markers of functional impairment in relation to cartilage loss and pathological changes in a post-traumatic mouse model of OA. We performed a battery of functional analyses in a mouse model of OA generated by cruciate ligament transection (CLT). The changes in functional analyses were linked to histological changes graded by OARSI standards, histological grading of synovitis, and volumetric changes of the articular cartilage and osteophytes quantified by phase contrast micro-computed tomography (μCT). OA generated by CLT led to decreased time on rotarod, delayed response on hotplate analysis, and altered gait starting from 4 weeks after surgery. Activity in open field analysis did not change at 4, 8, or 12 weeks after CLT. The magnitude of behavioral changes was directly correlated with higher OARSI histological scores of OA, synovitis in the knee joints, cartilage volume loss, and osteophyte formation. Our findings link functional analyses to histological grading, synovitis, comprehensive three-dimensional assessment of cartilage volume and osteophyte formation. This serves as a reference for a mouse model in predicting outcomes of OA treatment. Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

The contribution of mouse models to the understanding of constitutional thrombocytopenia.

PubMed

Léon, Catherine; Dupuis, Arnaud; Gachet, Christian; Lanza, François

2016-08-01

Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia. Copyright© Ferrata Storti Foundation.

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines

PubMed Central

Regla-Nava, Jose A.; Jimenez-Guardeño, Jose M.; Nieto-Torres, Jose L.; Gallagher, Thomas M.; Enjuanes, Luis; DeDiego, Marta L.

2013-01-01

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. PMID:23911968

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

PubMed Central

Zhen, Hanson H.; Oro, Anthony E.

2013-01-01

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models. PMID:23486463

Large-scale topology and the default mode network in the mouse connectome

PubMed Central

Stafford, James M.; Jarrett, Benjamin R.; Miranda-Dominguez, Oscar; Mills, Brian D.; Cain, Nicholas; Mihalas, Stefan; Lahvis, Garet P.; Lattal, K. Matthew; Mitchell, Suzanne H.; David, Stephen V.; Fryer, John D.; Nigg, Joel T.; Fair, Damien A.

2014-01-01

Noninvasive functional imaging holds great promise for serving as a translational bridge between human and animal models of various neurological and psychiatric disorders. However, despite a depth of knowledge of the cellular and molecular underpinnings of atypical processes in mouse models, little is known about the large-scale functional architecture measured by functional brain imaging, limiting translation to human conditions. Here, we provide a robust processing pipeline to generate high-resolution, whole-brain resting-state functional connectivity MRI (rs-fcMRI) images in the mouse. Using a mesoscale structural connectome (i.e., an anterograde tracer mapping of axonal projections across the mouse CNS), we show that rs-fcMRI in the mouse has strong structural underpinnings, validating our procedures. We next directly show that large-scale network properties previously identified in primates are present in rodents, although they differ in several ways. Last, we examine the existence of the so-called default mode network (DMN)—a distributed functional brain system identified in primates as being highly important for social cognition and overall brain function and atypically functionally connected across a multitude of disorders. We show the presence of a potential DMN in the mouse brain both structurally and functionally. Together, these studies confirm the presence of basic network properties and functional networks of high translational importance in structural and functional systems in the mouse brain. This work clears the way for an important bridge measurement between human and rodent models, enabling us to make stronger conclusions about how regionally specific cellular and molecular manipulations in mice relate back to humans. PMID:25512496

Anatomy and Histology of the Human and Murine Prostate.

PubMed

Ittmann, Michael

2018-05-01

The human and murine prostate glands have similar functional roles in the generation of seminal fluid to assist in reproduction. There are significant differences in the anatomy and histology of murine and human prostate and knowledge of the normal anatomy and histology of the murine prostate is essential to interpreting changes in genetically engineered mouse models. In this review, the normal anatomy and histology of both human and mouse prostate will be described. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Generation of a Slc39a8 hypomorph mouse: Markedly decreased ZIP8 Zn{sup 2+}/(HCO{sub 3}{sup -}){sub 2} transporter expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Bin; He, Lei; Dong, Hongbin

2011-07-01

Highlights: {yields} The mouse Slc39a8 gene encodes the ZIP8 transporter. {yields} ZIP8 functions endogenously as a electroneutral Zn{sup 2+}/(HCO{sub 3}{sup -}){sub 2} symporter. {yields} A Slc39a8(neo/neo) hypomorph mouse, due to retention of the neo mini-gene, has been created. {yields} ZIP8 expression in utero is {approx}90% decreased in all tissues examined. {yields} This mouse model will be useful for studying developmental and in utero physiological functions of ZIP8. -- Abstract: Previously this laboratory has identified the mouse Slc39a8 gene encoding the ZIP8 transporter, important in cadmium uptake. ZIP8 functions endogenously as a electroneutral Zn{sup 2+}/(HCO{sub 3}{sup -}){sub 2} symporter, moving bothmore » ions into the cell. The overall physiological importance of ZIP8 remains unclear. Herein we describe generation of a mouse line carrying the Slc39a8(neo) allele, containing the Frt-flanked neomycin-resistance (neo) mini-cassette in intron 3 and loxP sites in introns 3 and 6. Cre recombinase functions correctly in Escherichia coli and in adeno-Cre-infected mouse fetal fibroblasts, but does not function in the intact mouse for reasons not clear. Slc39a8(neo) is a hypomorphic allele, because Slc39a8(neo/neo) homozygotes exhibit dramatically decreased ZIP8 expression in embryo, fetus, and visceral yolk sac - in comparison to their littermate wild-type controls. This ZIP8 hypomorph will be instrumental in studying developmental and in utero physiological functions of the ZIP8 transporter.« less

Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy

PubMed Central

Freidl, Raphaela; Gstoettner, Antonia; Baranyi, Ulrike; Swoboda, Ines; Stolz, Frank; Focke-Tejkl, Margarete; Wekerle, Thomas; van Ree, Ronald; Valenta, Rudolf; Linhart, Birgit

2017-01-01

Background Fish is a frequent elicitor of severe IgE-mediated allergic reactions. Beside avoidance, there is currently no allergen-specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium-binding sites have been developed. Objectives This study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunization with a hypoallergenic mutant of the major carp allergen protect against allergic symptoms in sensitized mice. Methods C3H/HeJ mice were sensitized with recombinant wildtype Cyp c 1 or carp extract by intragastric gavage. Antibody, cellular immune responses, and epitope specificity in sensitized mice were investigated by ELISA, rat basophil leukemia assay, T-cell proliferation experiments using recombinant wildtype Cyp c 1, and overlapping peptides spanning the Cyp c 1 sequence. Anti-hypoallergenic Cyp c 1 mutant mouse and rabbit sera were tested for their ability to inhibit IgE recognition of Cyp c 1, Cyp c 1–specific basophil degranulation, and Cyp c 1–induced allergic symptoms in the mouse model. Results A mouse model of fish allergy mimicking human disease regarding IgE epitope recognition and symptoms as close as possible was established. Administration of antisera generated in mice and rabbits by immunization with a hypoallergenic Cyp c 1 mutant inhibited IgE binding to Cyp c 1, Cyp c 1–induced basophil degranulation, and allergic symptoms caused by allergen challenge in sensitized mice. Conclusions Antibodies induced by immunization with a hypoallergenic Cyp c 1 mutant protect against allergic reactions in a murine model of fish allergy. PMID:27876628

Blocking antibodies induced by immunization with a hypoallergenic parvalbumin mutant reduce allergic symptoms in a mouse model of fish allergy.

PubMed

Freidl, Raphaela; Gstoettner, Antonia; Baranyi, Ulrike; Swoboda, Ines; Stolz, Frank; Focke-Tejkl, Margarete; Wekerle, Thomas; van Ree, Ronald; Valenta, Rudolf; Linhart, Birgit

2017-06-01

Fish is a frequent elicitor of severe IgE-mediated allergic reactions. Beside avoidance, there is currently no allergen-specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium-binding sites have been developed. This study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunization with a hypoallergenic mutant of the major carp allergen protect against allergic symptoms in sensitized mice. C3H/HeJ mice were sensitized with recombinant wildtype Cyp c 1 or carp extract by intragastric gavage. Antibody, cellular immune responses, and epitope specificity in sensitized mice were investigated by ELISA, rat basophil leukemia assay, T-cell proliferation experiments using recombinant wildtype Cyp c 1, and overlapping peptides spanning the Cyp c 1 sequence. Anti-hypoallergenic Cyp c 1 mutant mouse and rabbit sera were tested for their ability to inhibit IgE recognition of Cyp c 1, Cyp c 1-specific basophil degranulation, and Cyp c 1-induced allergic symptoms in the mouse model. A mouse model of fish allergy mimicking human disease regarding IgE epitope recognition and symptoms as close as possible was established. Administration of antisera generated in mice and rabbits by immunization with a hypoallergenic Cyp c 1 mutant inhibited IgE binding to Cyp c 1, Cyp c 1-induced basophil degranulation, and allergic symptoms caused by allergen challenge in sensitized mice. Antibodies induced by immunization with a hypoallergenic Cyp c 1 mutant protect against allergic reactions in a murine model of fish allergy. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Expression of endogenous mouse APP modulates β-amyloid deposition in hAPP-transgenic mice.

PubMed

Steffen, Johannes; Krohn, Markus; Schwitlick, Christina; Brüning, Thomas; Paarmann, Kristin; Pietrzik, Claus U; Biverstål, Henrik; Jansone, Baiba; Langer, Oliver; Pahnke, Jens

2017-06-20

Amyloid-β (Aβ) deposition is one of the hallmarks of the amyloid hypothesis in Alzheimer's disease (AD). Mouse models using APP-transgene overexpression to generate amyloid plaques have shown to model only certain parts of the disease. The extent to which the data from mice can be transferred to man remains controversial. Several studies have shown convincing treatment results in reducing Aβ and enhancing cognition in mice but failed totally in human. One model-dependent factor has so far been almost completely neglected: the endogenous expression of mouse APP and its effects on the transgenic models and the readout for therapeutic approaches.Here, we report that hAPP-transgenic models of amyloidosis devoid of endogenous mouse APP expression (mAPP-knockout / mAPPko) show increased amounts and higher speed of Aβ deposition than controls with mAPP. The number of senile plaques and the level of aggregated hAβ were elevated in mAPPko mice, while the deposition in cortical blood vessels was delayed, indicating an alteration in the general aggregation propensity of hAβ together with endogenous mAβ. Furthermore, the cellular response to Aβ deposition was modulated: mAPPko mice developed a pronounced and age-dependent astrogliosis, while microglial association to amyloid plaques was diminished. The expression of human and murine aggregation-prone proteins with differing amino acid sequences within the same mouse model might not only alter the extent of deposition but also modulate the route of pathogenesis, and thus, decisively influence the study outcome, especially in translational research.

Tumor Tension Induces Persistent Inflammation and Promotes Breast Cancer Aggression

DTIC Science & Technology

2016-10-01

Task 2A. Generate the appropriate breeding scheme to build cohorts of tri- transgenic mice (MMTV-PyMT; Stat3C/+ mice and C3(1)/Tag; Stat3C/+ mice...simvastatin treatment on tumors in the C3(1)/TAg model ( transgenic , not orthotopic). I am also at the final stages of obtaining several breeding ...cytokines and degree of immunosuppression in LuBC and TNBC mouse models. Task 1A. Generate the appropriate breeding scheme to build cohorts of tri

Optoacoustic imaging of an animal model of prostate cancer

NASA Astrophysics Data System (ADS)

Patterson, Michelle P.; Arsenault, Michel; Riley, Chris; Kolios, Michael; Whelan, William M.

2010-02-01

Prostate cancer is currently the most common cancer among Canadian men. Due to an increase in public awareness and screening, prostate cancer is being detected at earlier stages and in much younger men. This is raising the need for better treatment monitoring approaches. Optoacoustic imaging is a new technique that involves exposing tissues to pulsed light and detecting the acoustic waves generated by the tissue. Optoacoustic images of a tumour bearing mouse and an agematched control were acquired for a 775 nm illumination using a reverse-mode imaging system. A murine model of prostate cancer, TRAMP (transgenetic adenocarcinoma of mouse prostate), was investigated. The results show an increase in optoacoustic signal generated by the tumour compared to that generated by the surrounding tissues with a contrast ratio of 3.5. The dimensions of the tumour in the optoacoustic image agreed with the true tumour dimensions to within 0.5 mm. In this study we show that there are detectable changes in optoacoustic signal strength that arise from the presence of a tumour in the prostate, which demonstrates the potential of optoacoustic imaging for the monitoring of prostate cancer therapy.

Efficient genome editing of differentiated renal epithelial cells.

PubMed

Hofherr, Alexis; Busch, Tilman; Huber, Nora; Nold, Andreas; Bohn, Albert; Viau, Amandine; Bienaimé, Frank; Kuehn, E Wolfgang; Arnold, Sebastian J; Köttgen, Michael

2017-02-01

Recent advances in genome editing technologies have enabled the rapid and precise manipulation of genomes, including the targeted introduction, alteration, and removal of genomic sequences. However, respective methods have been described mainly in non-differentiated or haploid cell types. Genome editing of well-differentiated renal epithelial cells has been hampered by a range of technological issues, including optimal design, efficient expression of multiple genome editing constructs, attainable mutation rates, and best screening strategies. Here, we present an easily implementable workflow for the rapid generation of targeted heterozygous and homozygous genomic sequence alterations in renal cells using transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR) system. We demonstrate the versatility of established protocols by generating novel cellular models for studying autosomal dominant polycystic kidney disease (ADPKD). Furthermore, we show that cell culture-validated genetic modifications can be readily applied to mouse embryonic stem cells (mESCs) for the generation of corresponding mouse models. The described procedure for efficient genome editing can be applied to any cell type to study physiological and pathophysiological functions in the context of precisely engineered genotypes.

Uric acid is released in the brain during seizure activity and increases severity of seizures in a mouse model for acute limbic seizures.

PubMed

Thyrion, Lisa; Raedt, Robrecht; Portelli, Jeanelle; Van Loo, Pieter; Wadman, Wytse J; Glorieux, Griet; Lambrecht, Bart N; Janssens, Sophie; Vonck, Kristl; Boon, Paul

2016-03-01

Recent evidence points at an important role of endogenous cell-damage induced pro-inflammatory molecules in the generation of epileptic seizures. Uric acid, under the form of monosodium urate crystals, has shown to have pro-inflammatory properties in the body, but less is known about its role in seizure generation. This study aimed to unravel the contribution of uric acid to seizure generation in a mouse model for acute limbic seizures. We measured extracellular levels of uric acid in the brain and modulated them using complementary pharmacological and genetic tools. Local extracellular uric acid levels increased three to four times during acute limbic seizures and peaked between 50 and 100 min after kainic acid infusion. Manipulating uric acid levels through administration of allopurinol or knock-out of urate oxidase significantly altered the number of generalized seizures, decreasing and increasing them by a twofold respectively. Taken together, our results consistently show that uric acid is released during limbic seizures and suggest that uric acid facilitates seizure generalization. Copyright © 2016 Elsevier Inc. All rights reserved.

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse)

PubMed Central

Zhou, Yihua; Xu, Bixiong C.; Maheshwari, Hiralal G.; He, Li; Reed, Michael; Lozykowski, Maria; Okada, Shigeru; Cataldo, Lori; Coschigamo, Karen; Wagner, Thomas E.; Baumann, Gerhard; Kopchick, John J.

1997-01-01

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans. PMID:9371826

A mammalian model for Laron syndrome produced by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron mouse).

PubMed

Zhou, Y; Xu, B C; Maheshwari, H G; He, L; Reed, M; Lozykowski, M; Okada, S; Cataldo, L; Coschigamo, K; Wagner, T E; Baumann, G; Kopchick, J J

1997-11-25

Laron syndrome [growth hormone (GH) insensitivity syndrome] is a hereditary dwarfism resulting from defects in the GH receptor (GHR) gene. GHR deficiency has not been reported in mammals other than humans. Many aspects of GHR dysfunction remain unknown because of ethical and practical limitations in studying humans. To create a mammalian model for this disease, we generated mice bearing a disrupted GHR/binding protein (GHR/BP) gene through a homologous gene targeting approach. Homozygous GHR/BP knockout mice showed severe postnatal growth retardation, proportionate dwarfism, absence of the GHR and GH binding protein, greatly decreased serum insulin-like growth factor I and elevated serum GH concentrations. These characteristics represent the phenotype typical of individuals with Laron syndrome. Animals heterozygous for the GHR/BP defect show only minimal growth impairment but have an intermediate biochemical phenotype, with decreased GHR and GH binding protein expression and slightly diminished insulin-like growth factor I levels. These findings indicate that the GHR/BP-deficient mouse (Laron mouse) is a suitable model for human Laron syndrome that will prove useful for the elucidation of many aspects of GHR/BP function that cannot be obtained in humans.

Loss of autocrine endothelial-derived VEGF significantly reduces hemangiosarcoma development in conditional p53-deficient mice

PubMed Central

Farhang Ghahremani, Morvarid; Radaelli, Enrico; Haigh, Katharina; Bartunkova, Sonia; Haenebalcke, Lieven; Marine, Jean-Christophe; Goossens, Steven; Haigh, Jody J

2014-01-01

Malignant transformation of the endothelium is rare, and hemangiosarcomas comprise only 1% of all sarcomas. For this reason and due to the lack of appropriate mouse models, the genetic mechanisms of malignant endothelial transformation are poorly understood. Here, we describe a hemangiosarcoma mouse model generated by deleting p53 specifically in the endothelial and hematopoietic lineages. This strategy led to a high incidence of hemangiosarcoma, with an average latency of 25 weeks. To study the in vivo roles of autocrine or endothelial cell autonomous VEGF signaling in the initiation and/or progression of hemangiosarcomas, we genetically deleted autocrine endothelial sources of VEGF in this mouse model. We found that loss of even a single conditional VEGF allele results in substantial rescue from endothelial cell transformation. These findings highlight the important role of threshold levels of autocrine VEGF signaling in endothelial malignancies and suggest a new approach for hemangiosarcoma treatment using targeted autocrine VEGF inhibition. PMID:24626176

Loss of autocrine endothelial-derived VEGF significantly reduces hemangiosarcoma development in conditional p53-deficient mice.

PubMed

Farhang Ghahremani, Morvarid; Radaelli, Enrico; Haigh, Katharina; Bartunkova, Sonia; Haenebalcke, Lieven; Marine, Jean-Christophe; Goossens, Steven; Haigh, Jody J

2014-01-01

Malignant transformation of the endothelium is rare, and hemangiosarcomas comprise only 1% of all sarcomas. For this reason and due to the lack of appropriate mouse models, the genetic mechanisms of malignant endothelial transformation are poorly understood. Here, we describe a hemangiosarcoma mouse model generated by deleting p53 specifically in the endothelial and hematopoietic lineages. This strategy led to a high incidence of hemangiosarcoma, with an average latency of 25 weeks. To study the in vivo roles of autocrine or endothelial cell autonomous VEGF signaling in the initiation and/or progression of hemangiosarcomas, we genetically deleted autocrine endothelial sources of VEGF in this mouse model. We found that loss of even a single conditional VEGF allele results in substantial rescue from endothelial cell transformation. These findings highlight the important role of threshold levels of autocrine VEGF signaling in endothelial malignancies and suggest a new approach for hemangiosarcoma treatment using targeted autocrine VEGF inhibition.

The second-generation ALK inhibitor alectinib effectively induces apoptosis in human neuroblastoma cells and inhibits tumor growth in a TH-MYCN transgenic neuroblastoma mouse model.

PubMed

Lu, Jiaxiong; Guan, Shan; Zhao, Yanling; Yu, Yang; Woodfield, Sarah E; Zhang, Huiyuan; Yang, Kristine L; Bieerkehazhi, Shayahati; Qi, Lin; Li, Xiaonan; Gu, Jerry; Xu, Xin; Jin, Jingling; Muscal, Jodi A; Yang, Tianshu; Xu, Guo-Tong; Yang, Jianhua

2017-08-01

Activating germline mutations of anaplastic lymphoma kinase (ALK) occur in most cases of hereditary neuroblastoma (NB) and the constitutively active kinase activity of ALK promotes cell proliferation and survival in NB. Therefore, ALK kinase is a potential therapeutic target for NB. In this study, we show that the novel ALK inhibitor alectinib effectively suppressed cell proliferation and induces apoptosis in NB cell lines with either wild-type ALK or mutated ALK (F1174L and D1091N) by blocking ALK-mediated PI3K/Akt/mTOR signaling. In addition, alectinib enhanced doxorubicin-induced cytotoxicity and apoptosis in NB cells. Furthermore, alectinib induced apoptosis in an orthotopic xenograft NB mouse model. Also, in the TH-MYCN transgenic mouse model, alectinib resulted in decreased tumor growth and prolonged survival time. These results indicate that alectinib may be a promising therapeutic agent for the treatment of NB. Copyright © 2017 Elsevier B.V. All rights reserved.

Prediction and generation of binary Markov processes: Can a finite-state fox catch a Markov mouse?

NASA Astrophysics Data System (ADS)

Ruebeck, Joshua B.; James, Ryan G.; Mahoney, John R.; Crutchfield, James P.

2018-01-01

Understanding the generative mechanism of a natural system is a vital component of the scientific method. Here, we investigate one of the fundamental steps toward this goal by presenting the minimal generator of an arbitrary binary Markov process. This is a class of processes whose predictive model is well known. Surprisingly, the generative model requires three distinct topologies for different regions of parameter space. We show that a previously proposed generator for a particular set of binary Markov processes is, in fact, not minimal. Our results shed the first quantitative light on the relative (minimal) costs of prediction and generation. We find, for instance, that the difference between prediction and generation is maximized when the process is approximately independently, identically distributed.

SAMHD1 knockout mice: modeling retrovirus restriction in vivo.

PubMed

Wu, Li

2013-11-20

The host dNTP hydrolase SAMHD1 acts as a viral restriction factor to inhibit the replication of several retroviruses and DNA viruses in non-cycling human immune cells. However, understanding the physiological role of mammalian SAMHD1 has been elusive due to the lack of an animal model. Two recent studies reported the generation of samhd1 knockout mouse models for investigating the restriction of HIV-1 vectors and endogenous retroviruses in vivo. Both studies suggest that SAMHD1 is important for regulating the intracellular dNTP pool and the intrinsic immunity against retroviral infection, despite different outcomes of HIV-1 vector transduction in these mouse models. Here I discuss the significance of these new findings and the future directions in studying SAMHD1-mediated retroviral restriction.

Characterization of a knock-in mouse model of the homozygous p.V37I variant in Gjb2.

PubMed

Chen, Ying; Hu, Lingxiang; Wang, Xueling; Sun, Changling; Lin, Xin; Li, Lei; Mei, Ling; Huang, Zhiwu; Yang, Tao; Wu, Hao

2016-09-13

The homozygous p.V37I variant in GJB2 is prevalent in East and Southeast Asians and may lead to mild-to-moderate hearing loss with reduced penetrance. To investigate the pathogenic mechanism underlying this variant, we generated a knock-in mouse model of homozygous p.V37I by an embryonic stem cell gene targeting method. Auditory brainstem response test showed that the knock-in mice developed progressive, mild-to-moderate hearing loss over the first 4-9 months. Overall no significant developmental and morphological abnormality was observed in the knock-in mouse cochlea, while confocal immunostaining and electron microscopic scanning revealed minor loss of the outer hair cells. Gene expression microarray analysis identified 105 up-regulated and 43 down-regulated genes in P5 knock-in mouse cochleae (P < 0.05 adjusted by the Benjamini &Hochberg method), among which four top candidate genes with the highest fold-changes or implication to deafness Fcer1g, Nnmt and Lars2 and Cuedc1 were verified by quantitative real-time PCR. Our study demonstrated that the homozygous p.V37I knock-in mouse modeled the hearing phenotype of the human patients and can serve as a useful animal model for further studies. The differentially expressed genes identified in this study may shed new insights into the understanding of the pathogenic mechanism and the phenotypic modification of homozygous p.V37I.

A fully humanized transgenic mouse model of Huntington disease

PubMed Central

Southwell, Amber L.; Warby, Simon C.; Carroll, Jeffrey B.; Doty, Crystal N.; Skotte, Niels H.; Zhang, Weining; Villanueva, Erika B.; Kovalik, Vlad; Xie, Yuanyun; Pouladi, Mahmoud A.; Collins, Jennifer A.; Yang, X. William; Franciosi, Sonia; Hayden, Michael R.

2013-01-01

Silencing the mutant huntingtin gene (muHTT) is a direct and simple therapeutic strategy for the treatment of Huntington disease (HD) in principle. However, targeting the HD mutation presents challenges because it is an expansion of a common genetic element (a CAG tract) that is found throughout the genome. Moreover, the HTT protein is important for neuronal health throughout life, and silencing strategies that also reduce the wild-type HTT allele may not be well tolerated during the long-term treatment of HD. Several HTT silencing strategies are in development that target genetic sites in HTT that are outside of the CAG expansion, including HD mutation-linked single-nucleotide polymorphisms and the HTT promoter. Preclinical testing of these genetic therapies has required the development of a new mouse model of HD that carries these human-specific genetic targets. To generate a fully humanized mouse model of HD, we have cross-bred BACHD and YAC18 on the Hdh−/− background. The resulting line, Hu97/18, is the first murine model of HD that fully genetically recapitulates human HD having two human HTT genes, no mouse Hdh genes and heterozygosity of the HD mutation. We find that Hu97/18 mice display many of the behavioral changes associated with HD including motor, psychiatric and cognitive deficits, as well as canonical neuropathological abnormalities. This mouse line will be useful for gaining additional insights into the disease mechanisms of HD as well as for testing genetic therapies targeting human HTT. PMID:23001568

Study on choroidal neovascularization with anti-VEGF treatment in the mouse retina using optical coherence tomography angiography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Park, Jang Ryul; Choi, WooJhon; Kim, Jaeryung; Hong, Hye Kyong; Kim, Yongjoo; Hwang, Yoonha; Park, Sang Jun; Woo, Se Joon; Kim, Pilhan; Park, Kyu Hyung; Koh, Gou Young; Oh, Wang-Yuhl

2017-02-01

To understand the pathogenesis of ophthalmic disease, utilizing small animal models such as mouse is necessary because of their ease of maintenance and availability. For identifying pathophysiology and drug development of retinal diseases in mouse model, optical coherence tomography angiography (OCTA) is promising imaging modality visualizing not only microstructure but also microvasculature. In this study, we serially imaged 3D structure and angiography of laser-induced choroidal neovascularization (CNV) in the mouse retina with/without anti-VEGF treatment. Also, the volume changes of CNV and avascular region in choroid layer are measured for identifying effects of anti-VEGF. A lab-built high-speed OCTA prototype using the wavelength-swept laser centered at 1040 nm with 230 kHz A-scan rate acquired 3-D volumetric data consisted of 1024 x 1024 x 3 A-scans. The OCTA scanned 1.7 mm x 1.7 mm area around ONH. For obtaining angiography, amplitude decorrelation from 3 consecutive B-scans at each position was generated. Seven days after the laser photocoagulation at mouse retina for generation of the laser-induced CNV, intravitreal administration of Fc and VEGF-Trap was given in the therapeutic arm. The OCTA were performed at 6, 14, 21 and 35 days after laser photocoagulation. Vasculatures of inner retina, outer retina and choroid layers were separately visualized after RPE flattening and layer segmentation. To investigate therapeutic effects of anti-VEGF treatment, the relative area and volume of CNV in outer retina layer is measured. Also, total volume of avascular zone surrounding the laser injury site in choroid layer is also analyzed.

A voxel-based mouse for internal dose calculations using Monte Carlo simulations (MCNP).

PubMed

Bitar, A; Lisbona, A; Thedrez, P; Sai Maurel, C; Le Forestier, D; Barbet, J; Bardies, M

2007-02-21

Murine models are useful for targeted radiotherapy pre-clinical experiments. These models can help to assess the potential interest of new radiopharmaceuticals. In this study, we developed a voxel-based mouse for dosimetric estimates. A female nude mouse (30 g) was frozen and cut into slices. High-resolution digital photographs were taken directly on the frozen block after each section. Images were segmented manually. Monoenergetic photon or electron sources were simulated using the MCNP4c2 Monte Carlo code for each source organ, in order to give tables of S-factors (in Gy Bq-1 s-1) for all target organs. Results obtained from monoenergetic particles were then used to generate S-factors for several radionuclides of potential interest in targeted radiotherapy. Thirteen source and 25 target regions were considered in this study. For each source region, 16 photon and 16 electron energies were simulated. Absorbed fractions, specific absorbed fractions and S-factors were calculated for 16 radionuclides of interest for targeted radiotherapy. The results obtained generally agree well with data published previously. For electron energies ranging from 0.1 to 2.5 MeV, the self-absorbed fraction varies from 0.98 to 0.376 for the liver, and from 0.89 to 0.04 for the thyroid. Electrons cannot be considered as 'non-penetrating' radiation for energies above 0.5 MeV for mouse organs. This observation can be generalized to radionuclides: for example, the beta self-absorbed fraction for the thyroid was 0.616 for I-131; absorbed fractions for Y-90 for left kidney-to-left kidney and for left kidney-to-spleen were 0.486 and 0.058, respectively. Our voxel-based mouse allowed us to generate a dosimetric database for use in preclinical targeted radiotherapy experiments.

Generation and Characterization of a Breast Cancer Resistance Protein Humanized Mouse Model.

PubMed

Dallas, Shannon; Salphati, Laurent; Gomez-Zepeda, David; Wanek, Thomas; Chen, Liangfu; Chu, Xiaoyan; Kunta, Jeevan; Mezler, Mario; Menet, Marie-Claude; Chasseigneaux, Stephanie; Declèves, Xavier; Langer, Oliver; Pierre, Esaie; DiLoreto, Karen; Hoft, Carolin; Laplanche, Loic; Pang, Jodie; Pereira, Tony; Andonian, Clara; Simic, Damir; Rode, Anja; Yabut, Jocelyn; Zhang, Xiaolin; Scheer, Nico

2016-05-01

Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murineBcrppromoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

A mouse model for the cystic fibrosis delta F508 mutation.

PubMed Central

van Doorninck, J H; French, P J; Verbeek, E; Peters, R H; Morreau, H; Bijman, J; Scholte, B J

1995-01-01

Most cystic fibrosis (CF) patients produce a mutant form (delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR), which is not properly processed in normal cells but is active as a chloride channel in several experimental systems. We used a double homologous recombination ('Hit and Run') procedure to generate a mouse model for the delta F508 mutation. Targeted embryonic stem (ES) cells (Hit clones) were found; of these either 80 or 20% of the clones had lost the delta F508 mutation, depending on the distance between the linearization site in the targeting construct and the delta F508 mutation. Correctly targeted clones underwent a second selection step resulting in ES cell clones (Run clones) heterozygous for the delta F508 mutation with an efficiency of 2-7%. Chimeric mice were generated and offspring homozygous for the delta F508 mutation showed electrophysiological abnormalities in nasal epithelium, gallbladder and in the intestine, and histological abnormalities in the intestine, typical of CF. Our data suggest that the delta F508 mice have residual delta F508 CFTR activity which would explain the mild pathology of the delta F508 mice. The delta F508 mouse may provide a useful model for the study of the processing defect of delta F508 CFTR and for the development of novel therapeutic approaches based on circumvention of the processing block. Images PMID:7556083

Extensive Chemical Modifications in the Primary Protein Structure of IgG1 Subvisible Particles Are Necessary for Breaking Immune Tolerance.

PubMed

Boll, Björn; Bessa, Juliana; Folzer, Emilien; Ríos Quiroz, Anacelia; Schmidt, Roland; Bulau, Patrick; Finkler, Christof; Mahler, Hanns-Christian; Huwyler, Jörg; Iglesias, Antonio; Koulov, Atanas V

2017-04-03

A current concern with the use of therapeutic proteins is the likely presence of aggregates and submicrometer, subvisible, and visible particles. It has been proposed that aggregates and particles may lead to unwanted increases in the immune response with a possible impact on safety or efficacy. The aim of this study was thus to evaluate the ability of subvisible particles of a therapeutic antibody to break immune tolerance in an IgG1 transgenic mouse model and to understand the particle attributes that might play a role in this process. We investigated the immunogenic properties of subvisible particles (unfractionated, mixed populations, and well-defined particle size fractions) using a transgenic mouse model expressing a mini-repertoire of human IgG1 (hIgG1 tg). Immunization with proteinaceous subvisible particles generated by artificial stress conditions demonstrated that only subvisible particles bearing very extensive chemical modifications within the primary amino acid structure could break immune tolerance in the hIgG1 transgenic mouse model. Protein particles exhibiting low levels of chemical modification were not immunogenic in this model.

Histopathology reveals correlative and unique phenotypes in a high-throughput mouse phenotyping screen

PubMed Central

Adissu, Hibret A.; Estabel, Jeanne; Sunter, David; Tuck, Elizabeth; Hooks, Yvette; Carragher, Damian M.; Clarke, Kay; Karp, Natasha A.; Project, Sanger Mouse Genetics; Newbigging, Susan; Jones, Nora; Morikawa, Lily; White, Jacqueline K.; McKerlie, Colin

2014-01-01

The Mouse Genetics Project (MGP) at the Wellcome Trust Sanger Institute aims to generate and phenotype over 800 genetically modified mouse lines over the next 5 years to gain a better understanding of mammalian gene function and provide an invaluable resource to the scientific community for follow-up studies. Phenotyping includes the generation of a standardized biobank of paraffin-embedded tissues for each mouse line, but histopathology is not routinely performed. In collaboration with the Pathology Core of the Centre for Modeling Human Disease (CMHD) we report the utility of histopathology in a high-throughput primary phenotyping screen. Histopathology was assessed in an unbiased selection of 50 mouse lines with (n=30) or without (n=20) clinical phenotypes detected by the standard MGP primary phenotyping screen. Our findings revealed that histopathology added correlating morphological data in 19 of 30 lines (63.3%) in which the primary screen detected a phenotype. In addition, seven of the 50 lines (14%) presented significant histopathology findings that were not associated with or predicted by the standard primary screen. Three of these seven lines had no clinical phenotype detected by the standard primary screen. Incidental and strain-associated background lesions were present in all mutant lines with good concordance to wild-type controls. These findings demonstrate the complementary and unique contribution of histopathology to high-throughput primary phenotyping of mutant mice. PMID:24652767

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong

Highlights: • ATM gene-targeted pigs were produced by somatic cell nuclear transfer. • A novel large animal model for ataxia telangiectasia was developed. • The new model may provide an alternative to the mouse model. - Abstract: Ataxia telangiectasia (A-T) is a recessive autosomal disorder associated with pleiotropic phenotypes, including progressive cerebellar degeneration, gonad atrophy, and growth retardation. Even though A-T is known to be caused by the mutations in the Ataxia telangiectasia mutated (ATM) gene, the correlation between abnormal cellular physiology caused by ATM mutations and the multiple symptoms of A-T disease has not been clearly determined. None ofmore » the existing ATM mouse models properly reflects the extent to which neurological degeneration occurs in human. In an attempt to provide a large animal model for A-T, we produced gene-targeted pigs with mutations in the ATM gene by somatic cell nuclear transfer. The disrupted allele in the ATM gene of cloned piglets was confirmed via PCR and Southern blot analysis. The ATM gene-targeted pigs generated in the present study may provide an alternative to the current mouse model for the study of mechanisms underlying A-T disorder and for the development of new therapies.« less

Modelling Human Regulatory Variation in Mouse: Finding the Function in Genome-Wide Association Studies and Whole-Genome Sequencing

PubMed Central

Schmouth, Jean-François; Bonaguro, Russell J.; Corso-Diaz, Ximena; Simpson, Elizabeth M.

2012-01-01

An increasing body of literature from genome-wide association studies and human whole-genome sequencing highlights the identification of large numbers of candidate regulatory variants of potential therapeutic interest in numerous diseases. Our relatively poor understanding of the functions of non-coding genomic sequence, and the slow and laborious process of experimental validation of the functional significance of human regulatory variants, limits our ability to fully benefit from this information in our efforts to comprehend human disease. Humanized mouse models (HuMMs), in which human genes are introduced into the mouse, suggest an approach to this problem. In the past, HuMMs have been used successfully to study human disease variants; e.g., the complex genetic condition arising from Down syndrome, common monogenic disorders such as Huntington disease and β-thalassemia, and cancer susceptibility genes such as BRCA1. In this commentary, we highlight a novel method for high-throughput single-copy site-specific generation of HuMMs entitled High-throughput Human Genes on the X Chromosome (HuGX). This method can be applied to most human genes for which a bacterial artificial chromosome (BAC) construct can be derived and a mouse-null allele exists. This strategy comprises (1) the use of recombineering technology to create a human variant–harbouring BAC, (2) knock-in of this BAC into the mouse genome using Hprt docking technology, and (3) allele comparison by interspecies complementation. We demonstrate the throughput of the HuGX method by generating a series of seven different alleles for the human NR2E1 gene at Hprt. In future challenges, we consider the current limitations of experimental approaches and call for a concerted effort by the genetics community, for both human and mouse, to solve the challenge of the functional analysis of human regulatory variation. PMID:22396661

Generation of Knock-in Mouse by Genome Editing.

PubMed

Fujii, Wataru

2017-01-01

Knock-in mice are useful for evaluating endogenous gene expressions and functions in vivo. Instead of the conventional gene-targeting method using embryonic stem cells, an exogenous DNA sequence can be inserted into the target locus in the zygote using genome editing technology. In this chapter, I describe the generation of epitope-tagged mice using engineered endonuclease and single-stranded oligodeoxynucleotide through the mouse zygote as an example of how to generate a knock-in mouse by genome editing.

Mouse Regenerating Myofibers Detected as False-Positive Donor Myofibers with Anti-Human Spectrin

PubMed Central

Rozkalne, Anete; Adkin, Carl; Meng, Jinhong; Lapan, Ariya; Morgan, Jennifer E.

2014-01-01

Abstract Stem cell transplantation is being tested as a potential therapy for a number of diseases. Stem cells isolated directly from tissue specimens or generated via reprogramming of differentiated cells require rigorous testing for both safety and efficacy in preclinical models. The availability of mice with immune-deficient background that carry additional mutations in specific genes facilitates testing the efficacy of cell transplantation in disease models. The muscular dystrophies are a heterogeneous group of disorders, of which Duchenne muscular dystrophy is the most severe and common type. Cell-based therapy for muscular dystrophy has been under investigation for several decades, with a wide selection of cell types being studied, including tissue-specific stem cells and reprogrammed stem cells. Several immune-deficient mouse models of muscular dystrophy have been generated, in which human cells obtained from various sources are injected to assess their preclinical potential. After transplantation, the presence of engrafted human cells is detected via immunofluorescence staining, using antibodies that recognize human, but not mouse, proteins. Here we show that one antibody specific to human spectrin, which is commonly used to evaluate the efficacy of transplanted human cells in mouse muscle, detects myofibers in muscles of NOD/Rag1nullmdx5cv, NOD/LtSz-scid IL2Rγnull mice, or mdx nude mice, irrespective of whether they were injected with human cells. These “reactive” clusters are regenerating myofibers, which are normally present in dystrophic tissue and the spectrin antibody is likely recognizing utrophin, which contains spectrin-like repeats. Therefore, caution should be used in interpreting data based on detection of single human-specific proteins, and evaluation of human stem cell engraftment should be performed using multiple human-specific labeling strategies. PMID:24152287

Mouse ENU Mutagenesis to Understand Immunity to Infection: Methods, Selected Examples, and Perspectives

PubMed Central

Caignard, Grégory; Eva, Megan M.; van Bruggen, Rebekah; Eveleigh, Robert; Bourque, Guillaume; Malo, Danielle; Gros, Philippe; Vidal, Silvia M.

2014-01-01

Infectious diseases are responsible for over 25% of deaths globally, but many more individuals are exposed to deadly pathogens. The outcome of infection results from a set of diverse factors including pathogen virulence factors, the environment, and the genetic make-up of the host. The completion of the human reference genome sequence in 2004 along with technological advances have tremendously accelerated and renovated the tools to study the genetic etiology of infectious diseases in humans and its best characterized mammalian model, the mouse. Advancements in mouse genomic resources have accelerated genome-wide functional approaches, such as gene-driven and phenotype-driven mutagenesis, bringing to the fore the use of mouse models that reproduce accurately many aspects of the pathogenesis of human infectious diseases. Treatment with the mutagen N-ethyl-N-nitrosourea (ENU) has become the most popular phenotype-driven approach. Our team and others have employed mouse ENU mutagenesis to identify host genes that directly impact susceptibility to pathogens of global significance. In this review, we first describe the strategies and tools used in mouse genetics to understand immunity to infection with special emphasis on chemical mutagenesis of the mouse germ-line together with current strategies to efficiently identify functional mutations using next generation sequencing. Then, we highlight illustrative examples of genes, proteins, and cellular signatures that have been revealed by ENU screens and have been shown to be involved in susceptibility or resistance to infectious diseases caused by parasites, bacteria, and viruses. PMID:25268389

Quantitative T2 combined with texture analysis of nuclear magnetic resonance images identify different degrees of muscle involvement in three mouse models of muscle dystrophy: mdx, Largemyd and mdx/Largemyd.

PubMed

Martins-Bach, Aurea B; Malheiros, Jackeline; Matot, Béatrice; Martins, Poliana C M; Almeida, Camila F; Caldeira, Waldir; Ribeiro, Alberto F; Loureiro de Sousa, Paulo; Azzabou, Noura; Tannús, Alberto; Carlier, Pierre G; Vainzof, Mariz

2015-01-01

Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant-T2-measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Largemyd mouse and the recently generated and worst double mutant mdx/Largemyd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Largemyd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Largemyd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Largemyd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Largemyd showed similar values than both the mild mdx and worst mdx/Largemyd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research.

Quantitative T2 Combined with Texture Analysis of Nuclear Magnetic Resonance Images Identify Different Degrees of Muscle Involvement in Three Mouse Models of Muscle Dystrophy: mdx, Largemyd and mdx/Largemyd

PubMed Central

Martins-Bach, Aurea B.; Malheiros, Jackeline; Matot, Béatrice; Martins, Poliana C. M.; Almeida, Camila F.; Caldeira, Waldir; Ribeiro, Alberto F.; Loureiro de Sousa, Paulo; Azzabou, Noura; Tannús, Alberto; Carlier, Pierre G.; Vainzof, Mariz

2015-01-01

Quantitative nuclear magnetic resonance imaging (MRI) has been considered a promising non-invasive tool for monitoring therapeutic essays in small size mouse models of muscular dystrophies. Here, we combined MRI (anatomical images and transverse relaxation time constant—T2—measurements) to texture analyses in the study of four mouse strains covering a wide range of dystrophic phenotypes. Two still unexplored mouse models of muscular dystrophies were analyzed: The severely affected Largemyd mouse and the recently generated and worst double mutant mdx/Largemyd mouse, as compared to the mildly affected mdx and normal mice. The results were compared to histopathological findings. MRI showed increased intermuscular fat and higher muscle T2 in the three dystrophic mouse models when compared to the wild-type mice (T2: mdx/Largemyd: 37.6±2.8 ms; mdx: 35.2±4.5 ms; Largemyd: 36.6±4.0 ms; wild-type: 29.1±1.8 ms, p<0.05), in addition to higher muscle T2 in the mdx/Largemyd mice when compared to mdx (p<0.05). The areas with increased muscle T2 in the MRI correlated spatially with the identified histopathological alterations such as necrosis, inflammation, degeneration and regeneration foci. Nevertheless, muscle T2 values were not correlated with the severity of the phenotype in the 3 dystrophic mouse strains, since the severely affected Largemyd showed similar values than both the mild mdx and worst mdx/Largemyd lineages. On the other hand, all studied mouse strains could be unambiguously identified with texture analysis, which reflected the observed differences in the distribution of signals in muscle MRI. Thus, combined T2 intensity maps and texture analysis is a powerful approach for the characterization and differentiation of dystrophic muscles with diverse genotypes and phenotypes. These new findings provide important noninvasive tools in the evaluation of the efficacy of new therapies, and most importantly, can be directly applied in human translational research. PMID:25710816

High-resolution detection of 13C multiplets from the conscious mouse brain by ex vivo NMR spectroscopy

PubMed Central

Marin-Valencia, Isaac; Good, Levi B.; Ma, Qian; Jeffrey, F. Mark; Malloy, Craig R.; Pascual, Juan M.

2011-01-01

Glucose readily supplies the brain with the majority of carbon needed to sustain neurotransmitter production and utilization., The rate of brain glucose metabolism can be computed using 13C nuclear magnetic resonance (NMR) spectroscopy by detecting changes in 13C contents of products generated by cerebral metabolism. As previously observed, scalar coupling between adjacent 13C carbons (multiplets) can provide additional information to 13C contents for the computation of metabolic rates. Most NMR studies have been conducted in large animals (often under anesthesia) because the mass of the target organ is a limiting factor for NMR. Yet, despite the challengingly small size of the mouse brain, NMR studies are highly desirable because the mouse constitutes a common animal model for human neurological disorders. We have developed a method for the ex vivo resolution of NMR multiplets arising from the brain of an awake mouse after the infusion of [1,6-13C2]glucose. NMR spectra obtained by this method display favorable signal-to-noise ratios. With this protocol, the 13C multiplets of glutamate, glutamine, GABA and aspartate achieved steady state after 150 min. The method enables the accurate resolution of multiplets over time in the awake mouse brain. We anticipate that this method can be broadly applicable to compute brain fluxes in normal and transgenic mouse models of neurological disorders. PMID:21946227

The replication of a mouse adapted SARS-CoV in a mouse cell line stably expressing the murine SARS-CoV receptor mACE2 efficiently induces the expression of proinflammatory cytokines.

PubMed

Regla-Nava, Jose A; Jimenez-Guardeño, Jose M; Nieto-Torres, Jose L; Gallagher, Thomas M; Enjuanes, Luis; DeDiego, Marta L

2013-11-01

Infection of conventional mice with a mouse adapted (MA15) severe acute respiratory syndrome (SARS) coronavirus (CoV) reproduces many aspects of human SARS such as pathological changes in lung, viremia, neutrophilia, and lethality. However, established mouse cell lines highly susceptible to mouse-adapted SARS-CoV infection are not available. In this work, efficiently transfectable mouse cell lines stably expressing the murine SARS-CoV receptor angiotensin converting enzyme 2 (ACE2) have been generated. These cells yielded high SARS-CoV-MA15 titers and also served as excellent tools for plaque assays. In addition, in these cell lines, SARS-CoV-MA15 induced the expression of proinflammatory cytokines and IFN-β, mimicking what has been observed in experimental animal models infected with SARS-CoV and SARS patients. These cell lines are valuable tools to perform in vitro studies in a mouse cell system that reflects the species used for in vivo studies of SARS-CoV-MA15 pathogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage.

PubMed

Li, Juan; Prins, Daniel; Park, Hyun Jung; Grinfeld, Jacob; Gonzalez-Arias, Carlos; Loughran, Stephen; Dovey, Oliver M; Klampfl, Thorsten; Bennett, Cavan; Hamilton, Tina L; Pask, Dean C; Sneade, Rachel; Williams, Matthew; Aungier, Juliet; Ghevaert, Cedric; Vassiliou, George S; Kent, David G; Green, Anthony R

2018-02-08

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALR del/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALR del/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALR del/+ HSCs were more proliferative in vitro, but neither CALR del/+ nor CALR del/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF. © 2018 by The American Society of Hematology.

Facilitation of endoglin-targeting cancer therapy by development/utilization of a novel genetically engineered mouse model expressing humanized endoglin (CD105).

PubMed

Toi, Hirofumi; Tsujie, Masanori; Haruta, Yuro; Fujita, Kanako; Duzen, Jill; Seon, Ben K

2015-01-15

Endoglin (ENG) is a TGF-β coreceptor and essential for vascular development and angiogenesis. A chimeric antihuman ENG (hENG) monoclonal antibody (mAb) c-SN6j (also known as TRC105) shows promising safety and clinical efficacy features in multiple clinical trials of patients with various advanced solid tumors. Here we developed a novel genetically engineered mouse model to optimize the ENG-targeting clinical trials. We designed a new targeting vector that contains exons 4-8 of hENG gene to generate novel genetically engineered mice (GEMs) expressing functional human/mouse chimeric (humanized) ENG with desired epitopes. Genotyping of the generated mice confirmed that we generated the desired GEMs. Immunohistochemical analysis demonstrated that humanized ENG protein of the GEMs expresses epitopes defined by 7 of our 8 anti-hENG mAbs tested. Surprisingly the homozygous GEMs develop normally and are healthy. Established breast and colon tumors as well as metastasis and tumor microvessels in the GEMs were effectively suppressed by systemic administration of anti-hENG mAbs. Additionally, test result indicates that synergistic potentiation of antitumor efficacy can be induced by simultaneous targeting of two distinct epitopes by anti-hENG mAbs. Sorafenib and capecitabine also showed antitumor efficacy in the GEMs. The presented novel GEMs are the first GEMs that express the targetable humanized ENG. Test results indicate utility of the GEMs for the clinically relevant studies. Additionally, we generated GEMs expressing a different humanized ENG containing exons 5-6 of hENG gene, and the homozygous GEMs develop normally and are healthy. © 2014 UICC.

Mouse forward genetics in the study of the peripheral nervous system and human peripheral neuropathy

PubMed Central

Douglas, Darlene S.; Popko, Brian

2009-01-01

Forward genetics, the phenotype-driven approach to investigating gene identity and function, has a long history in mouse genetics. Random mutations in the mouse transcend bias about gene function and provide avenues towards unique discoveries. The study of the peripheral nervous system is no exception; from historical strains such as the trembler mouse, which led to the identification of PMP22 as a human disease gene causing multiple forms of peripheral neuropathy, to the more recent identification of the claw paw and sprawling mutations, forward genetics has long been a tool for probing the physiology, pathogenesis, and genetics of the PNS. Even as spontaneous and mutagenized mice continue to enable the identification of novel genes, provide allelic series for detailed functional studies, and generate models useful for clinical research, new methods, such as the piggyBac transposon, are being developed to further harness the power of forward genetics. PMID:18481175

Generation and Characterization of a New Monoclonal Antibody Against CXCL4.

PubMed

Gao, Jing; Wu, Mingyuan; Gao, Jin; Wang, Xia; Zhang, Yang; Zhu, Shunying; Yu, Yan; Han, Wei

2015-04-01

CXCL4 plays important roles in numerous disease processes, which makes the CXCL4 signaling pathway a potential therapeutic target. In this study, we aimed to develop a neutralizing antibody against both human and mouse CXCL4. Rats were immunized with recombinant human CXCL4 (rhCXCL4). Hybridoma clones were created by fusion of the immunized rat spleen cells with mouse myeloma SP2/0 cells and screened using recombinant mouse CXCL4 (rmCXCL4) and rhCXCL4. The CXCL4 monoclonal antibody (CXCL4 MAb) produced by the 16D6-3 hybridoma clone was sequenced and characterized by Western blot and Biacore assays. It recognized both human and mouse CXCL4 with high affinity and neutralized the effect of rhCXCL4 in vitro. Thus, the antibody may be used in the studies of CXCL4 in murine disease models and as a template in the antibody humanization for clinical developments.

High-throughput mouse genotyping using robotics automation.

PubMed

Linask, Kaari L; Lo, Cecilia W

2005-02-01

The use of mouse models is rapidly expanding in biomedical research. This has dictated the need for the rapid genotyping of mutant mouse colonies for more efficient utilization of animal holding space. We have established a high-throughput protocol for mouse genotyping using two robotics workstations: a liquid-handling robot to assemble PCR and a microfluidics electrophoresis robot for PCR product analysis. This dual-robotics setup incurs lower start-up costs than a fully automated system while still minimizing human intervention. Essential to this automation scheme is the construction of a database containing customized scripts for programming the robotics workstations. Using these scripts and the robotics systems, multiple combinations of genotyping reactions can be assembled simultaneously, allowing even complex genotyping data to be generated rapidly with consistency and accuracy. A detailed protocol, database, scripts, and additional background information are available at http://dir.nhlbi.nih.gov/labs/ldb-chd/autogene/.

The Small GTPase Rif Is Dispensable for Platelet Filopodia Generation in Mice

PubMed Central

Goggs, Robert; Savage, Joshua S.; Mellor, Harry; Poole, Alastair W.

2013-01-01

Background Formation of filopodia and other shape change events are vital for platelet hemostatic function. The mechanisms regulating filopodia formation by platelets are incompletely understood however. In particular the small GTPase responsible for initiating filopodia formation by platelets remains elusive. The canonical pathway involving Cdc42 is not essential for filopodia formation in mouse platelets. The small GTPase Rif (RhoF) provides an alternative route to filopodia generation in other cell types and is expressed in both human and mouse platelets. Hypothesis/Objective We hypothesized that Rif might be responsible for generating filopodia by platelets and generated a novel knockout mouse model to investigate the functional role of Rif in platelets. Methodology/Principal Findings Constitutive RhoF−/− mice are viable and have normal platelet, leukocyte and erythrocyte counts and indices. RhoF−/− platelets form filopodia and spread normally on various agonist surfaces in static conditions and under arterial shear. In addition, RhoF−/− platelets have normal actin dynamics, are able to activate and aggregate normally and secrete from alpha and dense granules in response to collagen related peptide and thrombin stimulation. Conclusions The small GTPase Rif does not appear to be critical for platelet function in mice. Functional overlap between Rif and other small GTPases may be responsible for the non-essential role of Rif in platelets. PMID:23359340

New Anti-Nephrin Antibody Mediated Podocyte Injury Model Using a C57BL/6 Mouse Strain.

PubMed

Takeuchi, Kazuhiro; Naito, Shokichi; Kawashima, Nagako; Ishigaki, Naoko; Sano, Takashi; Kamata, Kouju; Takeuchi, Yasuo

2018-01-01

Focal segmental glomerulosclerosis (FSGS) is considered a subset of the podocytopathies. The molecular pathogenesis of podocytopathy is still unknown. There has not been an experimental animal model of isolated podocytopathy induced by antibody in C57BL/6 strain, which is widely used as the genetic background. Nephrin is closely associated with the slit diaphragm of the glomerular podocyte, and has recently received attention as a potential therapeutic target. The function of nephrin, especially its role in FSGS development via podocytopathy, is being elucidated. We report our experience with a C57BL/6 FSGS model induced by polyclonal rabbit anti-mouse nephrin antibody (α-mNep Ab). α-mNep Ab, which was generated by genetic immunization, was administered into C57BL/6 mice at once, intravenously. Urinary protein excretion, the development of glomerulosclerosis and the number of podocyte in mouse kidney were evaluated. The α-mNep Ab-induced FSGS was associated with massive proteinuria and nephrotic syndrome. In periodic acid-Schiff staining, FSGS was observed from day 7 after antibody injection. Podocyte numbers and podocyte marker (anti-Wilms tumor 1 and anti-synaptopodin)-positive areas were clearly decreased. These results suggest that this FSGS mouse model reliably reproduces the human nephrotic syndrome and FSGS. We succeeded in making the nephrotic syndrome model mice induced by α-mNep Ab using C57BL/6. This model may be useful for studying the mechanisms of podocytopathy. © 2017 S. Karger AG, Basel.

Tissue morphodynamics shaping the early mouse embryo.

PubMed

Sutherland, Ann E

2016-07-01

Generation of the elongated vertebrate body plan from the initially radially symmetrical embryo requires comprehensive changes to tissue form. These shape changes are generated by specific underlying cell behaviors, coordinated in time and space. Major principles and also specifics are emerging, from studies in many model systems, of the cell and physical biology of how region-specific cell behaviors produce regional tissue morphogenesis, and how these, in turn, are integrated at the level of the embryo. New technical approaches have made it possible more recently, to examine the morphogenesis of the mouse embryo in depth, and to elucidate the underlying cellular mechanisms. This review focuses on recent advances in understanding the cellular basis for the early fundamental events that establish the basic form of the embryo. Copyright © 2016 Elsevier Ltd. All rights reserved.

Analysis of enamel development using murine model systems: approaches and limitations

PubMed Central

Pugach, Megan K.; Gibson, Carolyn W.

2014-01-01

A primary goal of enamel research is to understand and potentially treat or prevent enamel defects related to amelogenesis imperfecta (AI). Rodents are ideal models to assist our understanding of how enamel is formed because they are easily genetically modified, and their continuously erupting incisors display all stages of enamel development and mineralization. While numerous methods have been developed to generate and analyze genetically modified rodent enamel, it is crucial to understand the limitations and challenges associated with these methods in order to draw appropriate conclusions that can be applied translationally, to AI patient care. We have highlighted methods involved in generating and analyzing rodent enamel and potential approaches to overcoming limitations of these methods: (1) generating transgenic, knockout, and knockin mouse models, and (2) analyzing rodent enamel mineral density and functional properties (structure and mechanics) of mature enamel. There is a need for a standardized workflow to analyze enamel phenotypes in rodent models so that investigators can compare data from different studies. These methods include analyses of gene and protein expression, developing enamel histology, enamel pigment, degree of mineralization, enamel structure, and mechanical properties. Standardization of these methods with regard to stage of enamel development and sample preparation is crucial, and ideally investigators can use correlative and complementary techniques with the understanding that developing mouse enamel is dynamic and complex. PMID:25278900

The artificial gene Jazz, a transcriptional regulator of utrophin, corrects the dystrophic pathology in mdx mice.

PubMed

Di Certo, Maria Grazia; Corbi, Nicoletta; Strimpakos, Georgios; Onori, Annalisa; Luvisetto, Siro; Severini, Cinzia; Guglielmotti, Angelo; Batassa, Enrico Maria; Pisani, Cinzia; Floridi, Aristide; Benassi, Barbara; Fanciulli, Maurizio; Magrelli, Armando; Mattei, Elisabetta; Passananti, Claudio

2010-03-01

The absence of the cytoskeletal protein dystrophin results in Duchenne muscular dystrophy (DMD). The utrophin protein is the best candidate for dystrophin replacement in DMD patients. To obtain therapeutic levels of utrophin expression in dystrophic muscle, we developed an alternative strategy based on the use of artificial zinc finger transcription factors (ZF ATFs). The ZF ATF 'Jazz' was recently engineered and tested in vivo by generating a transgenic mouse specifically expressing Jazz at the muscular level. To validate the ZF ATF technology for DMD treatment we generated a second mouse model by crossing Jazz-transgenic mice with dystrophin-deficient mdx mice. Here, we show that the artificial Jazz protein restores sarcolemmal integrity and prevents the development of the dystrophic disease in mdx mice. This exclusive animal model establishes the notion that utrophin-based therapy for DMD can be efficiently developed using ZF ATF technology and candidates Jazz as a novel therapeutic molecule for DMD therapy.

Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential.

PubMed

Bolton, Helen; Graham, Sarah J L; Van der Aa, Niels; Kumar, Parveen; Theunis, Koen; Fernandez Gallardo, Elia; Voet, Thierry; Zernicka-Goetz, Magdalena

2016-03-29

Most human pre-implantation embryos are mosaics of euploid and aneuploid cells. To determine the fate of aneuploid cells and the developmental potential of mosaic embryos, here we generate a mouse model of chromosome mosaicism. By treating embryos with a spindle assembly checkpoint inhibitor during the four- to eight-cell division, we efficiently generate aneuploid cells, resulting in embryo death during peri-implantation development. Live-embryo imaging and single-cell tracking in chimeric embryos, containing aneuploid and euploid cells, reveal that the fate of aneuploid cells depends on lineage: aneuploid cells in the fetal lineage are eliminated by apoptosis, whereas those in the placental lineage show severe proliferative defects. Overall, the proportion of aneuploid cells is progressively depleted from the blastocyst stage onwards. Finally, we show that mosaic embryos have full developmental potential, provided they contain sufficient euploid cells, a finding of significance for the assessment of embryo vitality in the clinic.

An animal model for Norrie disease (ND): gene targeting of the mouse ND gene.

PubMed

Berger, W; van de Pol, D; Bächner, D; Oerlemans, F; Winkens, H; Hameister, H; Wieringa, B; Hendriks, W; Ropers, H H

1996-01-01

In order to elucidate the cellular and molecular processes which are involved in Norrie disease (ND), we have used gene targeting technology to generate ND mutant mice. The murine homologue of the ND gene was cloned and shown to encode a polypeptide that shares 94% of the amino acid sequence with its human counterpart. RNA in situ hybridization revealed expression in retina, brain and the olfactory bulb and epithelium of 2 week old mice. Hemizygous mice carrying a replacement mutation in exon 2 of the ND gene developed retrolental structures in the vitreous body and showed an overall disorganization of the retinal ganglion cell layer. The outer plexiform layer disappears occasionally, resulting in a juxtaposed inner and outer nuclear layer. At the same regions, the outer segments of the photoreceptor cell layer are no longer present. These ocular findings are consistent with observations in ND patients and the generated mouse line provides a faithful model for study of early pathogenic events in this severe X-linked recessive neurological disorder.

NF-κB gene signature predicts prostate cancer progression

PubMed Central

Jin, Renjie; Yi, Yajun; Yull, Fiona E.; Blackwell, Timothy S.; Clark, Peter E.; Koyama, Tatsuki; Smith, Joseph A.; Matusik, Robert J.

2014-01-01

In many prostate cancer (PCa) patients, the cancer will be recurrent and eventually progress to lethal metastatic disease after primary treatment, such as surgery or radiation therapy. Therefore, it would be beneficial to better predict which patients with early-stage PCa would progress or recur after primary definitive treatment. In addition, many studies indicate that activation of NF-κB signaling correlates with PCa progression; however, the precise underlying mechanism is not fully understood. Our studies show that activation of NF-κB signaling via deletion of one allele of its inhibitor, IκBα, did not induce prostatic tumorigenesis in our mouse model. However, activation of NF-κB signaling did increase the rate of tumor progression in the Hi-Myc mouse PCa model when compared to Hi-Myc alone. Using the non-malignant NF-κB activated androgen depleted mouse prostate, a NF-κB Activated Recurrence Predictor 21 (NARP21) gene signature was generated. The NARP21 signature successfully predicted disease-specific survival and distant metastases-free survival in patients with PCa. This transgenic mouse model derived gene signature provides a useful and unique molecular profile for human PCa prognosis, which could be used on a prostatic biopsy to predict indolent versus aggressive behavior of the cancer after surgery. PMID:24686169

Synaptic Vesicle Recycling Is Unaffected in the Ts65Dn Mouse Model of Down Syndrome.

PubMed

Marland, Jamie R K; Smillie, Karen J; Cousin, Michael A

2016-01-01

Down syndrome (DS) is the most common genetic cause of intellectual disability, and arises from trisomy of human chromosome 21. Accumulating evidence from studies of both DS patient tissue and mouse models has suggested that synaptic dysfunction is a key factor in the disorder. The presence of several genes within the DS trisomy that are either directly or indirectly linked to synaptic vesicle (SV) endocytosis suggested that presynaptic dysfunction could underlie some of these synaptic defects. Therefore we determined whether SV recycling was altered in neurons from the Ts65Dn mouse, the best characterised model of DS to date. We found that SV exocytosis, the size of the SV recycling pool, clathrin-mediated endocytosis, activity-dependent bulk endocytosis and SV generation from bulk endosomes were all unaffected by the presence of the Ts65Dn trisomy. These results were obtained using battery of complementary assays employing genetically-encoded fluorescent reporters of SV cargo trafficking, and fluorescent and morphological assays of fluid-phase uptake in primary neuronal culture. The absence of presynaptic dysfunction in central nerve terminals of the Ts65Dn mouse suggests that future research should focus on the established alterations in excitatory / inhibitory balance as a potential route for future pharmacotherapy.

Synaptic Vesicle Recycling Is Unaffected in the Ts65Dn Mouse Model of Down Syndrome

PubMed Central

Marland, Jamie R. K.; Smillie, Karen J.; Cousin, Michael A.

2016-01-01

Down syndrome (DS) is the most common genetic cause of intellectual disability, and arises from trisomy of human chromosome 21. Accumulating evidence from studies of both DS patient tissue and mouse models has suggested that synaptic dysfunction is a key factor in the disorder. The presence of several genes within the DS trisomy that are either directly or indirectly linked to synaptic vesicle (SV) endocytosis suggested that presynaptic dysfunction could underlie some of these synaptic defects. Therefore we determined whether SV recycling was altered in neurons from the Ts65Dn mouse, the best characterised model of DS to date. We found that SV exocytosis, the size of the SV recycling pool, clathrin-mediated endocytosis, activity-dependent bulk endocytosis and SV generation from bulk endosomes were all unaffected by the presence of the Ts65Dn trisomy. These results were obtained using battery of complementary assays employing genetically-encoded fluorescent reporters of SV cargo trafficking, and fluorescent and morphological assays of fluid-phase uptake in primary neuronal culture. The absence of presynaptic dysfunction in central nerve terminals of the Ts65Dn mouse suggests that future research should focus on the established alterations in excitatory / inhibitory balance as a potential route for future pharmacotherapy. PMID:26808141

Development of the mouse cochlea database (MCD).

PubMed

Santi, Peter A; Rapson, Ian; Voie, Arne

2008-09-01

The mouse cochlea database (MCD) provides an interactive, image database of the mouse cochlea for learning its anatomy and data mining of its resources. The MCD website is hosted on a centrally maintained, high-speed server at the following URL: (http://mousecochlea.umn.edu). The MCD contains two types of image resources, serial 2D image stacks and 3D reconstructions of cochlear structures. Complete image stacks of the cochlea from two different mouse strains were obtained using orthogonal plane fluorescence optical microscopy (OPFOS). 2D images of the cochlea are presented on the MCD website as: viewable images within a stack, 2D atlas of the cochlea, orthogonal sections, and direct volume renderings combined with isosurface reconstructions. In order to assess cochlear structures quantitatively, "true" cross-sections of the scala media along the length of the basilar membrane were generated by virtual resectioning of a cochlea orthogonal to a cochlear structure, such as the centroid of the basilar membrane or the scala media. 3D images are presented on the MCD website as: direct volume renderings, movies, interactive QuickTime VRs, flythrough, and isosurface 3D reconstructions of different cochlear structures. 3D computer models can also be used for solid model fabrication by rapid prototyping and models from different cochleas can be combined to produce an average 3D model. The MCD is the first comprehensive image resource on the mouse cochlea and is a new paradigm for understanding the anatomy of the cochlea, and establishing morphometric parameters of cochlear structures in normal and mutant mice.

An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers

PubMed Central

Cho, Lily Ting-yin; Andrews, Robert; Carroll, Thomas; Iyer, Vivek; Tate, Peri; Rosen, Barry; Stunnenberg, Hendrik G.; Fisher, Amanda G.; Skarnes, William C.

2017-01-01

Abstract Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC ‘knockout-first’ ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the ‘knockout-first’ allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency ‘2i’ media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. PMID:28981838

Parent-of-origin effects on schizophrenia-relevant behaviours of type III neuregulin 1 mutant mice.

PubMed

Shang, Kani; Talmage, David A; Karl, Tim

2017-08-14

A robust, disease-relevant phenotype is paramount to the validity of genetic mouse models, which are an important tool in understanding complex diseases. Recent evidence from genome-wide association studies suggests the genetic contribution of parents to offspring is not equivalent. Despite this, few studies to date have examined the potential impact of parent genotype (i.e. origin of mutation) on the offspring of disease-relevant genetic mouse models. To elucidate the potential impact of the sex of the mutant parent on offspring phenotype, we characterized male and female offspring of an established schizophrenia mouse model, which had been generated using two different breeding schemes, in a range of disease-relevant behaviours. We compared heterozygous type III neuregulin 1 mutant (type III Nrg1 +/- ) and wild type-like control (WT) offspring from mutant father x WT mother pairings with offspring from mutant mother x WT father pairings. Offspring were tested in schizophrenia-relevant paradigms including the elevated plus maze (EPM), fear conditioning (FC), prepulse inhibition (PPI), social interaction (SI), and open field (OF). We found type III Nrg1 +/- males from mutant fathers, but not mutant mothers, showed deficits in contextual fear-associated memory and exhibited increased social interaction, compared to their WT littermates. Type III Nrg1 +/- females across breeding colonies only exhibited a subtle change to their acoustic startle response and sensorimotor gating. These results suggest a paternal-dependent transmission of genetically induced behavioural characteristics. Though the mechanisms governing this phenomenon are unclear, our results show that parental origin of mutation can alter the behavioural phenotype of genetic mouse models. Thus, researchers should carefully consider their breeding scheme when dealing with genetic mouse models of diseases such as schizophrenia. Copyright © 2017. Published by Elsevier B.V.

Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction coupling

PubMed Central

Gao, Junyuan; Sun, Xiurong; Moore, Leon C.; White, Thomas W.; Brink, Peter R.

2011-01-01

We recently modeled fluid flow through gap junction channels coupling the pigmented and nonpigmented layers of the ciliary body. The model suggested the channels could transport the secretion of aqueous humor, but flow would be driven by hydrostatic pressure rather than osmosis. The pressure required to drive fluid through a single layer of gap junctions might be just a few mmHg and difficult to measure. In the lens, however, there is a circulation of Na+ that may be coupled to intracellular fluid flow. Based on this hypothesis, the fluid would cross hundreds of layers of gap junctions, and this might require a large hydrostatic gradient. Therefore, we measured hydrostatic pressure as a function of distance from the center of the lens using an intracellular microelectrode-based pressure-sensing system. In wild-type mouse lenses, intracellular pressure varied from ∼330 mmHg at the center to zero at the surface. We have several knockout/knock-in mouse models with differing levels of expression of gap junction channels coupling lens fiber cells. Intracellular hydrostatic pressure in lenses from these mouse models varied inversely with the number of channels. When the lens’ circulation of Na+ was either blocked or reduced, intracellular hydrostatic pressure in central fiber cells was either eliminated or reduced proportionally. These data are consistent with our hypotheses: fluid circulates through the lens; the intracellular leg of fluid circulation is through gap junction channels and is driven by hydrostatic pressure; and the fluid flow is generated by membrane transport of sodium. PMID:21624945

Theranostic gas-generating nanoparticles for targeted ultrasound imaging and treatment of neuroblastoma.

PubMed

Lee, Jangwook; Min, Hyun-Su; You, Dong Gil; Kim, Kwangmeyung; Kwon, Ick Chan; Rhim, Taiyoun; Lee, Kuen Yong

2016-02-10

The development of safe and efficient diagnostic/therapeutic agents for treating cancer in clinics remains challenging due to the potential toxicity of conventional agents. Although the annual incidence of neuroblastoma is not that high, the disease mainly occurs in children, a population vulnerable to toxic contrast agents and therapeutics. We demonstrate here that cancer-targeting, gas-generating polymeric nanoparticles are useful as a theranostic tool for ultrasound (US) imaging and treating neuroblastoma. We encapsulated calcium carbonate using poly(d,l-lactide-co-glycolide) and created gas-generating polymer nanoparticles (GNPs). These nanoparticles release carbon dioxide bubbles under acidic conditions and enhance US signals. When GNPs are modified using rabies virus glycoprotein (RVG) peptide, a targeting moiety to neuroblastoma, RVG-GNPs effectively accumulate at the tumor site and substantially enhance US signals in a tumor-bearing mouse model. Intravenous administration of RVG-GNPs also reduces tumor growth in the mouse model without the use of conventional therapeutic agents. This approach to developing theranostic agents with disease-targeting ability may provide useful strategy for the detection and treatment of cancers, allowing safe and efficient clinical applications with fewer side effects than may occur with conventional agents. Copyright © 2015 Elsevier B.V. All rights reserved.

Effect of tumor properties on energy absorption, temperature mapping, and thermal dose in 13.56-MHz radiofrequency hyperthermia.

PubMed

Prasad, Bibin; Kim, Subin; Cho, Woong; Kim, Suzy; Kim, Jung Kyung

2018-05-01

Computational techniques can enhance personalized hyperthermia-treatment planning by calculating tissue energy absorption and temperature distribution. This study determined the effect of tumor properties on energy absorption, temperature mapping, and thermal dose distribution in mild radiofrequency hyperthermia using a mouse xenograft model. We used a capacitive-heating radiofrequency hyperthermia system with an operating frequency of 13.56 MHz for in vivo mouse experiments and performed simulations on a computed tomography mouse model. Additionally, we measured the dielectric properties of the tumors and considered temperature dependence for thermal properties, metabolic heat generation, and perfusion. Our results showed that dielectric property variations were more dominant than thermal properties and other parameters, and that the measured dielectric properties provided improved temperature-mapping results relative to the property values taken from previous study. Furthermore, consideration of temperature dependency in the bio heat-transfer model allowed elucidation of precise thermal-dose calculations. These results suggested that this method might contribute to effective thermoradiotherapy planning in clinics. Copyright © 2018 Elsevier Ltd. All rights reserved.

Photodynamic therapy stimulates anti-tumor immune response in mouse models: the role of regulatory Tcells, anti-tumor antibodies, and immune attacks on brain metastases

NASA Astrophysics Data System (ADS)

Vatansever, Fatma; Kawakubo, Masayoshi; Chung, Hoon; Hamblin, Michael R.

2013-02-01

We have previously shown that photodynamic therapy mediated by a vascular regimen of benzoporphyrin derivative and 690nm light is capable of inducing a robust immune response in the mouse CT26.CL25 tumor model that contains a tumor-rejection antigen, beta-galactosidase (β-gal). For the first time we show that PDT can stimulate the production of serum IgG antibodies against the β-gal antigen. It is known that a common cause of death from cancer, particularly lung cancer, is brain metastases; especially the inoperable ones that do not respond to traditional cytotoxic therapies either. We asked whether PDT of a primary tumor could stimulate immune response that could attack the distant brain metastases. We have developed a mouse model of generating brain metastases by injecting CT26.CL25 tumor cells into the brain as well as injecting the same cancer cells under the skin at the same time. When the subcutaneous tumor was treated with PDT, we observed a survival advantage compared to mice that had untreated brain metastases alone.

Mouse genetic corneal disease resulting from transgenic insertional mutagenesis

PubMed Central

Ramalho, J S; Gregory-Evans, K; Huxley, C; Seabra, M C

2004-01-01

Background/aims: To report the generation of a new mouse model for a genetically determined corneal abnormality that occurred in transgenesis experiments. Methods: Transgenic mice expressing mutant forms of Rab27a, a GTPase that has been implicated in the pathogenesis of choroideremia, were generated. Results: Only one transgenic line (T27aT15) exhibited an unexpected eye phenotype. T27aT15 mice developed corneal opacities, usually unilateral, and cataracts, resulting in some cases in phthisical eyes. Histologically, the corneal stroma was thickened and vacuolated, and both epithelium and endothelium were thinned. The posterior segment of the eye was also affected with abnormal pigmentation, vessel narrowing, and abnormal leakage of dye upon angiography but was histologically normal. Conclusion: Eye abnormality in T27aT15 mice results from random insertional mutagenesis of the transgene as it was only observed in one line. The corneal lesion observed in T27aT15 mice most closely resembles posterior polymorphous corneal dystrophy and might result from the disruption of the equivalent mouse locus. PMID:14977782

Production of cloned mice by somatic cell nuclear transfer.

PubMed

Kishigami, Satoshi; Wakayama, Sayaka; Thuan, Nguyen Van; Ohta, Hiroshi; Mizutani, Eiji; Hikichi, Takafusa; Bui, Hong-Thuy; Balbach, Sebastian; Ogura, Atsuo; Boiani, Michele; Wakayama, Teruhiko

2006-01-01

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), the success rate for producing live offspring by cloning remains < 5%. Nevertheless, the techniques have potential as important tools for future research in basic biology. We have been able to develop a stable NT method in the mouse, in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Although manipulation of the piezo unit is complex, once mastered it is of great help not only in NT experiments but also in almost all other forms of micromanipulation. In addition to this technique, embryonic stem (ES) cell lines established from somatic cell nuclei by NT can be generated relatively easily from a variety of mouse genotypes and cell types. Such NT-ES cells can be used not only for experimental models of human therapeutic cloning but also as a backup of the donor cell's genome. Our most recent protocols for mouse cloning, as described here, will allow the production of cloned mice in > or = 3 months.

Targeting vector construction through recombineering.

PubMed

Malureanu, Liviu A

2011-01-01

Gene targeting in mouse embryonic stem cells is an essential, yet still very expensive and highly time-consuming, tool and method to study gene function at the organismal level or to create mouse models of human diseases. Conventional cloning-based methods have been largely used for generating targeting vectors, but are hampered by a number of limiting factors, including the variety and location of restriction enzymes in the gene locus of interest, the specific PCR amplification of repetitive DNA sequences, and cloning of large DNA fragments. Recombineering is a technique that exploits the highly efficient homologous recombination function encoded by λ phage in Escherichia coli. Bacteriophage-based recombination can recombine homologous sequences as short as 30-50 bases, allowing manipulations such as insertion, deletion, or mutation of virtually any genomic region. The large availability of mouse genomic bacterial artificial chromosome (BAC) libraries covering most of the genome facilitates the retrieval of genomic DNA sequences from the bacterial chromosomes through recombineering. This chapter describes a successfully applied protocol and aims to be a detailed guide through the steps of generation of targeting vectors through recombineering.

A Mouse Geneticist’s Practical Guide to CRISPR Applications

PubMed Central

Singh, Priti; Schimenti, John C.; Bolcun-Filas, Ewelina

2015-01-01

CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Even for the laboratory mouse, a model that has thrived under the benefits of embryonic stem (ES) cell knockout capabilities for nearly three decades, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology enables one to manipulate the genome with unprecedented simplicity and speed. It allows generation of null, conditional, precisely mutated, reporter, or tagged alleles in mice. Moreover, it holds promise for other applications beyond genome editing. The crux of this system is the efficient and targeted introduction of DNA breaks that are repaired by any of several pathways in a predictable but not entirely controllable manner. Thus, further optimizations and improvements are being developed. Here, we summarize current applications and provide a practical guide to use the CRISPR/Cas9 system for mouse mutagenesis, based on published reports and our own experiences. We discuss critical points and suggest technical improvements to increase efficiency of RNA-guided genome editing in mouse embryos and address practical problems such as mosaicism in founders, which complicates genotyping and phenotyping. We describe a next-generation sequencing strategy for simultaneous characterization of on- and off-target editing in mice derived from multiple CRISPR experiments. Additionally, we report evidence that elevated frequency of precise, homology-directed editing can be achieved by transient inhibition of the Ligase IV-dependent nonhomologous end-joining pathway in one-celled mouse embryos. PMID:25271304

Midbrain stimulation-evoked lumbar spinal activity in the adult decerebrate mouse.

PubMed

Stecina, Katinka

2017-08-15

Genetic techniques rendering murine models a popular choice for neuroscience research has led to important insights on neural networks controlling locomotor function. Using genetically altered mouse models for in vivo, electrophysiological studies in the adult state could validate key principles of locomotor network organization that have been described in neonatal, in vitro preparations. The experimental model presented here describes a decerebrate, in vivo adult mouse preparation in which focal, electrical midbrain stimulation was combined with monitoring lumbar neural activity and motor output after pre-collicular decerebration and neuromuscular blockade. Lumbar cord dorsum potentials (in 9/10 animals) and motoneuron output (in 3/5 animals) including fictive locomotion, was achieved by focal midbrain stimulation. The stimulation electrode locations could be reconstructed (in 6/7 animals) thereby allowing anatomical identification of the stimulated supraspinal regions. This preparation allows for concomitant recording or stimulation in the spinal cord and in the mid/hindbrain of adult mice. It differs from other methods used in the past with adult mice as it does not require pharmacological manipulation of neural excitability in order to generate motor output. Midbrain stimulation can consistently be used for inducing lumbar neural activity in adult mice under neuromuscular blockade. This model is suited for examination of brain-spinal connectivity and it may benefit a wide range of fields depending on the features of the genetically modified mouse models used in combination with the presented methods. Copyright © 2017 Elsevier B.V. All rights reserved.

Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo

PubMed Central

Najm, Fadi J.; Madhavan, Mayur; Zaremba, Anita; Shick, Elizabeth; Karl, Robert T.; Factor, Daniel C.; Miller, Tyler E.; Nevin, Zachary S.; Kantor, Christopher; Sargent, Alex; Quick, Kevin L.; Schlatzer, Daniela M.; Tang, Hong; Papoian, Ruben; Brimacombe, Kyle R.; Shen, Min; Boxer, Matthew B.; Jadhav, Ajit; Robinson, Andrew P.; Podojil, Joseph R.; Miller, Stephen D.; Miller, Robert H.; Tesar, Paul J.

2015-01-01

Multiple sclerosis (MS) involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system (CNS). Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells (OPCs) are stem cells in the CNS and the principal source of myelinating oligodendrocytes1. OPCs are abundant in demyelinated regions of MS patients, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention2. To discover therapeutic compounds for enhancing myelination from endogenous OPCs, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem cell (EpiSC)-derived OPCs3–5. We identified seven drugs that functioned at nanomolar doses to selectively enhance the generation of mature oligodendrocytes from OPCs in vitro. Two drugs, miconazole and clobetasol, were effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increased the number of new oligodendrocytes and enhanced remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in the experimental autoimmune encephalomyelitis (EAE) mouse model of chronic progressive MS resulted in striking reversal of disease severity. Immune response assays showed that miconazole functioned directly as a remyelinating drug with no effect on the immune system, whereas clobetasol was a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies showed that miconazole and clobetasol functioned in OPCs through mitogen-activated protein kinase (MAPK) and glucocorticoid receptor (GR) signaling, respectively. Furthermore, both drugs enhanced the generation of human oligodendrocytes from human OPCs in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally-modified derivatives, to enhance remyelination in patients. PMID:25896324

PM-07LOSS OF ATRX DECREASES SURVIVAL AND IMPROVES RESPONSE TO DNA DAMAGING AGENTS IN A NOVEL MOUSE MODEL OF GLIOBLASTOMA

PubMed Central

Koschmann, Carl; Calinescu, Alexandra; Thomas, Daniel; Kamran, Neha; Nunez-Aguilera, Felipe; Dzaman, Marta; Lemons, Rosie; Li, Youping; Roh, Haeji; Lowenstein, Pedro; Castro, Maria

2014-01-01

Pediatric glioblastoma (GBM) remains one of the most difficult childhood tumors to treat. ATRX is a histone chaperone protein that is mutated primarily in younger patients with GBM. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. We cloned an ATRX knockdown sequence into a Sleeping Beauty (SB) transposase-responsive plasmid (shATRX) for insertion into host genomic DNA. Glioblastomas were induced in mice by injecting plasmids encoding SB transposase/ luciferase, shp53 and NRAS, with or without shATRX, into the ventricle of neonatal mice. Tumors in both groups (with or without shATRX) showed histological hallmarks of human glioblastoma. The loss of ATRX was specifically localized only within tumors generated with the shATRX plasmid and not in the adjacent cortex. Notably, loss of ATRX reduced median survival of mice by 43% (p = 0.012). ATRX-deficient tumors were significantly more likely to develop microsatellite instability (p = 0.014), a hallmark of impaired DNA-damage repair. Analysis of three human GBM sequencing datasets confirmed increased number of somatic nucleotide mutations in ATRX-deficient tumors. Treatment of primary cell cultures generated from mouse GBMs showed that ATRX-deficient tumor cells are significantly more sensitive to DNA damaging agents. In addition, mice with ATRX-deficient GBM treated with whole brain irradiation had trend towards improved survival (p= 0.06), with some long-term survivors. Treated ATRX-deficient tumor cells showed greater evidence of double-stranded DNA breakage, by gH2A.X. In summary, this mouse model prospectively validates ATRX as a tumor suppressor in human GBM for the first time in an animal model. In addition, loss of ATRX leads to increased mutation frequency and response to DNA-damaging therapy. We have generated the hypothesis that ATRX loss leads to a genetically unstable tumor; which is more aggressive when untreated, but more responsive to DNA-damaging therapy, ultimately resulting in equivalent or improved overall survival.

Generation and gene expression profiling of 48 transcription-factor-inducible mouse embryonic stem cell lines.

PubMed

Yamamizu, Kohei; Sharov, Alexei A; Piao, Yulan; Amano, Misa; Yu, Hong; Nishiyama, Akira; Dudekula, Dawood B; Schlessinger, David; Ko, Minoru S H

2016-05-06

Mouse embryonic stem cells (ESCs) can differentiate into a wide range - and possibly all cell types in vitro, and thus provide an ideal platform to study systematically the action of transcription factors (TFs) in cell differentiation. Previously, we have generated and analyzed 137 TF-inducible mouse ESC lines. As an extension of this "NIA Mouse ESC Bank," we generated and characterized 48 additional mouse ESC lines, in which single TFs in each line could be induced in a doxycycline-controllable manner. Together, with the previous ESC lines, the bank now comprises 185 TF-manipulable ESC lines (>10% of all mouse TFs). Global gene expression (transcriptome) profiling revealed that the induction of individual TFs in mouse ESCs for 48 hours shifts their transcriptomes toward specific differentiation fates (e.g., neural lineages by Myt1 Isl1, and St18; mesodermal lineages by Pitx1, Pitx2, Barhl2, and Lmx1a; white blood cells by Myb, Etv2, and Tbx6, and ovary by Pitx1, Pitx2, and Dmrtc2). These data also provide and lists of inferred target genes of each TF and possible functions of these TFs. The results demonstrate the utility of mouse ESC lines and their transcriptome data for understanding the mechanism of cell differentiation and the function of TFs.

Cross-species genomics matches driver mutations and cell compartments to model ependymoma

PubMed Central

Johnson, Robert A.; Wright, Karen D.; Poppleton, Helen; Mohankumar, Kumarasamypet M.; Finkelstein, David; Pounds, Stanley B.; Rand, Vikki; Leary, Sarah E.S.; White, Elsie; Eden, Christopher; Hogg, Twala; Northcott, Paul; Mack, Stephen; Neale, Geoffrey; Wang, Yong-Dong; Coyle, Beth; Atkinson, Jennifer; DeWire, Mariko; Kranenburg, Tanya A.; Gillespie, Yancey; Allen, Jeffrey C.; Merchant, Thomas; Boop, Fredrick A.; Sanford, Robert. A.; Gajjar, Amar; Ellison, David W.; Taylor, Michael D.; Grundy, Richard G.; Gilbertson, Richard J.

2010-01-01

Understanding the biology that underlies histologically similar but molecularly distinct subgroups of cancer has proven difficult since their defining genetic alterations are often numerous, and the cellular origins of most cancers remain unknown1–3. We sought to decipher this heterogeneity by integrating matched genetic alterations and candidate cells of origin to generate accurate disease models. First, we identified subgroups of human ependymoma, a form of neural tumor that arises throughout the central nervous system (CNS). Subgroup specific alterations included amplifications and homozygous deletions of genes not yet implicated in ependymoma. To select cellular compartments most likely to give rise to subgroups of ependymoma, we matched the transcriptomes of human tumors to those of mouse neural stem cells (NSCs), isolated from different regions of the CNS at different developmental stages, with an intact or deleted Ink4a/Arf locus. The transcriptome of human cerebral ependymomas with amplified EPHB2 and deleted INK4A/ARF matched only that of embryonic cerebral Ink4a/Arf−/− NSCs. Remarkably, activation of Ephb2 signaling in these, but not other NSCs, generated the first mouse model of ependymoma, which is highly penetrant and accurately models the histology and transcriptome of one subgroup of human cerebral tumor. Further comparative analysis of matched mouse and human tumors revealed selective deregulation in the expression and copy number of genes that control synaptogenesis, pinpointing disruption of this pathway as a critical event in the production of this ependymoma subgroup. Our data demonstrate the power of cross-species genomics to meticulously match subgroup specific driver mutations with cellular compartments to model and interrogate cancer subgroups. PMID:20639864

Dissecting the Functions of Autophagy in Breast Cancer Associated Fibroblasts

DTIC Science & Technology

2014-10-01

compound transgenic mouse model of mammary cancer (MMTV-PyMT) harboring genetic deletion of Atg12 in stromal fibroblasts using the fibroblast specific...Cre;MMTV-PyMT mice (months 2-18). Using the breeding strategy outlined in Figure 1, we have successfully generated these quadruple transgenic mice...could then use for generating lysate and interrogation by Western blot (Fig. 7). However, our data suggest that the autophagy incompetent MMFs (from

Reduced generation of lung tissue–resident memory T cells during infancy

PubMed Central

Zens, Kyra D.; Chen, Jun Kui; Wu, Felix L.; Cvetkovski, Filip

2017-01-01

Infants suffer disproportionately from respiratory infections and generate reduced vaccine responses compared with adults, although the underlying mechanisms remain unclear. In adult mice, lung-localized, tissue-resident memory T cells (TRMs) mediate optimal protection to respiratory pathogens, and we hypothesized that reduced protection in infancy could be due to impaired establishment of lung TRM. Using an infant mouse model, we demonstrate generation of lung-homing, virus-specific T effectors after influenza infection or live-attenuated vaccination, similar to adults. However, infection during infancy generated markedly fewer lung TRMs, and heterosubtypic protection was reduced compared with adults. Impaired TRM establishment was infant–T cell intrinsic, and infant effectors displayed distinct transcriptional profiles enriched for T-bet–regulated genes. Notably, mouse and human infant T cells exhibited increased T-bet expression after activation, and reduction of T-bet levels in infant mice enhanced lung TRM establishment. Our findings reveal that infant T cells are intrinsically programmed for short-term responses, and targeting key regulators could promote long-term, tissue-targeted protection at this critical life stage. PMID:28855242

Development of a novel mouse glioma model using lentiviral vectors

PubMed Central

Marumoto, Tomotoshi; Tashiro, Ayumu; Friedmann-Morvinski, Dinorah; Scadeng, Miriam; Soda, Yasushi; Gage, Fred H; Verma, Inder M

2009-01-01

We report the development of a new method to induce glioblastoma multiforme in adult immunocompetent mice by injecting Cre-loxP–controlled lentiviral vectors expressing oncogenes. Cell type- or region-specific expression of activated forms of the oncoproteins Harvey-Ras and AKT in fewer than 60 glial fibrillary acidic protein–positive cells in the hippocampus, subventricular zone or cortex of mice heterozygous for the gene encoding the tumor suppressor Tp53 were tested. Mice developed glioblastoma multiforme when transduced either in the subventricular zone or the hippocampus. However, tumors were rarely detected when the mice were transduced in the cortex. Transplantation of brain tumor cells into naive recipient mouse brain resulted in the formation of glioblastoma multiforme–like tumors, which contained CD133+ cells, formed tumorspheres and could differentiate into neurons and astrocytes. We suggest that the use of Cre-loxP–controlled lentiviral vectors is a novel way to generate a mouse glioblastoma multiforme model in a region- and cell type-specific manner in adult mice. PMID:19122659

Disease Model Discovery from 3,328 Gene Knockouts by The International Mouse Phenotyping Consortium

PubMed Central

Meehan, Terrence F.; Conte, Nathalie; West, David B.; Jacobsen, Julius O.; Mason, Jeremy; Warren, Jonathan; Chen, Chao-Kung; Tudose, Ilinca; Relac, Mike; Matthews, Peter; Karp, Natasha; Santos, Luis; Fiegel, Tanja; Ring, Natalie; Westerberg, Henrik; Greenaway, Simon; Sneddon, Duncan; Morgan, Hugh; Codner, Gemma F; Stewart, Michelle E; Brown, James; Horner, Neil; Haendel, Melissa; Washington, Nicole; Mungall, Christopher J.; Reynolds, Corey L; Gallegos, Juan; Gailus-Durner, Valerie; Sorg, Tania; Pavlovic, Guillaume; Bower, Lynette R; Moore, Mark; Morse, Iva; Gao, Xiang; Tocchini-Valentini, Glauco P; Obata, Yuichi; Cho, Soo Young; Seong, Je Kyung; Seavitt, John; Beaudet, Arthur L.; Dickinson, Mary E.; Herault, Yann; Wurst, Wolfgang; de Angelis, Martin Hrabe; Lloyd, K.C. Kent; Flenniken, Ann M; Nutter, Lauryl MJ; Newbigging, Susan; McKerlie, Colin; Justice, Monica J.; Murray, Stephen A.; Svenson, Karen L.; Braun, Robert E.; White, Jacqueline K.; Bradley, Allan; Flicek, Paul; Wells, Sara; Skarnes, William C.; Adams, David J.; Parkinson, Helen; Mallon, Ann-Marie; Brown, Steve D.M.; Smedley, Damian

2017-01-01

Although next generation sequencing has revolutionised the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by our lack of knowledge of function and pathobiological mechanism for most genes. To address this challenge, the International Mouse Phenotyping Consortium (IMPC) is creating a genome- and phenome-wide catalogue of gene function by characterizing new knockout mouse strains across diverse biological systems through a broad set of standardised phenotyping tests, with all mice made readily available to the biomedical community. Analysing the first 3328 genes reveals models for 360 diseases including the first for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations are novel, providing the first functional evidence for 1092 genes and candidates in unsolved diseases such as Arrhythmogenic Right Ventricular Dysplasia 3. Finally, we describe our role in variant functional validation with the 100,000 Genomes and other projects. PMID:28650483

Colonoscopy-based colorectal cancer modeling in mice with CRISPR-Cas9 genome editing and organoid transplantation.

PubMed

Roper, Jatin; Tammela, Tuomas; Akkad, Adam; Almeqdadi, Mohammad; Santos, Sebastian B; Jacks, Tyler; Yilmaz, Ömer H

2018-02-01

Most genetically engineered mouse models (GEMMs) of colorectal cancer are limited by tumor formation in the small intestine, a high tumor burden that limits metastasis, and the need to generate and cross mutant mice. Cell line or organoid transplantation models generally produce tumors in ectopic locations-such as the subcutaneous space, kidney capsule, or cecal wall-that do not reflect the native stromal environment of the colon mucosa. Here, we describe detailed protocols to rapidly and efficiently induce site-directed tumors in the distal colon of mice that are based on colonoscopy-guided mucosal injection. These techniques can be adapted to deliver viral vectors carrying Cre recombinase, CRISPR-Cas9 components, CRISPR-engineered mouse tumor organoids, or human cancer organoids to mice to model the adenoma-carcinoma-metastasis sequence of tumor progression. The colonoscopy injection procedure takes ∼15 min, including preparation. In our experience, anyone with reasonable hand-eye coordination can become proficient with mouse colonoscopy and mucosal injection with a few hours of practice. These approaches are ideal for a wide range of applications, including assessment of gene function in tumorigenesis, examination of tumor-stroma interactions, studies of cancer metastasis, and translational research with patient-derived cancers.

Advantages of using the CRISPR/Cas9 system of genome editing to investigate male reproductive mechanisms using mouse models.

PubMed

Young, Samantha A M; Aitken, R John; Ikawa, Masahito

2015-01-01

Gene disruption technology has long been beneficial for the study of male reproductive biology. However, because of the time and cost involved, this technology was not a viable method except in specialist laboratories. The advent of the CRISPR/Cas9 system of gene disruption has ushered in a new era of genetic investigation. Now, it is possible to generate gene-disrupted mouse models in very little time and at very little cost. This Highlight article discusses the application of this technology to study the genetics of male fertility and looks at some of the future uses of this system that could be used to reveal the essential and nonessential genetic components of male reproductive mechanisms.

Nicotinamide N‐methyltransferase expression decreases in iron overload, exacerbating toxicity in mouse hepatocytes

PubMed Central

Koppe, Tiago; Patchen, Bonnie; Cheng, Aaron; Bhasin, Manoj; Vulpe, Chris; Schwartz, Robert E.; Moreno‐Navarrete, Jose Maria; Fernandez‐Real, Jose Manuel

2017-01-01

Iron overload causes the generation of reactive oxygen species that can lead to lasting damage to the liver and other organs. The goal of this study was to identify genes that modify the toxicity of iron overload. We studied the effect of iron overload on the hepatic transcriptional and metabolomic profile in mouse models using a dietary model of iron overload and a genetic model, the hemojuvelin knockout mouse. We then evaluated the correlation of nicotinamide N‐methyltransferase (NNMT) expression with body iron stores in human patients and the effect of NNMT knockdown on gene expression and viability in primary mouse hepatocytes. We found that iron overload induced significant changes in the expression of genes and metabolites involved in glucose and nicotinamide metabolism and that NNMT, an enzyme that methylates nicotinamide and regulates hepatic glucose and cholesterol metabolism, is one of the most strongly down‐regulated genes in the liver in both genetic and dietary iron overload. We found that hepatic NNMT expression is inversely correlated with serum ferritin levels and serum transferrin saturation in patients who are obese, suggesting that body iron stores regulate human liver NNMT expression. Furthermore, we demonstrated that adenoviral knockdown of NNMT in primary mouse hepatocytes exacerbates iron‐induced hepatocyte toxicity and increases expression of transcriptional markers of oxidative and endoplasmic reticulum stress, while overexpression of NNMT partially reversed these effects. Conclusion: Iron overload alters glucose and nicotinamide transcriptional and metabolic pathways in mouse hepatocytes and decreases NNMT expression, while NNMT deficiency worsens the toxic effect of iron overload. For these reasons, NNMT may be a drug target for the prevention of iron‐induced hepatotoxicity. (Hepatology Communications 2017;1:803–815) PMID:29404495

EMMA—mouse mutant resources for the international scientific community

PubMed Central

Wilkinson, Phil; Sengerova, Jitka; Matteoni, Raffaele; Chen, Chao-Kung; Soulat, Gaetan; Ureta-Vidal, Abel; Fessele, Sabine; Hagn, Michael; Massimi, Marzia; Pickford, Karen; Butler, Richard H.; Marschall, Susan; Mallon, Ann-Marie; Pickard, Amanda; Raspa, Marcello; Scavizzi, Ferdinando; Fray, Martin; Larrigaldie, Vanessa; Leyritz, Johan; Birney, Ewan; Tocchini-Valentini, Glauco P.; Brown, Steve; Herault, Yann; Montoliu, Lluis; de Angelis, Martin Hrabé; Smedley, Damian

2010-01-01

The laboratory mouse is the premier animal model for studying human disease and thousands of mutants have been identified or produced, most recently through gene-specific mutagenesis approaches. High throughput strategies by the International Knockout Mouse Consortium (IKMC) are producing mutants for all protein coding genes. Generating a knock-out line involves huge monetary and time costs so capture of both the data describing each mutant alongside archiving of the line for distribution to future researchers is critical. The European Mouse Mutant Archive (EMMA) is a leading international network infrastructure for archiving and worldwide provision of mouse mutant strains. It operates in collaboration with the other members of the Federation of International Mouse Resources (FIMRe), EMMA being the European component. Additionally EMMA is one of four repositories involved in the IKMC, and therefore the current figure of 1700 archived lines will rise markedly. The EMMA database gathers and curates extensive data on each line and presents it through a user-friendly website. A BioMart interface allows advanced searching including integrated querying with other resources e.g. Ensembl. Other resources are able to display EMMA data by accessing our Distributed Annotation System server. EMMA database access is publicly available at http://www.emmanet.org. PMID:19783817

Comprehensive behavioral and molecular characterization of a new knock-in mouse model of Huntington's disease: zQ175.

PubMed

Menalled, Liliana B; Kudwa, Andrea E; Miller, Sam; Fitzpatrick, Jon; Watson-Johnson, Judy; Keating, Nicole; Ruiz, Melinda; Mushlin, Richard; Alosio, William; McConnell, Kristi; Connor, David; Murphy, Carol; Oakeshott, Steve; Kwan, Mei; Beltran, Jose; Ghavami, Afshin; Brunner, Dani; Park, Larry C; Ramboz, Sylvie; Howland, David

2012-01-01

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, cognitive and psychiatric manifestations. Since the mutation responsible for the disease was identified as an unstable expansion of CAG repeats in the gene encoding the huntingtin protein in 1993, numerous mouse models of HD have been generated to study disease pathogenesis and evaluate potential therapeutic approaches. Of these, knock-in models best mimic the human condition from a genetic perspective since they express the mutation in the appropriate genetic and protein context. Behaviorally, however, while some abnormal phenotypes have been detected in knock-in mouse models, a model with an earlier and more robust phenotype than the existing models is required. We describe here for the first time a new mouse line, the zQ175 knock-in mouse, derived from a spontaneous expansion of the CAG copy number in our CAG 140 knock-in colony [1]. Given the inverse relationship typically observed between age of HD onset and length of CAG repeat, since this new mouse line carries a significantly higher CAG repeat length it was expected to be more significantly impaired than the parent line. Using a battery of behavioral tests we evaluated both heterozygous and homozygous zQ175 mice. Homozygous mice showed motor and grip strength abnormalities with an early onset (8 and 4 weeks of age, respectively), which were followed by deficits in rotarod and climbing activity at 30 weeks of age and by cognitive deficits at around 1 year of age. Of particular interest for translational work, we also found clear behavioral deficits in heterozygous mice from around 4.5 months of age, especially in the dark phase of the diurnal cycle. Decreased body weight was observed in both heterozygotes and homozygotes, along with significantly reduced survival in the homozygotes. In addition, we detected an early and significant decrease of striatal gene markers from 12 weeks of age. These data suggest that the zQ175 knock-in line could be a suitable model for the evaluation of therapeutic approaches and early events in the pathogenesis of HD.

Developing a Mouse Model of Sensory and Cognitive Deficits for Multiple Sclerosis

DTIC Science & Technology

2013-07-01

the plot is essentially horizontal, indicating that these mutants cannot localize sounds in space. A well-known characteristic of signal flow ...the five characteristic peaks and troughs of the ABR arising from generators in the eighth cranial nerve, cochlear nucleus, SOC, lateral lemniscus and

High-Frequency Ultrasound for the Study of Early Mouse Embryonic Cardiovascular System.

PubMed

Greco, Adelaide; Coda, Anna Rita Daniela; Albanese, Sandra; Ragucci, Monica; Liuzzi, Raffaele; Auletta, Luigi; Gargiulo, Sara; Lamagna, Francesco; Salvatore, Marco; Mancini, Marcello

2015-12-01

An accurate diagnosis of congenital heart defects during fetal development is critical for interventional planning. Mice can be used to generate animal models with heart defects, and high-frequency ultrasound (HFUS) imaging enables in utero imaging of live mouse embryos. A wide range of physiological measurements is possible using Doppler-HFUS imaging; limitations of any single measurement warrant a multiparameter approach to characterize cardiovascular function. Doppler-HFUS was used to explore the embryonic (heart, aorta) and extraembryonic (umbilical blood flow) circulatory systems to create a database in normal mouse embryos between 9.5 and 16.5 days of gestation. Multivariate analyses were performed to explore correlations between gestational age and embryo echocardiographic parameters. Heart rate and peak velocity in the aorta were positively correlated with gestational time, whereas cardiac cycle length, isovolumetric relaxation time, myocardial performance index, and arterial deceleration time of the umbilical cord were negatively correlated with it. Doppler-HFUS facilitated detailed characterization of the embryonic mouse circulation and represents a useful tool for investigation of the early mouse embryonic cardiovascular system. © The Author(s) 2015.

Practical use of advanced mouse models for lung cancer.

PubMed

Safari, Roghaiyeh; Meuwissen, Ralph

2015-01-01

To date a variety of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) mouse models have been developed that mimic human lung cancer. Chemically induced or spontaneous lung cancer in susceptible inbred strains has been widely used, but the more recent genetically engineered somatic mouse models recapitulate much better the genotype-phenotype correlations found in human lung cancer. Additionally, improved orthotopic transplantation of primary human cancer tissue fragments or cells into lungs of immune-compromised mice can be valuable tools for preclinical research such as antitumor drug tests. Here we give a short overview of most somatic mouse models for lung cancer that are currently in use. We accompany each different model with a description of its practical use and application for all major lung tumor types, as well as the intratracheal injection or direct injection of fresh or freeze-thawed tumor cells or tumor cell lines into lung parenchyma of recipient mice. All here presented somatic mouse models are based on the ability to (in) activate specific alleles at a time, and in a tissue-specific cell type, of choice. This spatial-temporal controlled induction of genetic lesions allows the selective introduction of main genetic lesions in an adult mouse lung as found in human lung cancer. The resulting conditional somatic mouse models can be used as versatile powerful tools in basic lung cancer research and preclinical translational studies alike. These distinctively advanced lung cancer models permit us to investigate initiation (cell of origin) and progression of lung cancer, along with response and resistance to drug therapy. Cre/lox or FLP/frt recombinase-mediated methods are now well-used techniques to develop tissue-restricted lung cancer in mice with tumor-suppressor gene and/or oncogene (in)activation. Intranasal or intratracheal administration of engineered adenovirus-Cre or lentivirus-Cre has been optimized for introducing Cre recombinase activity into pulmonary tissues, and we discuss here the different techniques underlying these applications. Concomitant with Cre/Flp recombinase-based models are the tetracycline (Tet)-inducible bitransgenic systems in which presence or absence of doxycycline can turn the expression of a specific oncogene on or off. The use of several Tet-inducible lung cancer models for NSCLC is presented here in which the reversal of oncogene expression led to complete tumor regression and provided us with important insight of how oncogene dependence influence lung cancer survival and growth. As alternative to Tet-inducible models, we discuss the application of reversible expressed, transgenic mutant estrogen receptor (ER) fusion proteins, which are regulated via systemic tamoxifen administration. Most of the various lung cancer models can be combined through the generation of transgenic compound mice so that the use of these somatic mouse models can be even more enhanced for the study of specific molecular pathways that facilitate growth and maintenance of lung cancer. Finally, this description of the practical application and methodology of mouse models for lung cancer should be helpful in assisting researchers to make the best choices and optimal use of (existing) somatic models that suits the specific experimental needs in their study of lung cancer.

The Next Generation of Orthotopic Thyroid Cancer Models: Immunocompetent Orthotopic Mouse Models of BRAFV600E-Positive Papillary and Anaplastic Thyroid Carcinoma

PubMed Central

Vanden Borre, Pierre; McFadden, David G.; Gunda, Viswanath; Sadow, Peter M.; Varmeh, Shohreh; Bernasconi, Maria; Jacks, Tyler

2014-01-01

Background: While the development of new treatments for aggressive thyroid cancer has advanced in the last 10 years, progress has trailed headways made with other malignancies. A lack of reliable authenticated human cell lines and reproducible animal models is one major roadblock to preclinical testing of novel therapeutics. Existing xenograft and orthotopic mouse models of aggressive thyroid cancer rely on the implantation of highly passaged human thyroid carcinoma lines in immunodeficient mice. Genetically engineered models of papillary and undifferentiated (anaplastic) thyroid carcinoma (PTC and ATC) are immunocompetent; however, slow and stochastic tumor development hinders high-throughput testing. Novel models of PTC and ATC in which tumors arise rapidly and synchronously in immunocompetent mice would facilitate the investigation of novel therapeutics and approaches. Methods: We characterized and utilized mouse cell lines derived from PTC and ATC tumors arising in genetically engineered mice with thyroid-specific expression of endogenous BrafV600E/WT and deletion of either Trp53 (p53) or Pten. These murine thyroid cancer cells were transduced with luciferase- and GFP-expressing lentivirus and implanted into the thyroid glands of immunocompetent syngeneic B6129SF1/J mice in which the growth characteristics were assessed. Results: Large locally aggressive thyroid tumors form within one week of implantation. Tumors recapitulate their histologic subtype, including well-differentiated PTC and ATC, and exhibit CD3+, CD8+, B220+, and CD163+ immune cell infiltration. Tumor progression can be followed in vivo using luciferase and ex vivo using GFP. Metastatic spread is not detected at early time points. Conclusions: We describe the development of the next generation of murine orthotopic thyroid cancer models. The implantation of genetically defined murine BRAF-mutated PTC and ATC cell lines into syngeneic mice results in rapid and synchronous tumor formation. This model allows for preclinical investigation of novel therapeutics and/or therapeutic combinations in the context of a functional immune system. PMID:24295207

A pink mouse reports the switch from red to green fluorescence upon Cre-mediated recombination.

PubMed

Hartwich, Heiner; Satheesh, Somisetty V; Nothwang, Hans Gerd

2012-06-14

Targeted genetic modification in the mouse becomes increasingly important in biomedical and basic science. This goal is most often achieved by use of the Cre/loxP system and numerous Cre-driver mouse lines are currently generated. Their initial characterization requires reporter mouse lines to study the in vivo spatiotemporal activity of Cre. Here, we report a dual fluorescence reporter mouse line, which switches expression from the red fluorescent protein mCherry to eGFP after Cre-mediated recombination. Both fluorescent proteins are expressed from the ubiquitously active and strong CAGGS promoter. Among the founders, we noticed a pink mouse line, expressing high levels of the red fluorescent protein mCherry throughout the entire body. Presence of mCherry in the living animal as well as in almost all organs was clearly visible without optical equipment. Upon Cre-activity, mCherry expression was switched to eGFP, demonstrating functionality of this reporter mouse line. The pink mouse presented here is an attractive novel reporter line for fluorescence-based monitoring of Cre-activity. The high expression of mCherry, which is visible to the naked eye, facilitates breeding and crossing, as no genotyping is required to identify mice carrying the reporter allele. The presence of two fluorescent proteins allows in vivo monitoring of recombined and non-recombined cells. Finally, the pink mouse is an eye-catching animal model to demonstrate the power of transgenic techniques in teaching courses.

Effects of WIN 55,212-2 (a synthetic cannabinoid CB1 and CB2 receptor agonist) on the anticonvulsant activity of various novel antiepileptic drugs against 6 Hz-induced psychomotor seizures in mice.

PubMed

Florek-Luszczki, Magdalena; Wlaz, Aleksandra; Zagaja, Mirosław; Andres-Mach, Marta; Kondrat-Wrobel, Maria W; Luszczki, Jarogniew J

2015-03-01

The purpose of this study was to determine the influence of WIN 55,212-2 mesylate (WIN-a non-selective cannabinoid CB1 and CB2 receptor agonist) on the anticonvulsant activity of various second- and third-generation antiepileptic drugs (i.e., gabapentin, lacosamide, levetiracetam, oxcarbazepine, pregabalin and tiagabine) in the mouse 6 Hz-induced psychomotor seizure model. Psychomotor seizures were evoked in albino Swiss mice by a current (32 mA, 6 Hz, 3s stimulus duration) delivered via ocular electrodes. Additionally, total brain antiepileptic drug concentrations were measured. Results indicate that WIN (5 mg/kg, administered i.p.) significantly potentiated the anticonvulsant action of gabapentin (P < 0.05) and levetiracetam (P < 0.01), but not that of lacosamide, oxcarbazepine, pregabalin or tiagabine in the mouse psychomotor seizure model. Moreover, WIN (2.5 mg/kg) had no significant effect on the anticonvulsant activity of all tested antiepileptic drugs in the 6 Hz test in mice. Measurement of total brain antiepileptic drug concentrations revealed that WIN (5 mg/kg) had no impact on gabapentin or levetiracetam total brain concentrations, indicating the pharmacodynamic nature of interaction between these antiepileptic drugs in the mouse 6Hz model. In conclusion, WIN in combination with gabapentin and levetiracetam exerts beneficial anticonvulsant pharmacodynamic interactions in the mouse psychomotor seizure model. Copyright © 2015 Elsevier Inc. All rights reserved.

Characterization of a genetically engineered mouse model of hemophilia A with complete deletion of the F8 gene.

PubMed

Chao, B N; Baldwin, W H; Healey, J F; Parker, E T; Shafer-Weaver, K; Cox, C; Jiang, P; Kanellopoulou, C; Lollar, P; Meeks, S L; Lenardo, M J

2016-02-01

ESSENTIALS: Anti-factor VIII (FVIII) inhibitory antibody formation is a severe complication in hemophilia A therapy. We genetically engineered and characterized a mouse model with complete deletion of the F8 coding region. F8(TKO) mice exhibit severe hemophilia, express no detectable F8 mRNA, and produce FVIII inhibitors. The defined background and lack of FVIII in F8(TKO) mice will aid in studying FVIII inhibitor formation. The most important complication in hemophilia A treatment is the development of inhibitory anti-Factor VIII (FVIII) antibodies in patients after FVIII therapy. Patients with severe hemophilia who express no endogenous FVIII (i.e. cross-reacting material, CRM) have the greatest incidence of inhibitor formation. However, current mouse models of severe hemophilia A produce low levels of truncated FVIII. The lack of a corresponding mouse model hampers the study of inhibitor formation in the complete absence of FVIII protein. We aimed to generate and characterize a novel mouse model of severe hemophilia A (designated the F8(TKO) strain) lacking the complete coding sequence of F8 and any FVIII CRM. Mice were created on a C57BL/6 background using Cre-Lox recombination and characterized using in vivo bleeding assays, measurement of FVIII activity by coagulation and chromogenic assays, and anti-FVIII antibody production using ELISA. All F8 exonic coding regions were deleted from the genome and no F8 mRNA was detected in F8(TKO) mice. The bleeding phenotype of F8(TKO) mice was comparable to E16 mice by measurements of factor activity and tail snip assay. Similar levels of anti-FVIII antibody titers after recombinant FVIII injections were observed between F8(TKO) and E16 mice. We describe a new C57BL/6 mouse model for severe hemophilia A patients lacking CRM. These mice can be directly bred to the many C57BL/6 strains of genetically engineered mice, which is valuable for studying the impact of a wide variety of genes on FVIII inhibitor formation on a defined genetic background. © 2015 International Society on Thrombosis and Haemostasis.

Generation and Characterization of a New Monoclonal Antibody Against CXCL4

PubMed Central

Gao, Jing; Wu, Mingyuan; Gao, Jin; Wang, Xia; Zhang, Yang; Zhu, Shunying; Yu, Yan

2015-01-01

CXCL4 plays important roles in numerous disease processes, which makes the CXCL4 signaling pathway a potential therapeutic target. In this study, we aimed to develop a neutralizing antibody against both human and mouse CXCL4. Rats were immunized with recombinant human CXCL4 (rhCXCL4). Hybridoma clones were created by fusion of the immunized rat spleen cells with mouse myeloma SP2/0 cells and screened using recombinant mouse CXCL4 (rmCXCL4) and rhCXCL4. The CXCL4 monoclonal antibody (CXCL4 MAb) produced by the 16D6-3 hybridoma clone was sequenced and characterized by Western blot and Biacore assays. It recognized both human and mouse CXCL4 with high affinity and neutralized the effect of rhCXCL4 in vitro. Thus, the antibody may be used in the studies of CXCL4 in murine disease models and as a template in the antibody humanization for clinical developments. PMID:25897609

Experimental evidence showing that no mitotically active female germline progenitors exist in postnatal mouse ovaries.

PubMed

Zhang, Hua; Zheng, Wenjing; Shen, Yan; Adhikari, Deepak; Ueno, Hiroo; Liu, Kui

2012-07-31

It has been generally accepted for more than half a century that, in most mammalian species, oocytes cannot renew themselves in postnatal or adult life, and that the number of oocytes is already fixed in fetal or neonatal ovaries. This assumption, however, has been challenged over the past decade. In this study, we have taken an endogenous genetic approach to this question and generated a multiple fluorescent Rosa26(rbw/+);Ddx4-Cre germline reporter mouse model for in vivo and in vitro tracing of the development of female germline cell lineage. Through live cell imaging and de novo folliculogenesis experiments, we show that the Ddx4-expressing cells from postnatal mouse ovaries did not enter mitosis, nor did they contribute to oocytes during de novo folliculogenesis. Our results provide evidence that supports the traditional view that no postnatal follicular renewal occurs in mammals, and no mitotically active Ddx4-expressing female germline progenitors exist in postnatal mouse ovaries.

A mouse model for MERS coronavirus-induced acute respiratory distress syndrome.

PubMed

Cockrell, Adam S; Yount, Boyd L; Scobey, Trevor; Jensen, Kara; Douglas, Madeline; Beall, Anne; Tang, Xian-Chun; Marasco, Wayne A; Heise, Mark T; Baric, Ralph S

2016-11-28

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel virus that emerged in 2012, causing acute respiratory distress syndrome (ARDS), severe pneumonia-like symptoms and multi-organ failure, with a case fatality rate of ∼36%. Limited clinical studies indicate that humans infected with MERS-CoV exhibit pathology consistent with the late stages of ARDS, which is reminiscent of the disease observed in patients infected with severe acute respiratory syndrome coronavirus. Models of MERS-CoV-induced severe respiratory disease have been difficult to achieve, and small-animal models traditionally used to investigate viral pathogenesis (mouse, hamster, guinea-pig and ferret) are naturally resistant to MERS-CoV. Therefore, we used CRISPR-Cas9 gene editing to modify the mouse genome to encode two amino acids (positions 288 and 330) that match the human sequence in the dipeptidyl peptidase 4 receptor, making mice susceptible to MERS-CoV infection and replication. Serial MERS-CoV passage in these engineered mice was then used to generate a mouse-adapted virus that replicated efficiently within the lungs and evoked symptoms indicative of severe ARDS, including decreased survival, extreme weight loss, decreased pulmonary function, pulmonary haemorrhage and pathological signs indicative of end-stage lung disease. Importantly, therapeutic countermeasures comprising MERS-CoV neutralizing antibody treatment or a MERS-CoV spike protein vaccine protected the engineered mice against MERS-CoV-induced ARDS.

Activation and inhibition of mouse muscle and neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

PubMed

Papke, Roger L; Wecker, Lynn; Stitzel, Jerry A

2010-05-01

Transgenic mouse models with nicotinic acetylcholine receptor (nAChR) knockouts and knockins have provided important insights into the molecular substrates of addiction and disease. However, most studies of heterologously expressed neuronal nAChR have used clones obtained from other species, usually human or rat. In this work, we use mouse clones expressed in Xenopus oocytes to provide a relatively comprehensive characterization of the three primary classes of nAChR: muscle-type receptors, heteromeric neuronal receptors, and homomeric alpha7-type receptors. We evaluated the activation of these receptor subtypes with acetylcholine and cytisine-related compounds, including varenicline. We also characterized the activity of classic nAChR antagonists, confirming the utility of mecamylamine and dihydro-beta-erythroidine as selective antagonists in mouse models of alpha3beta4 and alpha4beta2 receptors, respectively. We also conducted an in-depth analysis of decamethonium and hexamethonium on muscle and neuronal receptor subtypes. Our data indicate that, as with receptors cloned from other species, pairwise expression of neuronal alpha and beta subunits in oocytes generates heterogeneous populations of receptors, most likely caused by variations in subunit stoichiometry. Coexpression of the mouse alpha5 subunit had varying effects, depending on the other subunits expressed. The properties of cytisine-related compounds are similar for mouse, rat, and human nAChR, except that varenicline produced greater residual inhibition of mouse alpha4beta2 receptors than with human receptors. We confirm that decamethonium is a partial agonist, selective for muscle-type receptors, but also note that it is a nondepolarizing antagonist for neuronal-type receptors. Hexamethonium was a relatively nonselective antagonist with mixed competitive and noncompetitive activity.

Activation and Inhibition of Mouse Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes

PubMed Central

Wecker, Lynn; Stitzel, Jerry A.

2010-01-01

Transgenic mouse models with nicotinic acetylcholine receptor (nAChR) knockouts and knockins have provided important insights into the molecular substrates of addiction and disease. However, most studies of heterologously expressed neuronal nAChR have used clones obtained from other species, usually human or rat. In this work, we use mouse clones expressed in Xenopus oocytes to provide a relatively comprehensive characterization of the three primary classes of nAChR: muscle-type receptors, heteromeric neuronal receptors, and homomeric α7-type receptors. We evaluated the activation of these receptor subtypes with acetylcholine and cytisine-related compounds, including varenicline. We also characterized the activity of classic nAChR antagonists, confirming the utility of mecamylamine and dihydro-β-erythroidine as selective antagonists in mouse models of α3β4 and α4β2 receptors, respectively. We also conducted an in-depth analysis of decamethonium and hexamethonium on muscle and neuronal receptor subtypes. Our data indicate that, as with receptors cloned from other species, pairwise expression of neuronal α and β subunits in oocytes generates heterogeneous populations of receptors, most likely caused by variations in subunit stoichiometry. Coexpression of the mouse α5 subunit had varying effects, depending on the other subunits expressed. The properties of cytisine-related compounds are similar for mouse, rat, and human nAChR, except that varenicline produced greater residual inhibition of mouse α4β2 receptors than with human receptors. We confirm that decamethonium is a partial agonist, selective for muscle-type receptors, but also note that it is a nondepolarizing antagonist for neuronal-type receptors. Hexamethonium was a relatively nonselective antagonist with mixed competitive and noncompetitive activity. PMID:20100906

An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers.

PubMed

Fisher, Cynthia L; Marks, Hendrik; Cho, Lily Ting-Yin; Andrews, Robert; Wormald, Sam; Carroll, Thomas; Iyer, Vivek; Tate, Peri; Rosen, Barry; Stunnenberg, Hendrik G; Fisher, Amanda G; Skarnes, William C

2017-12-01

Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivating both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele inactivation in conditional-ready IKMC 'knockout-first' ES cell lines. We applied our technology to several epigenetic regulators, recovering bi-allelic targeted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the 'knockout-first' allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibility of our system, enabling tamoxifen inducible conditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or culturing in naive pluripotency '2i' media, suggesting that the self-renewal defect is mediated through pluripotency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Yanlan; Chen, Yicheng; Ding, Guoqing

The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacymore » and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.« less

Hypoxic pulmonary vasoconstriction in isolated mouse pulmonary arterial vessels.

PubMed

Strielkov, Ievgen; Krause, Nicole Catherine; Sommer, Natasha; Schermuly, Ralph Theo; Ghofrani, Hossein Ardeschir; Grimminger, Friedrich; Gudermann, Thomas; Dietrich, Alexander; Weissmann, Norbert

2018-06-19

What is the central question of this study? Hypoxic pulmonary vasoconstriction has never been characterized in isolated mouse pulmonary arteries of different generations in detail. What is the main finding and its importance? We found that only small intrapulmonary arteries (80 - 200 μm in diameter) exhibit hypoxic pulmonary vasoconstriction. The observed response was sustained, significantly potentiated by depolarization-induced preconstriction, and not dependent on endothelium and TRPC6 channels. Hypoxic pulmonary vasoconstriction (HPV) is a physiological response of pulmonary arteries, which adapts lung perfusion to regional ventilation. Properties of hypoxic pulmonary vasoconstriction (HPV) vary significantly between animal species. Despite extensive use of mouse models in studies of HPV, this physiological response has never been characterized in isolated mouse pulmonary arteries in detail. We investigated the effect of 80-min exposure to hypoxia on tone in mouse pulmonary arteries of different generations in the presence and absence of preconstriction using wire myography. Hypoxia induced a sustained relaxation in non-preconstricted extrapulmonary arteries (500 - 700 μm in diameter), but not in the presence of KCl-induced preconstriction. Large intrapulmonary arteries (450 - 650 μm) did not exhibit a significant response to the hypoxic challenge. By contrast, in small intrapulmonary arteries (80 - 200 μm), hypoxia elicited a slowly developing sustained constriction, which was independent of endothelium. The response was significantly potentiated in arteries preconstricted with KCl, but not with U46619. HPV was not altered in pulmonary arteries of TRPC6-deficient mice, which suggests that this response corresponds to the sustained phase of biphasic HPV observed earlier in isolated, buffer-perfused, and ventilated mouse lungs. In conclusion, we have established the protocol allowing to study sustained HPV in isolated mouse pulmonary arteries. The obtained data may be useful for future studies of HPV mechanisms in mice. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A novel humanized mouse model of Huntington disease for preclinical development of therapeutics targeting mutant huntingtin alleles.

PubMed

Southwell, Amber L; Skotte, Niels H; Villanueva, Erika B; Østergaard, Michael E; Gu, Xiaofeng; Kordasiewicz, Holly B; Kay, Chris; Cheung, Daphne; Xie, Yuanyun; Waltl, Sabine; Dal Cengio, Louisa; Findlay-Black, Hailey; Doty, Crystal N; Petoukhov, Eugenia; Iworima, Diepiriye; Slama, Ramy; Ooi, Jolene; Pouladi, Mahmoud A; Yang, X William; Swayze, Eric E; Seth, Punit P; Hayden, Michael R

2017-03-15

Huntington disease (HD) is a neurodegenerative disease caused by a mutation in the huntingtin (HTT) gene. HTT is a large protein, interacts with many partners and is involved in many cellular pathways, which are perturbed in HD. Therapies targeting HTT directly are likely to provide the most global benefit. Thus there is a need for preclinical models of HD recapitulating human HTT genetics. We previously generated a humanized mouse model of HD, Hu97/18, by intercrossing BACHD and YAC18 mice with knockout of the endogenous mouse HD homolog (Hdh). Hu97/18 mice recapitulate the genetics of HD, having two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of Caucasian descent. We have now generated a companion model, Hu128/21, by intercrossing YAC128 and BAC21 mice on the Hdh-/- background. Hu128/21 mice have two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of East Asian descent and in a minority of patients from other ethnic groups. Hu128/21 mice display a wide variety of HD-like phenotypes that are similar to YAC128 mice. Additionally, both transgenes in Hu128/21 mice match the human HTT exon 1 reference sequence. Conversely, the BACHD transgene carries a floxed, synthetic exon 1 sequence. Hu128/21 mice will be useful for investigations of human HTT that cannot be addressed in Hu97/18 mice, for developing therapies targeted to exon 1, and for preclinical screening of personalized HTT lowering therapies in HD patients of East Asian descent. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Development of a Sox2 reporter system modeling cellular heterogeneity in glioma.

PubMed

Stoltz, Kevin; Sinyuk, Maksim; Hale, James S; Wu, Qiulian; Otvos, Balint; Walker, Kiera; Vasanji, Amit; Rich, Jeremy N; Hjelmeland, Anita B; Lathia, Justin D

2015-03-01

Malignant gliomas are complex systems containing a number of factors that drive tumor initiation and progression, including genetic aberrations that lead to extensive cellular heterogeneity within the neoplastic compartment. Mouse models recapitulate these genetic aberrations, but readily observable heterogeneity remains challenging. To interrogate cellular heterogeneity in mouse glioma models, we utilized a replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor/tumor virus A (RCAS-tva) system to generate spontaneous mouse gliomas that contained a Sox2-enhanced green fluorescent protein (EGFP) reporter. Glial fibrillary acidic protein-tva mice were crossed with Sox2-EGFP mice, and tumors were initiated that contained a subpopulation of Sox2-EGFP-high cells enriched for tumor-initiating cell properties such as self-renewal, multilineage differentiation potential, and perivascular localization. Following implantation into recipient mice, Sox2-EGFP-high cells generated tumors containing Sox2-EGFP-high and Sox2-EGFP-low cells. Kinomic analysis of Sox2-EGFP-high cells revealed activation of known glioma signaling pathways that are strongly correlated with patient survival including platelet-derived growth factor receptor beta, phosphoinositide-3 kinase, and vascular endothelial growth factor. Our functional analysis identified active feline sarcoma (Fes) signaling in Sox2-EGFP-high cells. Fes negatively correlated with glioma patient survival and was coexpressed with Sox2-positive cells in glioma xenografts and primary patient-derived tissue. Our RCAS-tva/Sox2-EGFP model will empower closer examination of cellular heterogeneity and will be useful for identifying novel glioma pathways as well as testing preclinical treatment efficacy. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Airway disease phenotypes in animal models of cystic fibrosis.

PubMed

McCarron, Alexandra; Donnelley, Martin; Parsons, David

2018-04-02

In humans, cystic fibrosis (CF) lung disease is characterised by chronic infection, inflammation, airway remodelling, and mucus obstruction. A lack of pulmonary manifestations in CF mouse models has hindered investigations of airway disease pathogenesis, as well as the development and testing of potential therapeutics. However, recently generated CF animal models including rat, ferret and pig models demonstrate a range of well characterised lung disease phenotypes with varying degrees of severity. This review discusses the airway phenotypes of currently available CF animal models and presents potential applications of each model in airway-related CF research.

Gad67 haploinsufficiency reduces amyloid pathology and rescues olfactory memory deficits in a mouse model of Alzheimer's disease.

PubMed

Wang, Yue; Wu, Zheng; Bai, Yu-Ting; Wu, Gang-Yi; Chen, Gong

2017-10-10

Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder, affecting millions of people worldwide. Although dysfunction of multiple neurotransmitter systems including cholinergic, glutamatergic and GABAergic systems has been associated with AD progression the underlying mechanisms remain elusive. We and others have recently found that GABA content is elevated in AD brains and linked to cognitive deficits in AD mouse models. The glutamic acid decarboxylase 67 (GAD67) is the major enzyme converting glutamate into GABA and has been implied in a number of neurological disorders such as epilepsy and schizophrenia. However, whether Gad67 is involved in AD pathology has not been well studied. Here, we investigate the functional role of GAD67 in an AD mouse model with Gad67 haploinsufficiency that is caused by replacing one allele of Gad67 with green fluorescent protein (GFP) gene during generation of GAD67-GFP mice. To genetically reduce GAD67 in AD mouse brains, we crossed the Gad67 haploinsufficient mice (GAD67-GFP +/- ) with 5xFAD mice (harboring 5 human familial AD mutations in APP and PS1 genes) to generate a new line of bigenic mice. Immunostaining, ELISA, electrophysiology and behavior test were applied to compare the difference between groups. We found that reduction of GAD67 resulted in a significant decrease of amyloid β production in 5xFAD mice. Concurrently, the abnormal astrocytic GABA and tonic GABA currents, as well as the microglial reactivity were significantly reduced in the 5xFAD mice with Gad67 haploinsufficiency. Importantly, the olfactory memory deficit of 5xFAD mice was rescued by Gad67 haploinsufficiency. Our results demonstrate that GAD67 plays an important role in AD pathology, suggesting that GAD67 may be a potential drug target for modulating the progress of AD.

Standardized Profiling of The Membrane-Enriched Proteome of Mouse Dorsal Root Ganglia (DRG) Provides Novel Insights Into Chronic Pain.

PubMed

Rouwette, Tom; Sondermann, Julia; Avenali, Luca; Gomez-Varela, David; Schmidt, Manuela

2016-06-01

Chronic pain is a complex disease with limited treatment options. Several profiling efforts have been employed with the aim to dissect its molecular underpinnings. However, generated results are often inconsistent and nonoverlapping, which is largely because of inherent technical constraints. Emerging data-independent acquisition (DIA)-mass spectrometry (MS) has the potential to provide unbiased, reproducible and quantitative proteome maps - a prerequisite for standardization among experiments. Here, we designed a DIA-based proteomics workflow to profile changes in the abundance of dorsal root ganglia (DRG) proteins in two mouse models of chronic pain, inflammatory and neuropathic. We generated a DRG-specific spectral library containing 3067 DRG proteins, which enables their standardized quantification by means of DIA-MS in any laboratory. Using this resource, we profiled 2526 DRG proteins in each biological replicate of both chronic pain models and respective controls with unprecedented reproducibility. We detected numerous differentially regulated proteins, the majority of which exhibited pain model-specificity. Our approach recapitulates known biology and discovers dozens of proteins that have not been characterized in the somatosensory system before. Functional validation experiments and analysis of mouse pain behaviors demonstrate that indeed meaningful protein alterations were discovered. These results illustrate how the application of DIA-MS can open new avenues to achieve the long-awaited standardization in the molecular dissection of pathologies of the somatosensory system. Therefore, our findings provide a valuable framework to qualitatively extend our understanding of chronic pain and somatosensation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Electron microscopy and in vitro deneddylation reveal similar architectures and biochemistry of isolated human and Flag-mouse COP9 signalosome complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rockel, Beate; Schmaler, Tilo; Huang, Xiaohua

2014-07-25

Highlights: • Deneddylation rates of human erythrocyte and mouse fibroblast CSN are very similar. • 3D models of native human and mouse CSN reveal common architectures. • The cryo-structure of native mammalian CSN shows a horseshoe subunit arrangement. - Abstract: The COP9 signalosome (CSN) is a regulator of the ubiquitin (Ub) proteasome system (UPS). In the UPS, proteins are Ub-labeled for degradation by Ub ligases conferring substrate specificity. The CSN controls a large family of Ub ligases called cullin-RING ligases (CRLs), which ubiquitinate cell cycle regulators, transcription factors and DNA damage response proteins. The CSN possesses structural similarities with themore » 26S proteasome Lid complex and the translation initiation complex 3 (eIF3) indicating similar ancestry and function. Initial structures were obtained 14 years ago by 2D electron microscopy (EM). Recently, first 3D molecular models of the CSN were created on the basis of negative-stain EM and single-particle analysis, mostly with recombinant complexes. Here, we compare deneddylating activity and structural features of CSN complexes purified in an elaborate procedure from human erythrocytes and efficiently pulled down from mouse Flag-CSN2 B8 fibroblasts. In an in vitro deneddylation assay both the human and the mouse CSN complexes deneddylated Nedd8-Cul1 with comparable rates. 3D structural models of the erythrocyte CSN as well as of the mouse Flag-CSN were generated by negative stain EM and by cryo-EM. Both complexes show a central U-shaped segment from which several arms emanate. This structure, called the horseshoe, is formed by the PCI domain subunits. CSN5 and CSN6 point away from the horseshoe. Compared to 3D models of negatively stained CSN complexes, densities assigned to CSN2 and CSN4 are better defined in the cryo-map. Because biochemical and structural results obtained with CSN complexes isolated from human erythrocytes and purified by Flag-CSN pulldown from mouse B8 fibroblasts are very similar, Flag-CSN pulldowns are a proper alternative to CSN preparation from erythrocytes.« less

Generating mouse models of degenerative diseases using Cre/lox-mediated in vivo mosaic cell ablation

PubMed Central

Fujioka, Masato; Tokano, Hisashi; Fujioka, Keiko Shiina; Okano, Hideyuki; Edge, Albert S.B.

2011-01-01

Most degenerative diseases begin with a gradual loss of specific cell types before reaching a threshold for symptomatic onset. However, the endogenous regenerative capacities of different tissues are difficult to study, because of the limitations of models for early stages of cell loss. Therefore, we generated a transgenic mouse line (Mos-iCsp3) in which a lox-mismatched Cre/lox cassette can be activated to produce a drug-regulated dimerizable caspase-3. Tissue-restricted Cre expression yielded stochastic Casp3 expression, randomly ablating a subset of specific cell types in a defined domain. The limited and mosaic cell loss led to distinct responses in 3 different tissues targeted using respective Cre mice: reversible, impaired glucose tolerance with normoglycemia in pancreatic β cells; wound healing and irreversible hair loss in the skin; and permanent moderate deafness due to the loss of auditory hair cells in the inner ear. These mice will be important for assessing the repair capacities of tissues and the potential effectiveness of new regenerative therapies. PMID:21576819

In vivo time-serial multi-modality optical imaging in a mouse model of ovarian tumorigenesis

PubMed Central

Watson, Jennifer M; Marion, Samuel L; Rice, Photini F; Bentley, David L; Besselsen, David G; Utzinger, Urs; Hoyer, Patricia B; Barton, Jennifer K

2014-01-01

Identification of the early microscopic changes associated with ovarian cancer may lead to development of a diagnostic test for high-risk women. In this study we use optical coherence tomography (OCT) and multiphoton microscopy (MPM) (collecting both two photon excited fluorescence [TPEF] and second harmonic generation [SHG]) to image mouse ovaries in vivo at multiple time points. We demonstrate the feasibility of imaging mouse ovaries in vivo during a long-term survival study and identify microscopic changes associated with early tumor development. These changes include alterations in tissue microstructure, as seen by OCT, alterations in cellular fluorescence and morphology, as seen by TPEF, and remodeling of collagen structure, as seen by SHG. These results suggest that a combined OCT-MPM system may be useful for early detection of ovarian cancer. PMID:24145178

In vivo three-dimensional optical coherence tomography and multiphoton microscopy in a mouse model of ovarian neoplasia

NASA Astrophysics Data System (ADS)

Watson, Jennifer M.; Marion, Samuel L.; Rice, Photini Faith; Bentley, David L.; Besselsen, David; Utzinger, Urs; Hoyer, Patricia B.; Barton, Jennifer K.

2013-03-01

Our goal is to use optical coherence tomography (OCT) and multiphoton microscopy (MPM) to detect early tumor development in a mouse model of ovarian neoplasia. We hope to use information regarding early tumor development to create a diagnostic test for high-risk patients. In this study we collect in vivo images using OCT, second harmonic generation and two-photon excited fluorescence from non-vinylcyclohexene diepoxide (VCD)-dosed and VCD-dosed mice. VCD causes follicular apoptosis (simulating menopause) and leads to tumor development. Using OCT and MPM we visualized the ovarian microstructure and were able to see differences between non-VCD-dosed and VCD-dosed animals. This leads us to believe that OCT and MPM may be useful for detecting changes due to early tumor development.

Role of catalase overproduction in drug resistance and virulence in Candida albicans.

PubMed

Román, Elvira; Prieto, Daniel; Martin, Ry; Correia, Inês; Mesa Arango, Ana Cecilia; Alonso-Monge, Rebeca; Zaragoza, Oscar; Pla, Jesús

2016-10-03

To investigate the role of Cat1 overproduction in Candida albicans. Strains overproducing the CAT1 gene were constructed. Cells overproducing CAT1 were found to be more resistant to some oxidants and mammalian phagocytic cells. They also showed reduced intracellular reactive oxygen species generated by amphotericin B or ciclopirox olamine. CAT1 overproduction did not change the minimum inhibitory concentration of fungal cells to fungistatic or fungicidal azoles nor to amphotericin B although increased twofold the minimum inhibitory concentration to caspofungin. The role of Cat1 overproduction in virulence and colonization was also analyzed in mouse models. The overproduction of Cat1 protects against oxidants, phagocytes and certain antifungals at subinhibitory concentration but does not increase virulence in a systemic infection mouse model.

Type I interferon signals in macrophages and dendritic cells control dengue virus infection: implications for a new mouse model to test dengue vaccines.

PubMed

Züst, Roland; Toh, Ying-Xiu; Valdés, Iris; Cerny, Daniela; Heinrich, Julia; Hermida, Lisset; Marcos, Ernesto; Guillén, Gerardo; Kalinke, Ulrich; Shi, Pei-Yong; Fink, Katja

2014-07-01

Dengue virus (DENV) infects an estimated 400 million people every year, causing prolonged morbidity and sometimes mortality. Development of an effective vaccine has been hampered by the lack of appropriate small animal models; mice are naturally not susceptible to DENV and only become infected if highly immunocompromised. Mouse models lacking both type I and type II interferon (IFN) receptors (AG129 mice) or the type I IFN receptor (IFNAR(-/-) mice) are susceptible to infection with mouse-adapted DENV strains but are severely impaired in mounting functional immune responses to the virus and thus are of limited use for study. Here we used conditional deletion of the type I IFN receptor (IFNAR) on individual immune cell subtypes to generate a minimally manipulated mouse model that is susceptible to DENV while retaining global immune competence. Mice lacking IFNAR expression on CD11c(+) dendritic cells and LysM(+) macrophages succumbed completely to DENV infection, while mice deficient in the receptor on either CD11c(+) or LysM(+) cells were susceptible to infection but often resolved viremia and recovered fully from infection. Conditional IFNAR mice responded with a swift and strong CD8(+) T-cell response to viral infection, compared to a weak response in IFNAR(-/-) mice. Furthermore, mice lacking IFNAR on either CD11c(+) or LysM(+) cells were also sufficiently immunocompetent to raise a protective immune response to a candidate subunit vaccine against DENV-2. These data demonstrate that mice with conditional deficiencies in expression of the IFNAR represent improved models for the study of DENV immunology and screening of vaccine candidates. Dengue virus infects 400 million people every year worldwide, causing 100 million clinically apparent infections, which can be fatal if untreated. Despite many years of research, there are no effective vaccine and no antiviral treatment available for dengue. Development of vaccines has been hampered in particular by the lack of a suitable small animal model. Mouse models used to test dengue vaccine are deficient in interferon (IFN) type I signaling and severely immunocompromised and therefore likely not ideal for the testing of vaccines. In this study, we explored alternative models lacking the IFN receptor only on certain cell types. We show that mice lacking the IFN receptor on either CD11c- or LysM-expressing cells (conditional IFNAR mice) are susceptible to dengue virus infection. Importantly, we demonstrate that conditional IFN receptor knockout mice generate a better immune response to live virus and a candidate dengue vaccine compared to IFNAR mice and are resistant to subsequent challenge. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Performing label-fusion-based segmentation using multiple automatically generated templates.

PubMed

Chakravarty, M Mallar; Steadman, Patrick; van Eede, Matthijs C; Calcott, Rebecca D; Gu, Victoria; Shaw, Philip; Raznahan, Armin; Collins, D Louis; Lerch, Jason P

2013-10-01

Classically, model-based segmentation procedures match magnetic resonance imaging (MRI) volumes to an expertly labeled atlas using nonlinear registration. The accuracy of these techniques are limited due to atlas biases, misregistration, and resampling error. Multi-atlas-based approaches are used as a remedy and involve matching each subject to a number of manually labeled templates. This approach yields numerous independent segmentations that are fused using a voxel-by-voxel label-voting procedure. In this article, we demonstrate how the multi-atlas approach can be extended to work with input atlases that are unique and extremely time consuming to construct by generating a library of multiple automatically generated templates of different brains (MAGeT Brain). We demonstrate the efficacy of our method for the mouse and human using two different nonlinear registration algorithms (ANIMAL and ANTs). The input atlases consist a high-resolution mouse brain atlas and an atlas of the human basal ganglia and thalamus derived from serial histological data. MAGeT Brain segmentation improves the identification of the mouse anterior commissure (mean Dice Kappa values (κ = 0.801), but may be encountering a ceiling effect for hippocampal segmentations. Applying MAGeT Brain to human subcortical structures improves segmentation accuracy for all structures compared to regular model-based techniques (κ = 0.845, 0.752, and 0.861 for the striatum, globus pallidus, and thalamus, respectively). Experiments performed with three manually derived input templates suggest that MAGeT Brain can approach or exceed the accuracy of multi-atlas label-fusion segmentation (κ = 0.894, 0.815, and 0.895 for the striatum, globus pallidus, and thalamus, respectively). Copyright © 2012 Wiley Periodicals, Inc.

Optimization of Protocols for Derivation of Mouse Embryonic Stem Cell Lines from Refractory Strains, Including the Non Obese Diabetic Mouse

PubMed Central

Davies, Timothy J.

2012-01-01

The derivation of pluripotent embryonic stem cells (ESCs) from a variety of genetic backgrounds remains a desirable objective in the generation of mice functionally deficient in genes of interest and the modeling of human disease. Nevertheless, disparity in the ease with which different strains of mice yield ESC lines has long been acknowledged. Indeed, the generation of bona fide ESCs from the non obese diabetic (NOD) mouse, a well-characterized model of human type I diabetes, has historically proved especially difficult to achieve. Here, we report the development of protocols for the derivation of novel ESC lines from C57Bl/6 mice based on the combined use of high concentrations of leukemia inhibitory factor and serum-replacement, which is equally applicable to fresh and cryo-preserved embryos. Further, we demonstrate the success of this approach using Balb/K and CBA/Ca mice, widely considered to be refractory strains. CBA/Ca ESCs contributed to the somatic germ layers of chimeras and displayed a very high competence at germline transmission. Importantly, we were able to use the same protocol for the derivation of ESC lines from nonpermissive NOD mice. These ESCs displayed a normal karyotype that was robustly stable during long-term culture, were capable of forming teratomas in vivo and germline competent chimeras after injection into recipient blastocysts. Further, these novel ESC lines efficiently formed embryoid bodies in vitro and could be directed in their differentiation along the dendritic cell lineage, thus illustrating their potential application to the generation of cell types of relevance to the pathogenesis of type I diabetes. PMID:21933027

Fertile offspring from sterile sex chromosome trisomic mice§

PubMed Central

Hirota, Takayuki; Ohta, Hiroshi; Powell, Benjamin E.; Mahadevaiah, Shantha K.; Ojarikre, Obah A.; Saitou, Mitinori; Turner, James M. A.

2017-01-01

Having the correct number of chromosomes is vital for normal development and health. Sex chromosome trisomy (SCT) affects 0.1% of the human population and is associated with infertility. We show that during reprogramming to induced pluripotent stem cells (iPSC), fibroblasts from sterile trisomic XXY and XYY mice lose the extra sex chromosome, by a phenomenon we term trisomy-biased chromosome loss (TCL). Resulting euploid XY iPSCs can be differentiated into the male germ cell lineage and functional sperm that can be used in intracytoplasmic sperm injection to produce chromosomally normal, fertile offspring. Sex chromosome loss is comparatively infrequent during mouse XX and XY iPSC generation. TCL also applies to other chromosomes, generating euploid iPSCs from cells of a Down syndrome mouse model. It can also create euploid iPSCs from human trisomic patient fibroblasts. The findings have relevance to overcoming infertility and other trisomic phenotypes. PMID:28818972

Infectious Progression of Canine Distemper Virus from Circulating Cerebrospinal Fluid into the Central Nervous System.

PubMed

Takenaka, Akiko; Sato, Hiroki; Ikeda, Fusako; Yoneda, Misako; Kai, Chieko

2016-10-15

In the current study, we generated recombinant chimeric canine distemper viruses (CDVs) by replacing the hemagglutinin (H) and/or phosphoprotein (P) gene in an avirulent strain expressing enhanced green fluorescent protein (EGFP) with those of a mouse-adapted neurovirulent strain. An in vitro experimental infection indicated that the chimeric CDVs possessing the H gene derived from the mouse-adapted CDV acquired infectivity for neural cells. These cells lack the CDV receptors that have been identified to date (SLAM and nectin-4), indicating that the H protein defines infectivity in various cell lines. The recombinant viruses were administered intracerebrally to 1-week-old mice. Fatal neurological signs of disease were observed only with a recombinant CDV that possessed both the H and P genes of the mouse-adapted strain, similar to the parental mouse-adapted strain, suggesting that both genes are important to drive virulence of CDV in mice. Using this recombinant CDV, we traced the intracerebral propagation of CDV by detecting EGFP. Widespread infection was observed in the cerebral hemispheres and brainstems of the infected mice. In addition, EGFP fluorescence in the brain slices demonstrated a sequential infectious progression in the central nervous system: CDV primarily infected the neuroependymal cells lining the ventricular wall and the neurons of the hippocampus and cortex adjacent to the ventricle, and it then progressed to an extensive infection of the brain surface, followed by the parenchyma and cortex. In the hippocampal formation, CDV spread in a unidirectional retrograde pattern along neuronal processes in the hippocampal formation from the CA1 region to the CA3 region and the dentate gyrus. Our mouse model demonstrated that the main target cells of CDV are neurons in the acute phase and that the virus spreads via neuronal transmission pathways in the hippocampal formation. CDV is the etiological agent of distemper in dogs and other carnivores, and in many respects, the pathogenesis of CDV infection in animals resembles that of measles virus infection in humans. We successfully generated a recombinant CDV containing the H and P genes from a mouse-adapted neurovirulent strain and expressing EGFP. The recombinant CDV exhibited severe neurovirulence with high mortality, comparable to the parental mouse-adapted strain. The mouse-infectious model could become a useful tool for analyzing CDV infection of the central nervous system subsequent to passing through the blood-cerebrospinal fluid barrier and infectious progression in the target cells in acute disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Infectious Progression of Canine Distemper Virus from Circulating Cerebrospinal Fluid into the Central Nervous System

PubMed Central

Takenaka, Akiko; Sato, Hiroki; Ikeda, Fusako; Yoneda, Misako

2016-01-01

ABSTRACT In the current study, we generated recombinant chimeric canine distemper viruses (CDVs) by replacing the hemagglutinin (H) and/or phosphoprotein (P) gene in an avirulent strain expressing enhanced green fluorescent protein (EGFP) with those of a mouse-adapted neurovirulent strain. An in vitro experimental infection indicated that the chimeric CDVs possessing the H gene derived from the mouse-adapted CDV acquired infectivity for neural cells. These cells lack the CDV receptors that have been identified to date (SLAM and nectin-4), indicating that the H protein defines infectivity in various cell lines. The recombinant viruses were administered intracerebrally to 1-week-old mice. Fatal neurological signs of disease were observed only with a recombinant CDV that possessed both the H and P genes of the mouse-adapted strain, similar to the parental mouse-adapted strain, suggesting that both genes are important to drive virulence of CDV in mice. Using this recombinant CDV, we traced the intracerebral propagation of CDV by detecting EGFP. Widespread infection was observed in the cerebral hemispheres and brainstems of the infected mice. In addition, EGFP fluorescence in the brain slices demonstrated a sequential infectious progression in the central nervous system: CDV primarily infected the neuroependymal cells lining the ventricular wall and the neurons of the hippocampus and cortex adjacent to the ventricle, and it then progressed to an extensive infection of the brain surface, followed by the parenchyma and cortex. In the hippocampal formation, CDV spread in a unidirectional retrograde pattern along neuronal processes in the hippocampal formation from the CA1 region to the CA3 region and the dentate gyrus. Our mouse model demonstrated that the main target cells of CDV are neurons in the acute phase and that the virus spreads via neuronal transmission pathways in the hippocampal formation. IMPORTANCE CDV is the etiological agent of distemper in dogs and other carnivores, and in many respects, the pathogenesis of CDV infection in animals resembles that of measles virus infection in humans. We successfully generated a recombinant CDV containing the H and P genes from a mouse-adapted neurovirulent strain and expressing EGFP. The recombinant CDV exhibited severe neurovirulence with high mortality, comparable to the parental mouse-adapted strain. The mouse-infectious model could become a useful tool for analyzing CDV infection of the central nervous system subsequent to passing through the blood-cerebrospinal fluid barrier and infectious progression in the target cells in acute disease. PMID:27489268

Effect of the Antioxidant Lipoic Acid in Aortic Phenotype in a Marfan Syndrome Mouse Model.

PubMed

Guido, Maria C; Debbas, Victor; Salemi, Vera M; Tavares, Elaine R; Meirelles, Thayna; Araujo, Thaís L S; Nolasco, Patricia; Ferreira-Filho, Julio C A; Takimura, Celso K; Pereira, Lygia V; Laurindo, Francisco R

2018-01-01

Marfan syndrome (MFS) cardiovascular manifestations such as aortic aneurysms and cardiomyopathy carry substantial morbidity/mortality. We investigated the effects of lipoic acid, an antioxidant, on ROS production and aortic remodeling in a MFS mgΔ loxPneo mouse model. MFS and WT (wild-type) 1-month-old mice were allocated to 3 groups: untreated, treated with losartan, and treated with lipoic acid. At 6 months old, echocardiography, ROS production, and morphological analysis of aortas were performed. Aortic ROS generation in 6-month-old MFS animals was higher at advanced stages of disease in MFS. An unprecedented finding in MFS mice analyzed by OCT was the occurrence of focal inhomogeneous regions in the aortic arch, either collagen-rich extremely thickened or collagen-poor hypotrophic regions. MFS animals treated with lipoic acid showed markedly reduced ROS production and lower ERK1/2 phosphorylation; meanwhile, aortic dilation and elastic fiber breakdown were unaltered. Of note, lipoic acid treatment associated with the absence of focal inhomogeneous regions in MFS animals. Losartan reduced aortic dilation and elastic fiber breakdown despite no change in ROS generation. In conclusion, oxidant generation by itself seems neutral with respect to aneurysm progression in MFS; however, lipoic acid-mediated reduction of inhomogeneous regions may potentially associate with less anisotropy and reduced chance of dissection/rupture.

Somatic cell hybrid mapping on mouse chromosome 11 (MMU11): Assignment of markers relative to two breakpoints in band D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morris, D.J.; Robinson, T.J.; Adler, I.D.

1993-02-01

Mouse [times] rat somatic cell hybrids were generated by fusing mouse cell lines that are heterozygous for reciprocal translocations involving the T42H and T9Ad breakpoints on mouse chromosome 11 (MMU11) to a thymidine kinase-negative (Tk[sup [minus

EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss.

PubMed

Sampson, John H; Choi, Bryan D; Sanchez-Perez, Luis; Suryadevara, Carter M; Snyder, David J; Flores, Catherine T; Schmittling, Robert J; Nair, Smita K; Reap, Elizabeth A; Norberg, Pamela K; Herndon, James E; Kuan, Chien-Tsun; Morgan, Richard A; Rosenberg, Steven A; Johnson, Laura A

2014-02-15

Chimeric antigen receptor (CAR) transduced T cells represent a promising immune therapy that has been shown to successfully treat cancers in mice and humans. However, CARs targeting antigens expressed in both tumors and normal tissues have led to significant toxicity. Preclinical studies have been limited by the use of xenograft models that do not adequately recapitulate the immune system of a clinically relevant host. A constitutively activated mutant of the naturally occurring epidermal growth factor receptor (EGFRvIII) is antigenically identical in both human and mouse glioma, but is also completely absent from any normal tissues. We developed a third-generation, EGFRvIII-specific murine CAR (mCAR), and performed tests to determine its efficacy in a fully immunocompetent mouse model of malignant glioma. At elevated doses, infusion with EGFRvIII mCAR T cells led to cures in all mice with brain tumors. In addition, antitumor efficacy was found to be dependent on lymphodepletive host conditioning. Selective blockade with EGFRvIII soluble peptide significantly abrogated the activity of EGFRvIII mCAR T cells in vitro and in vivo, and may offer a novel strategy to enhance the safety profile for CAR-based therapy. Finally, mCAR-treated, cured mice were resistant to rechallenge with EGFRvIII(NEG) tumors, suggesting generation of host immunity against additional tumor antigens. All together, these data support that third-generation, EGFRvIII-specific mCARs are effective against gliomas in the brain and highlight the importance of syngeneic, immunocompetent models in the preclinical evaluation of tumor immunotherapies. ©2013 AACR

Insights about Striatal Circuit Function and Schizophrenia from a Mouse Model of D2 Receptor Upregulation

PubMed Central

Simpson, Eleanor H.; Kellendonk, Christoph

2016-01-01

The dopamine hypothesis of schizophrenia is supported by a large number of imaging studies that have identified an increase in dopamine binding at the D2 receptor selectively in the striatum. Here we review a decade of work using a regionally restricted and temporally regulated transgenic mouse model to investigate the behavioral, molecular, electrophysiological, and anatomical consequences of selective D2 receptor upregulation in the striatum. These studies have identified new and potentially important biomarkers at the circuit and molecular level that can now be explored in patients with schizophrenia. They provide an example of how animal models and their detailed level of neurobiological analysis allow a deepening of our understanding of the relationship between neuronal circuit function and symptoms of schizophrenia, and as a consequence generate new hypotheses that are testable in patients. PMID:27720388

Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice.

PubMed

Murphy, Andrew J; Macdonald, Lynn E; Stevens, Sean; Karow, Margaret; Dore, Anthony T; Pobursky, Kevin; Huang, Tammy T; Poueymirou, William T; Esau, Lakeisha; Meola, Melissa; Mikulka, Warren; Krueger, Pamela; Fairhurst, Jeanette; Valenzuela, David M; Papadopoulos, Nicholas; Yancopoulos, George D

2014-04-08

Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.

Epidermal growth factor receptor as a novel molecular target for aggressive papillary tumors in the middle ear and temporal bone

PubMed Central

Kawabata, Shigeru; Christine Hollander, M; Munasinghe, Jeeva P.; Brinster, Lauren R.; Mercado-Matos, José R.; Li, Jie; Regales, Lucia; Pao, William; Jänne, Pasi A.; Wong, Kwok-Kin; Butman, John A.; Lonser, Russell R.; Hansen, Marlan R.; Gurgel, Richard K.; Vortmeyer, Alexander O.; Dennis, Phillip A.

2015-01-01

Adenomatous tumors in the middle ear and temporal bone are rare but highly morbid because they are difficult to detect prior to the development of audiovestibular dysfunction. Complete resection is often disfiguring and difficult because of location and the late stage at diagnosis, so identification of molecular targets and effective therapies is needed. Here, we describe a new mouse model of aggressive papillary ear tumor that was serendipitously discovered during the generation of a mouse model for mutant EGFR-driven lung cancer. Although these mice did not develop lung tumors, 43% developed head tilt and circling behavior. Magnetic resonance imaging (MRI) scans showed bilateral ear tumors located in the tympanic cavity. These tumors expressed mutant EGFR as well as active downstream targets such as Akt, mTOR and ERK1/2. EGFR-directed therapies were highly effective in eradicating the tumors and correcting the vestibular defects, suggesting these tumors are addicted to EGFR. EGFR activation was also observed in human ear neoplasms, which provides clinical relevance for this mouse model and rationale to test EGFR-targeted therapies in these rare neoplasms. PMID:26027747

Epidermal growth factor receptor as a novel molecular target for aggressive papillary tumors in the middle ear and temporal bone.

PubMed

Kawabata, Shigeru; Hollander, M Christine; Munasinghe, Jeeva P; Brinster, Lauren R; Mercado-Matos, José R; Li, Jie; Regales, Lucia; Pao, William; Jänne, Pasi A; Wong, Kwok-Kin; Butman, John A; Lonser, Russell R; Hansen, Marlan R; Gurgel, Richard K; Vortmeyer, Alexander O; Dennis, Phillip A

2015-05-10

Adenomatous tumors in the middle ear and temporal bone are rare but highly morbid because they are difficult to detect prior to the development of audiovestibular dysfunction. Complete resection is often disfiguring and difficult because of location and the late stage at diagnosis, so identification of molecular targets and effective therapies is needed. Here, we describe a new mouse model of aggressive papillary ear tumor that was serendipitously discovered during the generation of a mouse model for mutant EGFR-driven lung cancer. Although these mice did not develop lung tumors, 43% developed head tilt and circling behavior. Magnetic resonance imaging (MRI) scans showed bilateral ear tumors located in the tympanic cavity. These tumors expressed mutant EGFR as well as active downstream targets such as Akt, mTOR and ERK1/2. EGFR-directed therapies were highly effective in eradicating the tumors and correcting the vestibular defects, suggesting these tumors are addicted to EGFR. EGFR activation was also observed in human ear neoplasms, which provides clinical relevance for this mouse model and rationale to test EGFR-targeted therapies in these rare neoplasms.

Landscape resistance and habitat combine to provide an optimal model of genetic structure and connectivity at the range margin of a small mammal.

PubMed

Marrotte, R R; Gonzalez, A; Millien, V

2014-08-01

We evaluated the effect of habitat and landscape characteristics on the population genetic structure of the white-footed mouse. We develop a new approach that uses numerical optimization to define a model that combines site differences and landscape resistance to explain the genetic differentiation between mouse populations inhabiting forest patches in southern Québec. We used ecological distance computed from resistance surfaces with Circuitscape to infer the effect of the landscape matrix on gene flow. We calculated site differences using a site index of habitat characteristics. A model that combined site differences and resistance distances explained a high proportion of the variance in genetic differentiation and outperformed models that used geographical distance alone. Urban and agriculture-related land uses were, respectively, the most and the least resistant landscape features influencing gene flow. Our method detected the effect of rivers and highways as highly resistant linear barriers. The density of grass and shrubs on the ground best explained the variation in the site index of habitat characteristics. Our model indicates that movement of white-footed mouse in this region is constrained along routes of low resistance. Our approach can generate models that may improve predictions of future northward range expansion of this small mammal. © 2014 John Wiley & Sons Ltd.

Generation and characterisation of a parkin-Pacrg knockout mouse line and a Pacrg knockout mouse line.

PubMed

Stephenson, Sarah E M; Aumann, Timothy D; Taylor, Juliet M; Riseley, Jessica R; Li, Ruili; Mann, Jeffrey R; Tomas, Doris; Lockhart, Paul J

2018-05-14

Mutations in PARK2 (parkin) can result in Parkinson's disease (PD). Parkin shares a bidirectional promoter with parkin coregulated gene (PACRG) and the transcriptional start sites are separated by only ~200 bp. Bidirectionally regulated genes have been shown to function in common biological pathways. Mice lacking parkin have largely failed to recapitulate the dopaminergic neuronal loss and movement impairments seen in individuals with parkin-mediated PD. We aimed to investigate the function of PACRG and test the hypothesis that parkin and PACRG function in a common pathway by generating and characterizing two novel knockout mouse lines harbouring loss of both parkin and Pacrg or Pacrg alone. Successful modification of the targeted allele was confirmed at the genomic, transcriptional and steady state protein levels for both genes. At 18-20 months of age, there were no significant differences in the behaviour of parental and mutant lines when assessed by openfield, rotarod and balance beam. Subsequent neuropathological examination suggested there was no gross abnormality of the dopaminergic system in the substantia nigra and no significant difference in the number of dopaminergic neurons in either knockout model compared to wildtype mice.

Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models

PubMed Central

Ramalho, José S; Anders, Ross; Jaissle, Gesine B; Seeliger, Mathias W; Huxley, Clare; Seabra, Miguel C

2002-01-01

Background Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM), a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1) gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases) plays an essential role in the retinal degenerative process. Results To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal. Conclusions We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases. PMID:12401133

A methodology for the investigation of toughness and crack propagation in mouse bone.

PubMed

Carriero, Alessandra; Zimmermann, Elizabeth A; Shefelbine, Sandra J; Ritchie, Robert O

2014-11-01

Bone fracture is a health concern for those with aged bone and brittle bone diseases. Mouse bone is widely used as a model of human bone, especially to investigate preclinical treatment strategies. However, little is known about the mechanisms of mouse bone fracture and its similarities and differences from fracture in human bone. In this work we present a methodology to investigate the fracture toughness during crack initiation and crack propagation for mouse bone. Mouse femora were dissected, polished on their periosteal surface, notched on the posterior surface at their mid-diaphysis, and tested in three-point bending under displacement control at a rate of 0.1mm/min using an in situ loading stage within an environmental scanning electron microscope. We obtained high-resolution real-time imaging of the crack initiation and propagation in mouse bone. From the images we can measure the crack extension at each step of the crack growth and calculate the toughness of the bone (in terms of stress intensity factor (K) and work to fracture (Wf)) as a function of stable crack length (Δa), thus generating a resistance curve for the mouse bone. The technique presented here provides insight into the evolution of microdamage and the toughening mechanisms that resist crack propagation, which are essential for preclinical development of treatments to enhance bone quality and combat fracture risk. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evaluation of five diffeomorphic image registration algorithms for mouse brain magnetic resonance microscopy.

PubMed

Fu, Zhenrong; Lin, Lan; Tian, Miao; Wang, Jingxuan; Zhang, Baiwen; Chu, Pingping; Li, Shaowu; Pathan, Muhammad Mohsin; Deng, Yulin; Wu, Shuicai

2017-11-01

The development of genetically engineered mouse models for neuronal diseases and behavioural disorders have generated a growing need for small animal imaging. High-resolution magnetic resonance microscopy (MRM) provides powerful capabilities for noninvasive studies of mouse brains, while avoiding some limits associated with the histological procedures. Quantitative comparison of structural images is a critical step in brain imaging analysis, which highly relies on the performance of image registration techniques. Nowadays, there is a mushrooming growth of human brain registration algorithms, while fine-tuning of those algorithms for mouse brain MRMs is rarely addressed. Because of their topology preservation property and outstanding performance in human studies, diffeomorphic transformations have become popular in computational anatomy. In this study, we specially tuned five diffeomorphic image registration algorithms [DARTEL, geodesic shooting, diffeo-demons, SyN (Greedy-SyN and geodesic-SyN)] for mouse brain MRMs and evaluated their performance using three measures [volume overlap percentage (VOP), residual intensity error (RIE) and surface concordance ratio (SCR)]. Geodesic-SyN performed significantly better than the other methods according to all three different measures. These findings are important for the studies on structural brain changes that may occur in wild-type and transgenic mouse brains. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

Genome Editing in Mice Using TALE Nucleases.

PubMed

Wefers, Benedikt; Brandl, Christina; Ortiz, Oskar; Wurst, Wolfgang; Kühn, Ralf

2016-01-01

Gene engineering for generating targeted mouse mutants is a key technology for biomedical research. Using TALENs as sequence-specific nucleases to induce targeted double-strand breaks, the mouse genome can be directly modified in zygotes in a single step without the need for embryonic stem cells. By embryo microinjection of TALEN mRNAs and targeting vectors, knockout and knock-in alleles can be generated fast and efficiently. In this chapter we provide protocols for the application of TALENs in mouse zygotes.

Using Apex To Construct CPM-GOMS Models

NASA Technical Reports Server (NTRS)

John, Bonnie; Vera, Alonso; Matessa, Michael; Freed, Michael; Remington, Roger

2006-01-01

process for automatically generating computational models of human/computer interactions as well as graphical and textual representations of the models has been built on the conceptual foundation of a method known in the art as CPM-GOMS. This method is so named because it combines (1) the task decomposition of analysis according to an underlying method known in the art as the goals, operators, methods, and selection (GOMS) method with (2) a model of human resource usage at the level of cognitive, perceptual, and motor (CPM) operations. CPM-GOMS models have made accurate predictions about behaviors of skilled computer users in routine tasks, but heretofore, such models have been generated in a tedious, error-prone manual process. In the present process, CPM-GOMS models are generated automatically from a hierarchical task decomposition expressed by use of a computer program, known as Apex, designed previously to be used to model human behavior in complex, dynamic tasks. An inherent capability of Apex for scheduling of resources automates the difficult task of interleaving the cognitive, perceptual, and motor resources that underlie common task operators (e.g., move and click mouse). The user interface of Apex automatically generates Program Evaluation Review Technique (PERT) charts, which enable modelers to visualize the complex parallel behavior represented by a model. Because interleaving and the generation of displays to aid visualization are automated, it is now feasible to construct arbitrarily long sequences of behaviors. The process was tested by using Apex to create a CPM-GOMS model of a relatively simple human/computer-interaction task and comparing the time predictions of the model and measurements of the times taken by human users in performing the various steps of the task. The task was to withdraw $80 in cash from an automated teller machine (ATM). For the test, a Visual Basic mockup of an ATM was created, with a provision for input from (and measurement of the performance of) the user via a mouse. The times predicted by the automatically generated model turned out to approximate the measured times fairly well (see figure). While these results are promising, there is need for further development of the process. Moreover, it will also be necessary to test other, more complex models: The actions required of the user in the ATM task are too sequential to involve substantial parallelism and interleaving and, hence, do not serve as an adequate test of the unique strength of CPM-GOMS models to accommodate parallelism and interleaving.

The Ptch1DL mouse: a new model to study lambdoid craniosynostosis and basal cell nevus syndrome associated skeletal defects

PubMed Central

Feng, Weiguo; Choi, Irene; Clouthier, David E.; Niswander, Lee; Williams, Trevor

2013-01-01

Mouse models provide valuable opportunities for probing the underlying pathology of human birth defects. Employing an ENU-based screen for recessive mutations affecting craniofacial anatomy we isolated a mouse strain, Dogface-like (DL), with abnormal skull and snout morphology. Examination of the skull indicated that these mice developed craniosynostosis of the lambdoid suture. Further analysis revealed skeletal defects related to the pathology of basal cell nevus syndrome (BCNS) including defects in development of the limbs, scapula, ribcage, secondary palate, cranial base, and cranial vault. In humans, BCNS is often associated with mutations in the Hedgehog receptor PTCH1 and genetic mapping in DL identified a point mutation at a splice donor site in Ptch1. Using genetic complementation analysis we determined that DL is a hypomorphic allele of Ptch1, leading to increased Hedgehog signaling. Two aberrant transcripts are generated by the mutated Ptch1DL gene, which would be predicted to reduce significantly the levels of functional Patched1 protein. This new Ptch1 allele broadens the mouse genetic reagents available to study the Hedgehog pathway and provides a valuable means to study the underlying skeletal abnormalities in BCNS. In addition, these results strengthen the connection between elevated Hedgehog signaling and craniosynostosis. PMID:23897749

Four factors underlying mouse behavior in an open field

PubMed Central

Tanaka, Shoji; Young, Jared W.; Halberstadt, Adam L.; Masten, Virginia L.; Geyer, Mark A.

2012-01-01

The observation of the locomotor and exploratory behaviors of rodents in an open field is one of the most fundamental methods used in the field of behavioral pharmacology. A variety of behaviors can be recorded automatically and can readily generate a multivariate pattern of pharmacological effects. Nevertheless, the optimal ways to characterize observed behaviors and concomitant drug effects are still under development. The aim of this study was to extract meaningful behavioral factors that could explain variations in the observed variables from mouse exploration. Behavioral data were recorded from male C57BL/6J mice (n = 268) using the Behavioral Pattern Monitor (BPM). The BPM data were subjected to the exploratory factor analysis. The factor analysis extracted four factors: activity, sequential organization, diversive exploration, and inspective exploration. The activity factor and the two types of exploration factors correlated positively with one another, while the sequential organization factor negatively correlated with the remaining factors. The extracted factor structure constitutes a behavioral model of mouse exploration. This model will provide a platform on which one can assess the effects of psychoactive drugs and genetic manipulations on mouse exploratory behavior. Further studies are currently underway to examine the factor structure of similar multivariate data sets from humans tested in a human BPM. PMID:22569582

Four factors underlying mouse behavior in an open field.

PubMed

Tanaka, Shoji; Young, Jared W; Halberstadt, Adam L; Masten, Virginia L; Geyer, Mark A

2012-07-15

The observation of the locomotor and exploratory behaviors of rodents in an open field is one of the most fundamental methods used in the field of behavioral pharmacology. A variety of behaviors can be recorded automatically and can readily generate a multivariate pattern of pharmacological effects. Nevertheless, the optimal ways to characterize observed behaviors and concomitant drug effects are still under development. The aim of this study was to extract meaningful behavioral factors that could explain variations in the observed variables from mouse exploration. Behavioral data were recorded from male C57BL/6J mice (n=268) using the Behavioral Pattern Monitor (BPM). The BPM data were subjected to the exploratory factor analysis. The factor analysis extracted four factors: activity, sequential organization, diversive exploration, and inspective exploration. The activity factor and the two types of exploration factors correlated positively with one another, while the sequential organization factor negatively correlated with the remaining factors. The extracted factor structure constitutes a behavioral model of mouse exploration. This model will provide a platform on which one can assess the effects of psychoactive drugs and genetic manipulations on mouse exploratory behavior. Further studies are currently underway to examine the factor structure of similar multivariate data sets from humans tested in a human BPM. Copyright © 2012 Elsevier B.V. All rights reserved.

Role of AMACR (α-methylacyl-CoA racemase) and MFE-1 (peroxisomal multifunctional enzyme-1) in bile acid synthesis in mice.

PubMed

Autio, Kaija J; Schmitz, Werner; Nair, Remya R; Selkälä, Eija M; Sormunen, Raija T; Miinalainen, Ilkka J; Crick, Peter J; Wang, Yuqin; Griffiths, William J; Reddy, Janardan K; Baes, Myriam; Hiltunen, J Kalervo

2014-07-01

Cholesterol is catabolized to bile acids by peroxisomal β-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type 2), in which this β-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type 1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.

Generative models for network neuroscience: prospects and promise

PubMed Central

Betzel, Richard F.

2017-01-01

Network neuroscience is the emerging discipline concerned with investigating the complex patterns of interconnections found in neural systems, and identifying principles with which to understand them. Within this discipline, one particularly powerful approach is network generative modelling, in which wiring rules are algorithmically implemented to produce synthetic network architectures with the same properties as observed in empirical network data. Successful models can highlight the principles by which a network is organized and potentially uncover the mechanisms by which it grows and develops. Here, we review the prospects and promise of generative models for network neuroscience. We begin with a primer on network generative models, with a discussion of compressibility and predictability, and utility in intuiting mechanisms, followed by a short history on their use in network science, broadly. We then discuss generative models in practice and application, paying particular attention to the critical need for cross-validation. Next, we review generative models of biological neural networks, both at the cellular and large-scale level, and across a variety of species including Caenorhabditis elegans, Drosophila, mouse, rat, cat, macaque and human. We offer a careful treatment of a few relevant distinctions, including differences between generative models and null models, sufficiency and redundancy, inferring and claiming mechanism, and functional and structural connectivity. We close with a discussion of future directions, outlining exciting frontiers both in empirical data collection efforts as well as in method and theory development that, together, further the utility of the generative network modelling approach for network neuroscience. PMID:29187640

Binding of PLD2-Generated Phosphatidic Acid to KIF5B Promotes MT1-MMP Surface Trafficking and Lung Metastasis of Mouse Breast Cancer Cells.

PubMed

Wang, Ziqing; Zhang, Feng; He, Jingquan; Wu, Ping; Tay, Li Wei Rachel; Cai, Ming; Nian, Weiqi; Weng, Yuanyuan; Qin, Li; Chang, Jeffrey T; McIntire, Laura B; Di Paolo, Gilbert; Xu, Jianming; Peng, Junmin; Du, Guangwei

2017-10-23

Little is known about the cellular events promoting metastasis. We show that knockout of phospholipase D 2 (PLD2), which generates the signaling lipid phosphatidic acid (PA), inhibits lung metastases in the mammary tumor virus (MMTV)-Neu transgenic mouse breast cancer model. PLD2 promotes local invasion through the regulation of the plasma membrane targeting of MT1-MMP and its associated invadopodia. A liposome pull-down screen identifies KIF5B, the heavy chain of the motor protein kinesin-1, as a new PA-binding protein. In vitro assays reveal that PA specifically and directly binds to the C terminus of KIF5B. The binding between PLD2-generated PA and KIF5B is required for the vesicular association of KIF5B, surface localization of MT1-MMP, invadopodia, and invasion in cancer cells. Taken together, these results identify a role of PLD2-generated PA in the regulation of kinesin-1 motor functions and breast cancer metastasis and suggest PLD2 as a potential therapeutic target for metastatic breast cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

Construction of a viral T2A-peptide based knock-in mouse model for enhanced Cre recombinase activity and fluorescent labeling of podocytes.

PubMed

Koehler, Sybille; Brähler, Sebastian; Braun, Fabian; Hagmann, Henning; Rinschen, Markus M; Späth, Martin R; Höhne, Martin; Wunderlich, F Thomas; Schermer, Bernhard; Benzing, Thomas; Brinkkoetter, Paul T

2017-06-01

Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type -mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type and hNphs2.Cre mice to Phb2 fl/fl mice. The podocyte-specific Phb2 knockout by Nphs2 pod.T2A.ciCre.T2A.mTomato/wild-type mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Controlled insertional mutagenesis using a LINE-1 (ORFeus) gene-trap mouse model.

PubMed

O'Donnell, Kathryn A; An, Wenfeng; Schrum, Christina T; Wheelan, Sarah J; Boeke, Jef D

2013-07-16

A codon-optimized mouse LINE-1 element, ORFeus, exhibits dramatically higher retrotransposition frequencies compared with its native long interspersed element 1 counterpart. To establish a retrotransposon-mediated mouse model with regulatable and potent mutagenic capabilities, we generated a tetracycline (tet)-regulated ORFeus element harboring a gene-trap cassette. Here, we show that mice expressing tet-ORFeus broadly exhibit robust retrotransposition in somatic tissues when treated with doxycycline. Consistent with a significant mutagenic burden, we observed a reduced number of double transgenic animals when treated with high-level doxycycline during embryogenesis. Transgene induction in skin resulted in a white spotting phenotype due to somatic ORFeus-mediated mutations that likely disrupt melanocyte development. The data suggest a high level of transposition in melanocyte precursors and consequent mutation of genes important for melanoblast proliferation, differentiation, or migration. These findings reveal the utility of a retrotransposon-based mutagenesis system as an alternative to existing DNA transposon systems. Moreover, breeding these mice to different tet-transactivator/reversible tet-transactivator lines supports broad functionality of tet-ORFeus because of the potential for dose-dependent, tissue-specific, and temporal-specific mutagenesis.

Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

PubMed

Sjögren, Jonathan; Andersson, Linda; Mejàre, Malin; Olsson, Fredrik

2017-01-01

Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2.

PubMed

Fukuda, Tomokazu; Scott, Gregory; Komatsu, Yoshihiro; Araya, Runa; Kawano, Masako; Ray, Manas K; Yamada, Masahisa; Mishina, Yuji

2006-04-01

BMP signaling plays pleiotropic roles in various tissues. Transgenic mouse lines that overexpress BMP signaling in a tissue-specific manner would be beneficial; however, production of each tissue-specific transgenic mouse line is labor-intensive. Here, using a Cre-loxP system, we generated a conditionally overexpressing mouse line for BMP signaling through the type I receptor ALK2 (alternatively known as AVCRI, ActRI, or ActRIA). By mating this line with Cre-expression mouse lines, Cre-mediated recombination removes an intervening floxed lacZ expression cassette and thereby permits the expression of a constitutively active form of Alk2 (caAlk2) driven by a ubiquitous promoter, CAG. Tissue specificity of Cre recombination was monitored by a bicistronically expressed EGFP following Alk2 cDNA. Increased BMP signaling was confirmed by ectopic phosphorylation of SMAD1/5/8 in the areas where Cre recombination had occurred. The conditional overexpression system described here provides versatility in investigating gene functions in a tissue-specific manner without having to generate independent tissue-specific transgenic lines. Published 2006 Wiley-Liss, Inc.

Altered Expression of Ganglioside Metabolizing Enzymes Results in GM3 Ganglioside Accumulation in Cerebellar Cells of a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis.

PubMed

Somogyi, Aleksandra; Petcherski, Anton; Beckert, Benedikt; Huebecker, Mylene; Priestman, David A; Banning, Antje; Cotman, Susan L; Platt, Frances M; Ruonala, Mika O; Tikkanen, Ritva

2018-02-22

Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene. Most JNCL patients exhibit a 1.02 kb genomic deletion removing exons 7 and 8 of this gene, which results in a truncated CLN3 protein carrying an aberrant C-terminus. A genetically accurate mouse model ( Cln3 Δex7/8 mice) for this deletion has been generated. Using cerebellar precursor cell lines generated from wildtype and Cln3 Δex7/8 mice, we have here analyzed the consequences of the CLN3 deletion on levels of cellular gangliosides, particularly GM3, GM2, GM1a and GD1a. The levels of GM1a and GD1a were found to be significantly reduced by both biochemical and cytochemical methods. However, quantitative high-performance liquid chromatography analysis revealed a highly significant increase in GM3, suggesting a metabolic blockade in the conversion of GM3 to more complex gangliosides. Quantitative real-time PCR analysis revealed a significant reduction in the transcripts of the interconverting enzymes, especially of β-1,4- N -acetyl-galactosaminyl transferase 1 (GM2 synthase), which is the enzyme converting GM3 to GM2. Thus, our data suggest that the complex a-series gangliosides are reduced in Cln3 Δex7/8 mouse cerebellar precursor cells due to impaired transcription of the genes responsible for their synthesis.

Altered Expression of Ganglioside Metabolizing Enzymes Results in GM3 Ganglioside Accumulation in Cerebellar Cells of a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis

PubMed Central

Somogyi, Aleksandra; Petcherski, Anton; Beckert, Benedikt; Huebecker, Mylene; Priestman, David A.; Banning, Antje; Cotman, Susan L.; Platt, Frances M.; Ruonala, Mika O.

2018-01-01

Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene. Most JNCL patients exhibit a 1.02 kb genomic deletion removing exons 7 and 8 of this gene, which results in a truncated CLN3 protein carrying an aberrant C-terminus. A genetically accurate mouse model (Cln3Δex7/8 mice) for this deletion has been generated. Using cerebellar precursor cell lines generated from wildtype and Cln3Δex7/8 mice, we have here analyzed the consequences of the CLN3 deletion on levels of cellular gangliosides, particularly GM3, GM2, GM1a and GD1a. The levels of GM1a and GD1a were found to be significantly reduced by both biochemical and cytochemical methods. However, quantitative high-performance liquid chromatography analysis revealed a highly significant increase in GM3, suggesting a metabolic blockade in the conversion of GM3 to more complex gangliosides. Quantitative real-time PCR analysis revealed a significant reduction in the transcripts of the interconverting enzymes, especially of β-1,4-N-acetyl-galactosaminyl transferase 1 (GM2 synthase), which is the enzyme converting GM3 to GM2. Thus, our data suggest that the complex a-series gangliosides are reduced in Cln3Δex7/8 mouse cerebellar precursor cells due to impaired transcription of the genes responsible for their synthesis. PMID:29470438

Enhanced inflammation and attenuated tumor suppressor pathways are associated with oncogene-induced lung tumors in aged mice

USDA-ARS?s Scientific Manuscript database

Aging is often accompanied by a dramatic increase in cancer susceptibility. To gain insights into how aging affects tumor susceptibility, we generated a conditional mouse model in which oncogenic KrasG12D was activated specifically in lungs of young (3-5 months) and old (19-24 months) mice. Activati...

A null-mutation in the Znt7 gene accelerates prostate tumor formation in a transgenic adenocarcinoma mouse prostate model

USDA-ARS?s Scientific Manuscript database

Decrease of cellular zinc in the epithelium of the prostate has been implicated in the development of prostate cancer. To investigate whether ZnT7, a zinc transporter involved in intracellular zinc accumulation, plays a role in prostate cancer development, we have generated and characterized a trans...

p53 constrains progression to anaplastic thyroid carcinoma in a Braf-mutant mouse model of papillary thyroid cancer

PubMed Central

McFadden, David G.; Vernon, Amanda; Santiago, Philip M.; Martinez-McFaline, Raul; Bhutkar, Arjun; Crowley, Denise M.; McMahon, Martin; Sadow, Peter M.; Jacks, Tyler

2014-01-01

Anaplastic thyroid carcinoma (ATC) has among the worst prognoses of any solid malignancy. The low incidence of the disease has in part precluded systematic clinical trials and tissue collection, and there has been little progress in developing effective therapies. v-raf murine sarcoma viral oncogene homolog B (BRAF) and tumor protein p53 (TP53) mutations cooccur in a high proportion of ATCs, particularly those associated with a precursor papillary thyroid carcinoma (PTC). To develop an adult-onset model of BRAF-mutant ATC, we generated a thyroid-specific CreER transgenic mouse. We used a Cre-regulated BrafV600E mouse and a conditional Trp53 allelic series to demonstrate that p53 constrains progression from PTC to ATC. Gene expression and immunohistochemical analyses of murine tumors identified the cardinal features of human ATC including loss of differentiation, local invasion, distant metastasis, and rapid lethality. We used small-animal ultrasound imaging to monitor autochthonous tumors and showed that treatment with the selective BRAF inhibitor PLX4720 improved survival but did not lead to tumor regression or suppress signaling through the MAPK pathway. The combination of PLX4720 and the mapk/Erk kinase (MEK) inhibitor PD0325901 more completely suppressed MAPK pathway activation in mouse and human ATC cell lines and improved the structural response and survival of ATC-bearing animals. This model expands the limited repertoire of autochthonous models of clinically aggressive thyroid cancer, and these data suggest that small-molecule MAPK pathway inhibitors hold clinical promise in the treatment of advanced thyroid carcinoma. PMID:24711431

Measurement of generation-dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation.

PubMed

Akbarian, Vahe; Wang, Weijia; Audet, Julie

2012-05-01

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G(0) -G(5) ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G(2) to G(5) and higher death rates in G(2) , G(3) , and G(5) compared with SCF. In addition, proliferation rates tended to increase from G(1) to G(5) in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture. Copyright © 2012 International Society for Advancement of Cytometry.

A critical view of the use of genetic tools to unveil neural circuits: the case of leptin action in reproduction.

PubMed

Elias, Carol F

2014-01-01

The remarkable development and refinement of the Cre-loxP system coupled with the nonstop production of new mouse models and virus vectors have impelled the growth of various fields of investigation. In this article, I will discuss the data collected using these genetic tools in our area of interest, giving specific emphasis to the identification of the neuronal populations that relay leptin action in reproductive physiology. A series of mouse models that allow manipulation of the leptin receptor gene have been generated. Of those, I will discuss the use of two models of leptin receptor gene reexpression (LepR(neo/neo) and LepR(loxTB/loxTB)) and one model of leptin signaling blockade (LepR(flox/flox)). I will also highlight the differences of using stereotaxic delivery of virus vectors expressing DNA-recombinases (Flp and Cre) and mouse models expressing Cre-recombinase. Our findings indicate that leptin action in the ventral premammillary nucleus is sufficient, but not required, for leptin action in reproduction and that leptin action in Kiss1 neurons arises after pubertal maturation; therefore, direct leptin signaling in Kiss1 neurons is neither required nor sufficient for the permissive action of leptin in pubertal development. It also became evident that the full action of leptin in the reproductive neuroendocrine axis requires the engagement of an integrated circuitry, yet to be fully unveiled.

[The development of therapeutic vaccine for hepatitis C virus].

PubMed

Kimura, Kiminori; Kohara, Michinori

2012-10-01

Chronic hepatitis C caused by infection with the hepatitis C virus(HCV)is a global health problem. HCV causes persistent infection that can lead to chronic liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The therapeutic efficacy of antiviral drugs is not optimal in patients with chronic infection; furthermore, an effective vaccine has not yet been developed. To design an effective HCV vaccine, generation of a convenient animal model of HCV infection is necessary. Recently, we used the Cre/loxP switching system to generate an immunocompetent mouse model of HCV expression, thereby enabling the study of host immune responses against HCV proteins. At present vaccine has not yet been shown to be therapeutically effective against chronic HCV infection. We examined the therapeutic effects of a recombinant vaccinia virus(rVV)encoding HCV protein in a mouse model. we generated rVVs for 3 different HCV proteins and found that one of the recombinant viruses encoding a nonstructural protein(rVV-N25)resolved pathological chronic hepatitis C symptoms in the liver. We propose the possibility that rVV-N25 immunization has the potential for development of an effective therapeutic vaccine for HCV induced chronic hepatitis. The utilization of the therapeutic vaccine can protect progress to chronic hepatitis, and as a consequence, leads to eradication of hepatocellular carcinoma. In this paper, we summarized our current study for HCV therapeutic vaccine and review the vaccine development to date.

Reduced Expression of SARM in Mouse Spleen during Polymicrobial Sepsis.

PubMed

Gong, Yu; Zou, Lin; Cen, Dongzhi; Chao, Wei; Chen, Dunjin

2016-12-01

Objective Immune dysfunction, including prominent apoptosis of immune cells and decreased functioning of the remaining immune cells, plays a central role in the pathogenesis of sepsis. Sterile α and HEAT/armadillo motif-containing protein (SARM) is implicated in the regulation of immune cell apoptosis. This study aimed to elucidate SARM contributes to sepsis-induced immune cell death and immunosuppression. Methods A mouse model of polymicrobial sepsis was generated by cecum ligation and puncture (CLP). SARM gene and protein expression, caspase 3 cleavage and intracellular ATP production were measured in the mouse spleens. Results CLP-induced polymicrobial sepsis specifically attenuated both the gene and protein expression of SARM in the spleens. Moreover, the attenuation of SARM expression synchronized with splenocyte apoptosis, as evidenced by increased caspase 3 cleavage and ATP depletion. Conclusions These findings suggest that SARM is a potential regulator of sepsis-induced splenocyte apoptosis.

Enhanced microglial pro-inflammatory response to lipopolysaccharide correlates with brain infiltration and blood-brain barrier dysregulation in a mouse model of telomere shortening.

PubMed

Raj, Divya D A; Moser, Jill; van der Pol, Susanne M A; van Os, Ronald P; Holtman, Inge R; Brouwer, Nieske; Oeseburg, Hisko; Schaafsma, Wandert; Wesseling, Evelyn M; den Dunnen, Wilfred; Biber, Knut P H; de Vries, Helga E; Eggen, Bart J L; Boddeke, Hendrikus W G M

2015-12-01

Microglia are a proliferative population of resident brain macrophages that under physiological conditions self-renew independent of hematopoiesis. Microglia are innate immune cells actively surveying the brain and are the earliest responders to injury. During aging, microglia elicit an enhanced innate immune response also referred to as 'priming'. To date, it remains unknown whether telomere shortening affects the proliferative capacity and induces priming of microglia. We addressed this issue using early (first-generation G1 mTerc(-/-) )- and late-generation (third-generation G3 and G4 mTerc(-/-) ) telomerase-deficient mice, which carry a homozygous deletion for the telomerase RNA component gene (mTerc). Late-generation mTerc(-/-) microglia show telomere shortening and decreased proliferation efficiency. Under physiological conditions, gene expression and functionality of G3 mTerc(-/-) microglia are comparable with microglia derived from G1 mTerc(-/-) mice despite changes in morphology. However, after intraperitoneal injection of bacterial lipopolysaccharide (LPS), G3 mTerc(-/-) microglia mice show an enhanced pro-inflammatory response. Nevertheless, this enhanced inflammatory response was not accompanied by an increased expression of genes known to be associated with age-associated microglia priming. The increased inflammatory response in microglia correlates closely with increased peripheral inflammation, a loss of blood-brain barrier integrity, and infiltration of immune cells in the brain parenchyma in this mouse model of telomere shortening. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Quantitative Metaproteomics and Activity-Based Probe Enrichment Reveals Significant Alterations in Protein Expression from a Mouse Model of Inflammatory Bowel Disease.

PubMed

Mayers, Michael D; Moon, Clara; Stupp, Gregory S; Su, Andrew I; Wolan, Dennis W

2017-02-03

Tandem mass spectrometry based shotgun proteomics of distal gut microbiomes is exceedingly difficult due to the inherent complexity and taxonomic diversity of the samples. We introduce two new methodologies to improve metaproteomic studies of microbiome samples. These methods include the stable isotope labeling in mammals to permit protein quantitation across two mouse cohorts as well as the application of activity-based probes to enrich and analyze both host and microbial proteins with specific functionalities. We used these technologies to study the microbiota from the adoptive T cell transfer mouse model of inflammatory bowel disease (IBD) and compare these samples to an isogenic control, thereby limiting genetic and environmental variables that influence microbiome composition. The data generated highlight quantitative alterations in both host and microbial proteins due to intestinal inflammation and corroborates the observed phylogenetic changes in bacteria that accompany IBD in humans and mouse models. The combination of isotope labeling with shotgun proteomics resulted in the total identification of 4434 protein clusters expressed in the microbial proteomic environment, 276 of which demonstrated differential abundance between control and IBD mice. Notably, application of a novel cysteine-reactive probe uncovered several microbial proteases and hydrolases overrepresented in the IBD mice. Implementation of these methods demonstrated that substantial insights into the identity and dysregulation of host and microbial proteins altered in IBD can be accomplished and can be used in the interrogation of other microbiome-related diseases.

Analysis of mouse models carrying the I26T and R160C substitutions in the transcriptional repressor HESX1 as models for septo-optic dysplasia and hypopituitarism

PubMed Central

Sajedi, Ezat; Gaston-Massuet, Carles; Signore, Massimo; Andoniadou, Cynthia L.; Kelberman, Daniel; Castro, Sandra; Etchevers, Heather C.; Gerrelli, Dianne; Dattani, Mehul T.; Martinez-Barbera, Juan Pedro

2008-01-01

SUMMARY A homozygous substitution of the highly conserved isoleucine at position 26 by threonine (I26T) in the transcriptional repressor HESX1 has been associated with anterior pituitary hypoplasia in a human patient, with no forebrain or eye defects. Two individuals carrying a homozygous substitution of the conserved arginine at position 160 by cysteine (R160C) manifest septo-optic dysplasia (SOD), a condition characterised by pituitary abnormalities associated with midline telencephalic structure defects and optic nerve hypoplasia. We have generated two knock-in mouse models containing either the I26T or R160C substitution in the genomic locus. Hesx1I26T/I26T embryos show pituitary defects comparable with Hesx1−/− mouse mutants, with frequent occurrence of ocular abnormalities, although the telencephalon develops normally. Hesx1R160C/R160C mutants display forebrain and pituitary defects that are identical to those observed in Hesx1−/− null mice. We also show that the expression pattern of HESX1 during early human development is very similar to that described in the mouse, suggesting that the function of HESX1 is conserved between the two species. Together, these results suggest that the I26T mutation yields a hypomorphic allele, whereas R160C produces a null allele and, consequently, a more severe phenotype in both mice and humans. PMID:19093031

Micro-imaging of the Mouse Lung via MRI

NASA Astrophysics Data System (ADS)

Wang, Wei

Quantitative measurement of lung microstructure is of great significance in assessment of pulmonary disease, particularly in the earliest stages. Conventional stereological assessment of ex-vivo fixed tissue specimens under the microscope has a long and successful tradition and is regarded as a gold standard, but the invasive nature limits its applications and the practicality of use in longitudinal studies. The technique for diffusion MRI-based 3He lung morphometry was previously developed and validated for human lungs, and was recently extended to ex-vivo mouse lungs. The technique yields accurate, quantitative information about the microstructure and geometry of acinar airways. In this dissertation, the 3He lung morphometry technique is for the first time successfully implemented for in-vivo studies of mice. It can generate spatially-resolved maps of parameters that reveal the microstructure of mouse lung. Results in healthy mice indicate excellent agreement between in-vivo morphometry via 3He MRI and microscopic morphometry after sacrifice. The implementation and validation of 3He morphometry in healthy mice open up new avenues for application of the technique as a precise, noninvasive, in-vivo biomarker of changes in lung microstructure, within various mouse models of lung disease. We have applied 3He morphometry to the Sendai mouse model of lung disease. Specifically, the Sendai-virus model of chronic obstructive lung disease has demonstrated an innate immune response in mouse airways that exhibits similarities to the chronic airway inflammation in human COPD and asthma, but the effect on distal lung parenchyma had not been investigated. We imaged the time course and regional distribution of mouse lung microstructural changes in vivo after Sendai virus (SeV) infection with 1H and 3He diffusion MRI. 1H MR images detected the SeV-induced pulmonary inflammation in vivo and 3He lung morphometry showed modest increase in alveolar duct radius distal to airway inflammation, particularly in the lung periphery, indicating airspace enlargement after virus infection. Another important application of the imaging technique is the study of lung regeneration in a pneumonectomy (PNX) model. Partial resection of the lung by unilateral PNX is a robust model of compensatory lung growth. It is typically studied by postmortem morphometry in which longitudinal assessment in the same animal cannot be achieved. Here we successfully assess the microstructural changes and quantify the compensatory lung growth in vivo in the PNX mouse model via 1H and hyperpolarized 3He diffusion MRI. Our results show complete restoration in lung volume and total alveolar number with enlargement of alveolar size, which is consistent with prior histological studies conducted in different animals at various time points. This dissertation demonstrates that 3He lung morphometry has good sensitivity in quantifying small microstructural changes in the mouse lung and can be applied to a variety of mouse pulmonary models. Particularly, it has great potential to become a valuable tool in understanding the time course and the mechanism of lung growth in individual animals and may provide insight into post-natal lung growth and lung regeneration.

Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer

PubMed Central

Bortolussi, Giulia; Zentilin, Lorena; Baj, Gabriele; Giraudi, Pablo; Bellarosa, Cristina; Giacca, Mauro; Tiribelli, Claudio; Muro, Andrés F.

2012-01-01

Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.—Bortolussi, G., Zentilin, L., Baj, G., Giraudi, P., Bellarosa, C., Giacca, M., Tiribelli, C., Muro, A. F. Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer. PMID:22094718

The dynamics of gene expression changes in a mouse model of oral tumorigenesis may help refine prevention and treatment strategies in patients with oral cancer.

PubMed

Foy, Jean-Philippe; Tortereau, Antonin; Caulin, Carlos; Le Texier, Vincent; Lavergne, Emilie; Thomas, Emilie; Chabaud, Sylvie; Perol, David; Lachuer, Joël; Lang, Wenhua; Hong, Waun Ki; Goudot, Patrick; Lippman, Scott M; Bertolus, Chloé; Saintigny, Pierre

2016-06-14

A better understanding of the dynamics of molecular changes occurring during the early stages of oral tumorigenesis may help refine prevention and treatment strategies. We generated genome-wide expression profiles of microdissected normal mucosa, hyperplasia, dysplasia and tumors derived from the 4-NQO mouse model of oral tumorigenesis. Genes differentially expressed between tumor and normal mucosa defined the "tumor gene set" (TGS), including 4 non-overlapping gene subsets that characterize the dynamics of gene expression changes through different stages of disease progression. The majority of gene expression changes occurred early or progressively. The relevance of these mouse gene sets to human disease was tested in multiple datasets including the TCGA and the Genomics of Drug Sensitivity in Cancer project. The TGS was able to discriminate oral squamous cell carcinoma (OSCC) from normal oral mucosa in 3 independent datasets. The OSCC samples enriched in the mouse TGS displayed high frequency of CASP8 mutations, 11q13.3 amplifications and low frequency of PIK3CA mutations. Early changes observed in the 4-NQO model were associated with a trend toward a shorter oral cancer-free survival in patients with oral preneoplasia that was not seen in multivariate analysis. Progressive changes observed in the 4-NQO model were associated with an increased sensitivity to 4 different MEK inhibitors in a panel of 51 squamous cell carcinoma cell lines of the areodigestive tract. In conclusion, the dynamics of molecular changes in the 4-NQO model reveal that MEK inhibition may be relevant to prevention and treatment of a specific molecularly-defined subgroup of OSCC.

Modeling prostate cancer in mice: something old, something new, something premalignant, something metastatic.

PubMed

Irshad, Shazia; Abate-Shen, Cory

2013-06-01

More than 15 years ago, the first generation of genetically engineered mouse (GEM) models of prostate cancer was introduced. These transgenic models utilized prostate-specific promoters to express SV40 oncogenes specifically in prostate epithelium. Since the description of these initial models, there have been a plethora of GEM models of prostate cancer representing various perturbations of oncogenes or tumor suppressors, either alone or in combination. This review describes these GEM models, focusing on their relevance for human prostate cancer and highlighting their strengths and limitations, as well as opportunities for the future.

Dietary L-leucine improves the anemia in a mouse model for Diamond-Blackfan anemia.

PubMed

Jaako, Pekka; Debnath, Shubhranshu; Olsson, Karin; Bryder, David; Flygare, Johan; Karlsson, Stefan

2012-09-13

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Recently, a case study reported a patient who became transfusion-independent in response to treatment with the amino acid L-leucine. Therefore, we have validated the therapeutic effect of L-leucine using our recently generated mouse model for RPS19-deficient DBA. Administration of L-leucine significantly improved the anemia in Rps19-deficient mice (19% improvement in hemoglobin concentration; 18% increase in the number of erythrocytes), increased the bone marrow cellularity, and alleviated stress hematopoiesis. Furthermore, the therapeutic response to L-leucine appeared specific for Rps19-deficient hematopoiesis and was associated with down-regulation of p53 activity. Our study supports the rationale for clinical trials of L-leucine as a therapeutic agent for DBA.

Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model.

PubMed

Maggi, Elaine C; Gravina, Silvia; Cheng, Haiying; Piperdi, Bilal; Yuan, Ziqiang; Dong, Xiao; Libutti, Steven K; Vijg, Jan; Montagna, Cristina

2018-01-01

The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

A bioluminescent imaging mouse model for Marburg virus based on a pseudovirus system.

PubMed

Zhang, Li; Li, Qianqian; Liu, Qiang; Huang, Weijin; Nie, Jianhui; Wang, Youchun

2017-08-03

Marburg virus (MARV) can cause lethal hemorrhagic fever in humans. Handling of MARV is restricted to high-containment biosafety level 4 (BSL-4) facilities, which greatly impedes research into this virus. In this study, a high titer of MARV pseudovirus was generated through optimization of the HIV backbone vectors, the ratio of backbone vector to MARV glycoprotein expression vector, and the transfection reagents. An in vitro neutralization assay and an in vivo bioluminescent imaging mouse model for MARV were developed based on the pseudovirus. Protective serum against MARV was successfully induced in guinea pigs, which showed high neutralization activity in vitro and could also protect Balb/c mice from MARV pseudovirus infection in vivo. This system could be a convenient tool to enable the evaluation of vaccines and therapeutic drugs against MARV in non-BSL-4 laboratories.

A Naturally Fluorescent Mgp Transgenic Mouse for Angiogenesis and Glaucoma Longitudinal Studies

PubMed Central

Asokan, Priyadarsini; Mitra, Rajendra N.; Periasamy, Ramesh; Han, Zongchao

2018-01-01

Purpose Our goal was to generate and characterize a new mouse model in which only angiogenesis- and glaucoma-relevant tissues would be naturally fluorescent. The Matrix Gla (MGP) gene is highly expressed in vascular smooth muscle cells (VSMC) and trabecular meshwork (TM). We sought to direct our Mgp-Cre.KI mouse recombinase to VSMC/TM cells to produce their longitudinal fluorescent profiles. Methods Homozygous Mgp-Cre.KI mice were crossed with Ai9 homozygous reporter mice harboring a loxP-flanked STOP cassette preventing transcription of a DsRed fluorescent protein (tdTomato). The F1 double-heterozygous (Mgp-tdTomato) was examined by direct fluorescence, whole mount, histology, and fundus photography. Custom-made filters had 554/23 emission and 609/54 exciter nanometer wavelengths. Proof of concept of the model's usefulness was conducted by inducing guided imaging laser burns. Evaluation of a vessel's leakage and proliferation was followed by noninvasive angiography. Results The Mgp-tdTomato mouse was viable, fertile, with normal IOP and ERG. Its phenotype exhibited red paws and snout (cartilage expression), which precluded genotyping. A fluorescent red ring was seen at the limbus and confirmed to be TM expression by histology. The entire retinal vasculature was red fluorescent (VSMC) and directly visualized by fundus photography. Laser burns on the Mgp-tdTomato allowed separation of leakiness and neovascularization evaluation parameters. Conclusions The availability of a transgenic mouse naturally fluorescent in glaucoma-relevant tissues and retinal vasculature brings the unique opportunity to study a wide spectrum of single and combined glaucomatous conditions in vivo. Moreover, the Mgp-tdTomato mouse provides a new tool to study mechanisms and therapeutics of retinal angiogenesis longitudinally. PMID:29392320

A Naturally Fluorescent Mgp Transgenic Mouse for Angiogenesis and Glaucoma Longitudinal Studies.

PubMed

Asokan, Priyadarsini; Mitra, Rajendra N; Periasamy, Ramesh; Han, Zongchao; Borrás, Teresa

2018-02-01

Our goal was to generate and characterize a new mouse model in which only angiogenesis- and glaucoma-relevant tissues would be naturally fluorescent. The Matrix Gla (MGP) gene is highly expressed in vascular smooth muscle cells (VSMC) and trabecular meshwork (TM). We sought to direct our Mgp-Cre.KI mouse recombinase to VSMC/TM cells to produce their longitudinal fluorescent profiles. Homozygous Mgp-Cre.KI mice were crossed with Ai9 homozygous reporter mice harboring a loxP-flanked STOP cassette preventing transcription of a DsRed fluorescent protein (tdTomato). The F1 double-heterozygous (Mgp-tdTomato) was examined by direct fluorescence, whole mount, histology, and fundus photography. Custom-made filters had 554/23 emission and 609/54 exciter nanometer wavelengths. Proof of concept of the model's usefulness was conducted by inducing guided imaging laser burns. Evaluation of a vessel's leakage and proliferation was followed by noninvasive angiography. The Mgp-tdTomato mouse was viable, fertile, with normal IOP and ERG. Its phenotype exhibited red paws and snout (cartilage expression), which precluded genotyping. A fluorescent red ring was seen at the limbus and confirmed to be TM expression by histology. The entire retinal vasculature was red fluorescent (VSMC) and directly visualized by fundus photography. Laser burns on the Mgp-tdTomato allowed separation of leakiness and neovascularization evaluation parameters. The availability of a transgenic mouse naturally fluorescent in glaucoma-relevant tissues and retinal vasculature brings the unique opportunity to study a wide spectrum of single and combined glaucomatous conditions in vivo. Moreover, the Mgp-tdTomato mouse provides a new tool to study mechanisms and therapeutics of retinal angiogenesis longitudinally.

Expression of HLA Class II Molecules in Humanized NOD.Rag1KO.IL2RgcKO Mice Is Critical for Development and Function of Human T and B Cells

PubMed Central

Danner, Rebecca; Chaudhari, Snehal N.; Rosenberger, John; Surls, Jacqueline; Richie, Thomas L.; Brumeanu, Teodor-Doru; Casares, Sofia

2011-01-01

Background Humanized mice able to reconstitute a surrogate human immune system (HIS) can be used for studies on human immunology and may provide a predictive preclinical model for human vaccines prior to clinical trials. However, current humanized mouse models show sub-optimal human T cell reconstitution and limited ability to support immunoglobulin class switching by human B cells. This limitation has been attributed to the lack of expression of Human Leukocyte Antigens (HLA) molecules in mouse lymphoid organs. Recently, humanized mice expressing HLA class I molecules have been generated but showed little improvement in human T cell reconstitution and function of T and B cells. Methods We have generated NOD.Rag1KO.IL2RγcKO mice expressing HLA class II (HLA-DR4) molecules under the I-Ed promoter that were infused as adults with HLA-DR-matched human hematopoietic stem cells (HSC). Littermates lacking expression of HLA-DR4 molecules were used as control. Results HSC-infused HLA-DR4.NOD.Rag1KO.IL-2RγcKO mice developed a very high reconstitution rate (>90%) with long-lived and functional human T and B cells. Unlike previous humanized mouse models reported in the literature and our control mice, the HLA-DR4 expressing mice reconstituted serum levels (natural antibodies) of human IgM, IgG (all four subclasses), IgA, and IgE comparable to humans, and elicited high titers of specific human IgG antibodies upon tetanus toxoid vaccination. Conclusions Our study demonstrates the critical role of HLA class II molecules for development of functional human T cells able to support immunoglobulin class switching and efficiently respond to vaccination. PMID:21611197

The Mouse House: A brief history of the ORNL mouse-genetics program, 1947–2009

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russell, Liane B.

The large mouse genetics program at the Oak Ridge National Lab is often re-membered chiefly for the germ-cell mutation-rate data it generated and their uses in estimating the risk of heritable radiation damage. In fact, it soon became a multi-faceted research effort that, over a period of almost 60 years, generated a wealth of information in the areas of mammalian mutagenesis, basic genetics (later enriched by molecular techniques), cytogenetics, reproductive biology, biochemistry of germ cells, and teratology. Research in the area of germ-cell mutagenesis explored the important physical and biological factors that affect the frequency and nature of induced mutationsmore » and made several unexpected discoveries, such as the major importance of the perigametic interval (the zygote stage) for the origin of spontaneous mutations and for the sensitivity to induced genetic change. Of practical value was the discovery that ethylnitrosourea was a supermutagen for point mutations, making high-efficiency mutagenesis in the mouse feasible worldwide. Teratogenesis findings resulted in recommendations still generally accepted in radiological practice. Studies supporting the mutagenesis research added whole bodies of information about mammalian germ-cell development and about molecular targets in germ cells. The early decision to not merely count but propagate genetic variants of all sorts made possible further discoveries, such as the Y-Chromosome s importance in mammalian sex determination and the identification of rare X-autosome translocations, which, in turn, led to the formulation of the single-active-X hypothesis and provided tools for studies of functional mosaicism for autosomal genes, male sterility, and chromosome-pairing mechanism. Extensive genetic and then molecular analyses of large numbers of induced specific-locus mutants resulted in fine-structure physical and correlated functional mapping of significant portions of the mouse genome and constituted a valuable source of mouse models for human genetic disorders.« less

The Mouse House: a brief history of the ORNL mouse-genetics program, 1947-2009.

PubMed

Russell, Liane B

2013-01-01

The large mouse genetics program at the Oak Ridge National Laboratory (ORNL) is often remembered chiefly for the germ-cell mutation-rate data it generated and their uses in estimating the risk of heritable radiation damage. In fact, it soon became a multi-faceted research effort that, over a period of almost 60 years, generated a wealth of information in the areas of mammalian mutagenesis, basic genetics (later enriched by molecular techniques), cytogenetics, reproductive biology, biochemistry of germ cells, and teratology. Research in the area of germ-cell mutagenesis explored the important physical and biological factors that affect the frequency and nature of induced mutations and made several unexpected discoveries, such as the major importance of the perigametic interval (the zygote stage) for the origin of spontaneous mutations and for the sensitivity to induced genetic change. Of practical value was the discovery that ethylnitrosourea was a supermutagen for point mutations, making high-efficiency mutagenesis in the mouse feasible worldwide. Teratogenesis findings resulted in recommendations still generally accepted in radiological practice. Studies supporting the mutagenesis research added whole bodies of information about mammalian germ-cell development and about molecular targets in germ cells. The early decision to not merely count but propagate genetic variants of all sorts made possible further discoveries, such as the Y-chromosome's importance in mammalian sex determination and the identification of rare X-autosome translocations, which, in turn, led to the formulation of the single-active-X hypothesis and provided tools for studies of functional mosaicism for autosomal genes, male sterility, and chromosome-pairing mechanism. Extensive genetic and then molecular analyses of large numbers of induced specific-locus mutants resulted in fine-structure physical and correlated functional mapping of significant portions of the mouse genome and constituted a valuable source of mouse models for human genetic disorders. Copyright © 2013 Elsevier B.V. All rights reserved.

Finding mouse models of human lymphomas and leukemia's using the Jackson laboratory mouse tumor biology database.

PubMed

Begley, Dale A; Sundberg, John P; Krupke, Debra M; Neuhauser, Steven B; Bult, Carol J; Eppig, Janan T; Morse, Herbert C; Ward, Jerrold M

2015-12-01

Many mouse models have been created to study hematopoietic cancer types. There are over thirty hematopoietic tumor types and subtypes, both human and mouse, with various origins, characteristics and clinical prognoses. Determining the specific type of hematopoietic lesion produced in a mouse model and identifying mouse models that correspond to the human subtypes of these lesions has been a continuing challenge for the scientific community. The Mouse Tumor Biology Database (MTB; http://tumor.informatics.jax.org) is designed to facilitate use of mouse models of human cancer by providing detailed histopathologic and molecular information on lymphoma subtypes, including expertly annotated, on line, whole slide scans, and providing a repository for storing information on and querying these data for specific lymphoma models. Copyright © 2015 Elsevier Inc. All rights reserved.

Mouse models rarely mimic the transcriptome of human neurodegenerative diseases: A systematic bioinformatics-based critique of preclinical models.

PubMed

Burns, Terry C; Li, Matthew D; Mehta, Swapnil; Awad, Ahmed J; Morgan, Alexander A

2015-07-15

Translational research for neurodegenerative disease depends intimately upon animal models. Unfortunately, promising therapies developed using mouse models mostly fail in clinical trials, highlighting uncertainty about how well mouse models mimic human neurodegenerative disease at the molecular level. We compared the transcriptional signature of neurodegeneration in mouse models of Alzheimer׳s disease (AD), Parkinson׳s disease (PD), Huntington׳s disease (HD) and amyotrophic lateral sclerosis (ALS) to human disease. In contrast to aging, which demonstrated a conserved transcriptome between humans and mice, only 3 of 19 animal models showed significant enrichment for gene sets comprising the most dysregulated up- and down-regulated human genes. Spearman׳s correlation analysis revealed even healthy human aging to be more closely related to human neurodegeneration than any mouse model of AD, PD, ALS or HD. Remarkably, mouse models frequently upregulated stress response genes that were consistently downregulated in human diseases. Among potential alternate models of neurodegeneration, mouse prion disease outperformed all other disease-specific models. Even among the best available animal models, conserved differences between mouse and human transcriptomes were found across multiple animal model versus human disease comparisons, surprisingly, even including aging. Relative to mouse models, mouse disease signatures demonstrated consistent trends toward preserved mitochondrial function protein catabolism, DNA repair responses, and chromatin maintenance. These findings suggest a more complex and multifactorial pathophysiology in human neurodegeneration than is captured through standard animal models, and suggest that even among conserved physiological processes such as aging, mice are less prone to exhibit neurodegeneration-like changes. This work may help explain the poor track record of mouse-based translational therapies for neurodegeneration and provides a path forward to critically evaluate and improve animal models of human disease. Copyright © 2015 Elsevier B.V. All rights reserved.

Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes.

PubMed

Macdonald, Lynn E; Karow, Margaret; Stevens, Sean; Auerbach, Wojtek; Poueymirou, William T; Yasenchak, Jason; Frendewey, David; Valenzuela, David M; Giallourakis, Cosmas C; Alt, Frederick W; Yancopoulos, George D; Murphy, Andrew J

2014-04-08

Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome-based targeting vectors introduced into mouse ES cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and κ light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice.

[Programmed mouse genome modifications].

PubMed

Babinet, C

1998-02-01

The availability, in the mouse, of embryonic stem cells (ES cells) which have the ability to colonize the germ line of a developing embryo, has opened entirely new avenues to the genetic approach of embryonic development, physiology and pathology of this animal. Indeed, it is now possible, using homologous recombination in ES cells, to introduce mutations in any gene as long as it has been cloned. Thus, null as well as more subtle mutations can be created. Furthermore, scenarios are currently being derived which will allow one to generate conditional mutations. Taken together, these methods offer a tremendous tool to study gene function in vivo; they also open the way to creating murine models of human genetic diseases.

Development of isotope labeling liquid chromatography mass spectrometry for mouse urine metabolomics: quantitative metabolomic study of transgenic mice related to Alzheimer's disease.

PubMed

Peng, Jun; Guo, Kevin; Xia, Jianguo; Zhou, Jianjun; Yang, Jing; Westaway, David; Wishart, David S; Li, Liang

2014-10-03

Because of a limited volume of urine that can be collected from a mouse, it is very difficult to apply the common strategy of using multiple analytical techniques to analyze the metabolites to increase the metabolome coverage for mouse urine metabolomics. We report an enabling method based on differential isotope labeling liquid chromatography mass spectrometry (LC-MS) for relative quantification of over 950 putative metabolites using 20 μL of urine as the starting material. The workflow involves aliquoting 10 μL of an individual urine sample for ¹²C-dansylation labeling that target amines and phenols. Another 10 μL of aliquot was taken from each sample to generate a pooled sample that was subjected to ¹³C-dansylation labeling. The ¹²C-labeled individual sample was mixed with an equal volume of the ¹³C-labeled pooled sample. The mixture was then analyzed by LC-MS to generate information on metabolite concentration differences among different individual samples. The interday repeatability for the LC-MS runs was assessed, and the median relative standard deviation over 4 days was 5.0%. This workflow was then applied to a metabolomic biomarker discovery study using urine samples obtained from the TgCRND8 mouse model of early onset familial Alzheimer's disease (FAD) throughout the course of their pathological deposition of beta amyloid (Aβ). It was showed that there was a distinct metabolomic separation between the AD prone mice and the wild type (control) group. As early as 15-17 weeks of age (presymptomatic), metabolomic differences were observed between the two groups, and after the age of 25 weeks the metabolomic alterations became more pronounced. The metabolomic changes at different ages corroborated well with the phenotype changes in this transgenic mice model. Several useful candidate biomarkers including methionine, desaminotyrosine, taurine, N1-acetylspermidine, and 5-hydroxyindoleacetic acid were identified. Some of them were found in previous metabolomics studies in human cerebrospinal fluid or blood samples. This work illustrates the utility of this isotope labeling LC-MS method for biomarker discovery using mouse urine metabolomics.

Reconstitution of mouse oogenesis in a dish from pluripotent stem cells.

PubMed

Hayashi, Katsuhiko; Hikabe, Orie; Obata, Yayoi; Hirao, Yuji

2017-09-01

This protocol is an extension to: Nat. Protoc. 8, 1513-1524 (2013); doi: 10.1038/nprot.2013.090; published online 11 July 2013Generation of functional oocytes in culture from pluripotent stem cells should provide a useful model system for improving our understanding of the basic mechanisms underlying oogenesis. In addition, it has potential applications as an alternative source of oocytes for reproduction. Using the most advanced mouse model in regard to reproductive engineering and stem cell biology, we previously developed a culture method that produces functional primorial germ cells starting from pluripotent cells in culture and described it in a previous protocol. This Protocol Extension describes an adaptation of this existing Protocol in which oogenesis also occurs in vitro, thus substantially modifying the technique. Oocytes generated from embryonic stem cells (ESCs) or induced pluripotent stem cells give rise to healthy pups. Here, we describe the protocol for oocyte generation in culture. The protocol is mainly composed of three different culture stages: in vitro differentiation (IVDi), in vitro growth (IVG), and in vitro maturation (IVM), which in total take ∼5 weeks. In each culture period, there are several checkpoints that enable the number of oocytes being produced in the culture to be monitored. The basic structure of the culture system should provide a useful tool for clarifying the complicated sequence of oogenesis in mammals.

Effect of the Antioxidant Lipoic Acid in Aortic Phenotype in a Marfan Syndrome Mouse Model

PubMed Central

Debbas, Victor; Salemi, Vera M.; Tavares, Elaine R.; Meirelles, Thayna; Ferreira-Filho, Julio C. A.; Takimura, Celso K.; Pereira, Lygia V.; Laurindo, Francisco R.

2018-01-01

Marfan syndrome (MFS) cardiovascular manifestations such as aortic aneurysms and cardiomyopathy carry substantial morbidity/mortality. We investigated the effects of lipoic acid, an antioxidant, on ROS production and aortic remodeling in a MFS mgΔloxPneo mouse model. MFS and WT (wild-type) 1-month-old mice were allocated to 3 groups: untreated, treated with losartan, and treated with lipoic acid. At 6 months old, echocardiography, ROS production, and morphological analysis of aortas were performed. Aortic ROS generation in 6-month-old MFS animals was higher at advanced stages of disease in MFS. An unprecedented finding in MFS mice analyzed by OCT was the occurrence of focal inhomogeneous regions in the aortic arch, either collagen-rich extremely thickened or collagen-poor hypotrophic regions. MFS animals treated with lipoic acid showed markedly reduced ROS production and lower ERK1/2 phosphorylation; meanwhile, aortic dilation and elastic fiber breakdown were unaltered. Of note, lipoic acid treatment associated with the absence of focal inhomogeneous regions in MFS animals. Losartan reduced aortic dilation and elastic fiber breakdown despite no change in ROS generation. In conclusion, oxidant generation by itself seems neutral with respect to aneurysm progression in MFS; however, lipoic acid-mediated reduction of inhomogeneous regions may potentially associate with less anisotropy and reduced chance of dissection/rupture. PMID:29765495

A CTRP5 gene S163R mutation knock-in mouse model for late-onset retinal degeneration.

PubMed

Chavali, Venkata R M; Khan, Naheed W; Cukras, Catherine A; Bartsch, Dirk-Uwe; Jablonski, Monica M; Ayyagari, Radha

2011-05-15

Late-onset retinal macular degeneration (L-ORD) is an autosomal dominant inherited disorder caused by a single missense mutation (S163R) in the CTRP5/C1QTNF5 protein. Early phenotypic features of L-ORD include: dark adaptation abnormalities, nyctalopia, and drusen deposits in the peripheral macular region. Apart from posterior segment abnormalities, these patients also develop abnormally long anterior lens zonules. In the sixth decade of life the rod and cone function declines, accompanied by electroretinogram (ERG) abnormalities. Some patients also develop choroidal neovascularization and glaucoma. In order to understand the disease pathology and mechanisms involved in retinal dystrophy, we generated a knock-in (Ctrp5(+/-)) mouse model carrying the disease-associated mutation in the mouse Ctrp5/C1QTNF5 gene. These mice develop slower rod-b wave recovery consistent with early dark adaptation abnormalities, accumulation of hyperautofluorescence spots, retinal pigment epithelium abnormalities, drusen, Bruch's membrane abnormalities, loss of photoreceptors, and retinal vascular leakage. The Ctrp5(+/-) mice, which have most of the pathological features of age-related macular degeneration, are unique and may serve as a valuable model both to understand the molecular pathology of late-onset retinal degeneration and to evaluate therapies.

A CTRP5 gene S163R mutation knock-in mouse model for late-onset retinal degeneration

PubMed Central

Chavali, Venkata R.M.; Khan, Naheed W.; Cukras, Catherine A.; Bartsch, Dirk-Uwe; Jablonski, Monica M.; Ayyagari, Radha

2011-01-01

Late-onset retinal macular degeneration (L-ORD) is an autosomal dominant inherited disorder caused by a single missense mutation (S163R) in the CTRP5/C1QTNF5 protein. Early phenotypic features of L-ORD include: dark adaptation abnormalities, nyctalopia, and drusen deposits in the peripheral macular region. Apart from posterior segment abnormalities, these patients also develop abnormally long anterior lens zonules. In the sixth decade of life the rod and cone function declines, accompanied by electroretinogram (ERG) abnormalities. Some patients also develop choroidal neovascularization and glaucoma. In order to understand the disease pathology and mechanisms involved in retinal dystrophy, we generated a knock-in (Ctrp5+/−) mouse model carrying the disease-associated mutation in the mouse Ctrp5/C1QTNF5 gene. These mice develop slower rod-b wave recovery consistent with early dark adaptation abnormalities, accumulation of hyperautofluorescence spots, retinal pigment epithelium abnormalities, drusen, Bruch's membrane abnormalities, loss of photoreceptors, and retinal vascular leakage. The Ctrp5+/−mice, which have most of the pathological features of age-related macular degeneration, are unique and may serve as a valuable model both to understand the molecular pathology of late-onset retinal degeneration and to evaluate therapies. PMID:21349921

Mice with Inactivation of Aryl Hydrocarbon Receptor-Interacting Protein (Aip) Display Complete Penetrance of Pituitary Adenomas with Aberrant ARNT Expression

PubMed Central

Raitila, Anniina; Lehtonen, Heli J.; Arola, Johanna; Heliövaara, Elina; Ahlsten, Manuel; Georgitsi, Marianthi; Jalanko, Anu; Paetau, Anders; Aaltonen, Lauri A.; Karhu, Auli

2010-01-01

Mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene have been shown to predispose to pituitary adenoma predisposition, a condition characterized by growth hormone (GH)-secreting pituitary tumors. To study AIP-mediated tumorigenesis, we generated an Aip mouse model. Heterozygous mice developed normally but were prone to pituitary adenomas, in particular to those secreting GH. A complete loss of AIP was detected in these lesions, and full penetrance was reached at the age of 15 months. No excess of any other tumor type was found. Ki-67 analysis indicated that Aip-deficient tumors have higher proliferation rates compared with Aip-proficient tumors, suggesting a more aggressive disease. Similar to human AIP-deficient pituitary adenomas, immunohistochemical studies showed that expression of aryl hydrocarbon receptor nuclear translocator 1 or 2 (ARNT or ARNT2) protein was lost in the mouse tumors, suggesting that mechanisms of AIP-related tumorigenesis involve aberrant ARNT function. The Aip+/− mouse appears to be an excellent model for the respective human disease phenotype. This model constitutes a tool to further study AIP-associated pituitary tumorigenesis and may be potentially valuable in efforts to develop therapeutic strategies to treat pituitary adenomas. PMID:20709796

Genetic disruption of ankyrin-G in adult mouse forebrain causes cortical synapse alteration and behavior reminiscent of bipolar disorder.

PubMed

Zhu, Shanshan; Cordner, Zachary A; Xiong, Jiali; Chiu, Chi-Tso; Artola, Arabiye; Zuo, Yanning; Nelson, Andrew D; Kim, Tae-Yeon; Zaika, Natalya; Woolums, Brian M; Hess, Evan J; Wang, Xiaofang; Chuang, De-Maw; Pletnikov, Mikhail M; Jenkins, Paul M; Tamashiro, Kellie L; Ross, Christopher A

2017-09-26

Genome-wide association studies have implicated the ANK3 locus in bipolar disorder, a major human psychotic illness. ANK3 encodes ankyrin-G, which organizes the neuronal axon initial segment (AIS). We generated a mouse model with conditional disruption of ANK3 in pyramidal neurons of the adult forebrain (Ank-G cKO). This resulted in the expected loss of pyramidal neuron AIS voltage-gated sodium and potassium channels. There was also dramatic loss of markers of afferent GABAergic cartridge synapses, resembling the cortical microcircuitry changes in brains from psychotic patients, and suggesting disinhibition. Expression of c-fos was increased in cortical pyramidal neurons, consistent with increased neuronal activity due to disinhibition. The mice showed robust behavioral phenotypes reminiscent of aspects of human mania, ameliorated by antimania drugs lithium and valproate. Repeated social defeat stress resulted in repeated episodes of dramatic behavioral changes from hyperactivity to "depression-like" behavior, suggestive of some aspects of human bipolar disorder. Overall, we suggest that this Ank-G cKO mouse model recapitulates some of the core features of human bipolar disorder and indicates that cortical microcircuitry alterations during adulthood may be involved in pathogenesis. The model may be useful for studying disease pathophysiology and for developing experimental therapeutics.

Interspecies chimera between primate embryonic stem cells and mouse embryos: Monkey ESCs engraft into mouse embryos, but not post-implantation fetuses

PubMed Central

Simerly, Calvin; McFarland, Dave; Castro, Carlos; Lin, Chih-Cheng; Redinger, Carrie; Jacoby, Ethan; Mich-Basso, Jocelyn; Orwig, Kyle; Mills, Parker; Ahrens, Eric; Navara, Chris; Schatten, Gerald

2016-01-01

Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more naïve states in these inter-specific chimera assays will be an important future endeavor. PMID:21543277

Testis development, fertility, and survival in Ethanolamine kinase 2-deficient mice.

PubMed

Gustin, Sonja E; Western, Patrick S; McClive, Peter J; Harley, Vincent R; Koopman, Peter A; Sinclair, Andrew H

2008-12-01

Ethanolamine kinase 2 (Eki2) was previously isolated from a differential expression screen designed to identify candidate genes involved in testis development and differentiation. In mouse, Eki2 is specifically up-regulated in Sertoli cells of the developing testis at the time of sex determination. Based on this expression profile, Eki2 was considered a good candidate testis-determining gene. To investigate a possible role of Eki2 in testis development, we have generated a mouse with targeted disruption of the Eki2 gene by using an EGFP replacement strategy. No abnormalities were detected in the Eki2-deficient mice with regard to embryonic and adult testis morphology, differentiation, function, or fertility. Furthermore, no significant differences were observed in litter sizes, pup mortality rates, or distribution of the sexes among the offspring. Ethanolamine kinases are involved in the biosynthesis of phosphatidylethanolamine, a major membrane phospholipid. Expression analysis indicates that the absence of an apparent phenotype in the Eki2-deficient mice may be due to compensation by Eki2-family members or the activation of an alternative pathway to generate phosphatidylethanolamine. Expression of EGFP in this mouse model enabled the isolation of gonad cell populations, providing a useful resource from which to obtain relatively pure early steroidogenic cells for further studies.

Protective effect of 4,4'-diaminodiphenylsulfone against paraquat-induced mouse lung injury

PubMed Central

Cho, Sung Chun; Rhim, Ji Heon; Choi, Hae Ri; Son, Young Hoon; Lee, Seok Jin; Song, Kye-Yong

2011-01-01

Although 4,4'-diaminodiphenylsulfone (DDS, dapsone) has been used to treat several dermatologic conditions, including Hansen disease, for the past several decades, its mode of action has remained a topic of debate. We recently reported that DDS treatment significantly extends the lifespan of the nematode C. elegans by decreasing the generation of reactive oxygen species. Additionally, in in vitro experiments using non-phagocytic human fibroblasts, we found that DDS effectively counteracted the toxicity of paraquat (PQ). In the present study, we extended our work to test the protective effect of DDS against PQ in vivo using a mouse lung injury model. Oral administration of DDS to mice significantly attenuated the lung tissue damage caused by subsequent administration of PQ. Moreover, DDS reduced the local expression of mRNA transcripts encoding inflammation-related molecules, including endothelin-1 (ET-1), macrophage inflammatory protein-1α (MIP-1α), and transforming growth factor-β (TGF-β). In addition, DDS decreased the PQ-induced expression of NADPH oxidase mRNA and activation of protein kinase Cµ (PKCµ). DDS treatment also decreased the PQ-induced generation of superoxide anions in mouse lung fibroblasts. Taken together, these data suggest the novel efficacy of DDS as an effective protective agent against oxidative stress-induced tissue damages. PMID:21765237

Noise Enhances Action Potential Generation in Mouse Sensory Neurons via Stochastic Resonance.

PubMed

Onorato, Irene; D'Alessandro, Giuseppina; Di Castro, Maria Amalia; Renzi, Massimiliano; Dobrowolny, Gabriella; Musarò, Antonio; Salvetti, Marco; Limatola, Cristina; Crisanti, Andrea; Grassi, Francesca

2016-01-01

Noise can enhance perception of tactile and proprioceptive stimuli by stochastic resonance processes. However, the mechanisms underlying this general phenomenon remain to be characterized. Here we studied how externally applied noise influences action potential firing in mouse primary sensory neurons of dorsal root ganglia, modelling a basic process in sensory perception. Since noisy mechanical stimuli may cause stochastic fluctuations in receptor potential, we examined the effects of sub-threshold depolarizing current steps with superimposed random fluctuations. We performed whole cell patch clamp recordings in cultured neurons of mouse dorsal root ganglia. Noise was added either before and during the step, or during the depolarizing step only, to focus onto the specific effects of external noise on action potential generation. In both cases, step + noise stimuli triggered significantly more action potentials than steps alone. The normalized power norm had a clear peak at intermediate noise levels, demonstrating that the phenomenon is driven by stochastic resonance. Spikes evoked in step + noise trials occur earlier and show faster rise time as compared to the occasional ones elicited by steps alone. These data suggest that external noise enhances, via stochastic resonance, the recruitment of transient voltage-gated Na channels, responsible for action potential firing in response to rapid step-wise depolarizing currents.

Noise Enhances Action Potential Generation in Mouse Sensory Neurons via Stochastic Resonance

PubMed Central

Onorato, Irene; D'Alessandro, Giuseppina; Di Castro, Maria Amalia; Renzi, Massimiliano; Dobrowolny, Gabriella; Musarò, Antonio; Salvetti, Marco; Limatola, Cristina; Crisanti, Andrea; Grassi, Francesca

2016-01-01

Noise can enhance perception of tactile and proprioceptive stimuli by stochastic resonance processes. However, the mechanisms underlying this general phenomenon remain to be characterized. Here we studied how externally applied noise influences action potential firing in mouse primary sensory neurons of dorsal root ganglia, modelling a basic process in sensory perception. Since noisy mechanical stimuli may cause stochastic fluctuations in receptor potential, we examined the effects of sub-threshold depolarizing current steps with superimposed random fluctuations. We performed whole cell patch clamp recordings in cultured neurons of mouse dorsal root ganglia. Noise was added either before and during the step, or during the depolarizing step only, to focus onto the specific effects of external noise on action potential generation. In both cases, step + noise stimuli triggered significantly more action potentials than steps alone. The normalized power norm had a clear peak at intermediate noise levels, demonstrating that the phenomenon is driven by stochastic resonance. Spikes evoked in step + noise trials occur earlier and show faster rise time as compared to the occasional ones elicited by steps alone. These data suggest that external noise enhances, via stochastic resonance, the recruitment of transient voltage-gated Na channels, responsible for action potential firing in response to rapid step-wise depolarizing currents. PMID:27525414

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

PubMed

Perez-Cunningham, Jessica; Boyer, Scott W; Landon, Mark; Forsberg, E Camilla

2016-08-01

Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Generation of Mouse Lung Epithelial Cells.

PubMed

Kasinski, Andrea L; Slack, Frank J

2013-08-05

Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of Kras LSL-G12D/+ ; p53 LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following cre-recombinase dependent removal of a stop cassette in the transgenes and subsequent expression of Kra -G12D and p53 R172 . While this protocol may be useful for the generation of epithelial lines from other genetic backgrounds, it should be noted that the Kras; p53 cell line generated here is capable of proliferating in culture without any additional genetic manipulation that is often needed for less aggressive backgrounds.

Generation of improved humanized mouse models for human infectious diseases

PubMed Central

Brehm, Michael A.; Wiles, Michael V.; Greiner, Dale L.; Shultz, Leonard D.

2014-01-01

The study of human-specific infectious agents has been hindered by the lack of optimal small animal models. More recently development of novel strains of immunodeficient mice has begun to provide the opportunity to utilize small animal models for the study of many human-specific infectious agents. The introduction of a targeted mutation in the IL2 receptor common gamma chain gene (IL2rgnull) in mice already deficient in T and B cells led to a breakthrough in the ability to engraft hematopoietic stem cells, as well as functional human lymphoid cells and tissues, effectively creating human immune systems in immunodeficient mice. These humanized mice are becoming increasingly important as pre-clinical models for the study of human immunodeficiency virus-1 (HIV-1) and other human-specific infectious agents. However, there remain a number of opportunities to further improve humanized mouse models for the study of human-specific infectious agents. This is being done by the implementation of innovative technologies, which collectively will accelerate the development of new models of genetically modified mice, including; i) modifications of the host to reduce innate immunity, which impedes human cell engraftment; ii) genetic modification to provide human-specific growth factors and cytokines required for optimal human cell growth and function; iii) and new cell and tissue engraftment protocols. The development of “next generation” humanized mouse models continues to provide exciting opportunities for the establishment of robust small animal models to study the pathogenesis of human-specific infectious agents, as well as for testing the efficacy of therapeutic agents and experimental vaccines. PMID:24607601

A mouse model for fucosidosis recapitulates storage pathology and neurological features of the milder form of the human disease

PubMed Central

Wolf, Heike; Stroobants, Stijn; D'Hooge, Rudi; Hermans-Borgmeyer, Irm; Lüllmann-Rauch, Renate; Dierks, Thomas; Lübke, Torben

2016-01-01

ABSTRACT Fucosidosis is a rare lysosomal storage disorder caused by the inherited deficiency of the lysosomal hydrolase α-L-fucosidase, which leads to an impaired degradation of fucosylated glycoconjugates. Here, we report the generation of a fucosidosis mouse model, in which the gene for lysosomal α-L-fucosidase (Fuca1) was disrupted by gene targeting. Homozygous knockout mice completely lack α-L-fucosidase activity in all tested organs leading to highly elevated amounts of the core-fucosylated glycoasparagine Fuc(α1,6)-GlcNAc(β1-N)-Asn and, to a lesser extent, other fucosylated glycoasparagines, which all were also partially excreted in urine. Lysosomal storage pathology was observed in many visceral organs, such as in the liver, kidney, spleen and bladder, as well as in the central nervous system (CNS). On the cellular level, storage was characterized by membrane-limited cytoplasmic vacuoles primarily containing water-soluble storage material. In the CNS, cellular alterations included enlargement of the lysosomal compartment in various cell types, accumulation of secondary storage material and neuroinflammation, as well as a progressive loss of Purkinje cells combined with astrogliosis leading to psychomotor and memory deficits. Our results demonstrate that this new fucosidosis mouse model resembles the human disease and thus will help to unravel underlying pathological processes. Moreover, this model could be utilized to establish diagnostic and therapeutic strategies for fucosidosis. PMID:27491075

Multiplexed MRM-based assays for the quantitation of proteins in mouse plasma and heart tissue.

PubMed

Percy, Andrew J; Michaud, Sarah A; Jardim, Armando; Sinclair, Nicholas J; Zhang, Suping; Mohammed, Yassene; Palmer, Andrea L; Hardie, Darryl B; Yang, Juncong; LeBlanc, Andre M; Borchers, Christoph H

2017-04-01

The mouse is the most commonly used laboratory animal, with more than 14 million mice being used for research each year in North America alone. The number and diversity of mouse models is increasing rapidly through genetic engineering strategies, but detailed characterization of these models is still challenging because most phenotypic information is derived from time-consuming histological and biochemical analyses. To expand the biochemists' toolkit, we generated a set of targeted proteomic assays for mouse plasma and heart tissue, utilizing bottom-up LC/MRM-MS with isotope-labeled peptides as internal standards. Protein quantitation was performed using reverse standard curves, with LC-MS platform and curve performance evaluated by quality control standards. The assays comprising the final panel (101 peptides for 81 proteins in plasma; 227 peptides for 159 proteins in heart tissue) have been rigorously developed under a fit-for-purpose approach and utilize stable-isotope labeled peptides for every analyte to provide high-quality, precise relative quantitation. In addition, the peptides have been tested to be interference-free and the assay is highly multiplexed, with reproducibly determined protein concentrations spanning >4 orders of magnitude. The developed assays have been used in a small pilot study to demonstrate their application to molecular phenotyping or biomarker discovery/verification studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development and characterization of a mouse floxed Bmp2 osteoblast cell line that retains osteoblast genotype and phenotype

PubMed Central

Wu, Li-an; Feng, Junsheng; Wang, Lynn; Mu, Yan-dong; Baker, Andrew; Donly, Kevin J.; Harris, Stephen E.; MacDougall, Mary; Chen, Shuo

2011-01-01

Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways. PMID:21271257

Geometry Processing of Conventionally Produced Mouse Brain Slice Images.

PubMed

Agarwal, Nitin; Xu, Xiangmin; Gopi, M

2018-04-21

Brain mapping research in most neuroanatomical laboratories relies on conventional processing techniques, which often introduce histological artifacts such as tissue tears and tissue loss. In this paper we present techniques and algorithms for automatic registration and 3D reconstruction of conventionally produced mouse brain slices in a standardized atlas space. This is achieved first by constructing a virtual 3D mouse brain model from annotated slices of Allen Reference Atlas (ARA). Virtual re-slicing of the reconstructed model generates ARA-based slice images corresponding to the microscopic images of histological brain sections. These image pairs are aligned using a geometric approach through contour images. Histological artifacts in the microscopic images are detected and removed using Constrained Delaunay Triangulation before performing global alignment. Finally, non-linear registration is performed by solving Laplace's equation with Dirichlet boundary conditions. Our methods provide significant improvements over previously reported registration techniques for the tested slices in 3D space, especially on slices with significant histological artifacts. Further, as one of the application we count the number of neurons in various anatomical regions using a dataset of 51 microscopic slices from a single mouse brain. To the best of our knowledge the presented work is the first that automatically registers both clean as well as highly damaged high-resolutions histological slices of mouse brain to a 3D annotated reference atlas space. This work represents a significant contribution to this subfield of neuroscience as it provides tools to neuroanatomist for analyzing and processing histological data. Copyright © 2018 Elsevier B.V. All rights reserved.

Propagating Humanized BLT Mice for the Study of Human Immunology and Immunotherapy.

PubMed

Smith, Drake J; Lin, Levina J; Moon, Heesung; Pham, Alexander T; Wang, Xi; Liu, Siyuan; Ji, Sunjong; Rezek, Valerie; Shimizu, Saki; Ruiz, Marlene; Lam, Jennifer; Janzen, Deanna M; Memarzadeh, Sanaz; Kohn, Donald B; Zack, Jerome A; Kitchen, Scott G; An, Dong Sung; Yang, Lili

2016-12-15

The humanized bone marrow-liver-thymus (BLT) mouse model harbors a nearly complete human immune system, therefore providing a powerful tool to study human immunology and immunotherapy. However, its application is greatly limited by the restricted supply of human CD34 + hematopoietic stem cells and fetal thymus tissues that are needed to generate these mice. The restriction is especially significant for the study of human immune systems with special genetic traits, such as certain human leukocyte antigen (HLA) haplotypes or monogene deficiencies. To circumvent this critical limitation, we have developed a method to quickly propagate established BLT mice. Through secondary transfer of bone marrow cells and human thymus implants from BLT mice into NSG (NOD/SCID/IL-2Rγ -/- ) recipient mice, we were able to expand one primary BLT mouse into a colony of 4-5 proBLT (propagated BLT) mice in 6-8 weeks. These proBLT mice reconstituted human immune cells, including T cells, at levels comparable to those of their primary BLT donor mouse. They also faithfully inherited the human immune cell genetic traits from their donor BLT mouse, such as the HLA-A2 haplotype that is of special interest for studying HLA-A2-restricted human T cell immunotherapies. Moreover, an EGFP reporter gene engineered into the human immune system was stably passed from BLT to proBLT mice, making proBLT mice suitable for studying human immune cell gene therapy. This method provides an opportunity to overcome a critical hurdle to utilizing the BLT humanized mouse model and enables its more widespread use as a valuable preclinical research tool.

Long-term imaging of mouse embryos using adaptive harmonic generation microscopy

NASA Astrophysics Data System (ADS)

Thayil, Anisha; Watanabe, Tomoko; Jesacher, Alexander; Wilson, Tony; Srinivas, Shankar; Booth, Martin

2011-04-01

We present a detailed description of an adaptive harmonic generation (HG) microscope and culture techniques that permit long-term, three-dimensional imaging of mouse embryos. HG signal from both pre- and postimplantation stage (0.5-5.5 day-old) mouse embryos are fully characterized. The second HG images reveal central spindles during cytokinesis whereas third HG images show several features, such as lipid droplets, nucleoli, and plasma membranes. The embryos are found to develop normally during one-day-long discontinuous HG imaging, permitting the observation of several dynamic events, such as morula compaction and blastocyst formation.

Analysis of response to 20 generations of selection for body composition in mice: fit to infinitesimal model assumptions

PubMed Central

Martinez, Victor; Bünger, Lutz; Hill, William G

2000-01-01

Data were analysed from a divergent selection experiment for an indicator of body composition in the mouse, the ratio of gonadal fat pad to body weight (GFPR). Lines were selected for 20 generations for fat (F), lean (L) or were unselected (C), with three replicates of each. Selection was within full-sib families, 16 families per replicate for the first seven generations, eight subsequently. At generation 20, GFPR in the F lines was twice and in the L lines half that of C. A log transformation removed both asymmetry of response and heterogeneity of variance among lines, and so was used throughout. Estimates of genetic variance and heritability (approximately 50%) obtained using REML with an animal model were very similar, whether estimated from the first few generations of selection, or from all 20 generations, or from late generations having fitted pedigree. The estimates were also similar when estimated from selected or control lines. Estimates from REML also agreed with estimates of realised heritability. The results all accord with expectations under the infinitesimal model, despite the four-fold changes in mean. Relaxed selection lines, derived from generation 20, showed little regression in fatness after 40 generations without selection. PMID:14736404

MPHASYS: a mouse phenotype analysis system

PubMed Central

Calder, R Brent; Beems, Rudolf B; van Steeg, Harry; Mian, I Saira; Lohman, Paul HM; Vijg, Jan

2007-01-01

Background Systematic, high-throughput studies of mouse phenotypes have been hampered by the inability to analyze individual animal data from a multitude of sources in an integrated manner. Studies generally make comparisons at the level of genotype or treatment thereby excluding associations that may be subtle or involve compound phenotypes. Additionally, the lack of integrated, standardized ontologies and methodologies for data exchange has inhibited scientific collaboration and discovery. Results Here we introduce a Mouse Phenotype Analysis System (MPHASYS), a platform for integrating data generated by studies of mouse models of human biology and disease such as aging and cancer. This computational platform is designed to provide a standardized methodology for working with animal data; a framework for data entry, analysis and sharing; and ontologies and methodologies for ensuring accurate data capture. We describe the tools that currently comprise MPHASYS, primarily ones related to mouse pathology, and outline its use in a study of individual animal-specific patterns of multiple pathology in mice harboring a specific germline mutation in the DNA repair and transcription-specific gene Xpd. Conclusion MPHASYS is a system for analyzing multiple data types from individual animals. It provides a framework for developing data analysis applications, and tools for collecting and distributing high-quality data. The software is platform independent and freely available under an open-source license [1]. PMID:17553167

A mouse model for human anal cancer.

PubMed

Stelzer, Marie K; Pitot, Henry C; Liem, Amy; Schweizer, Johannes; Mahoney, Charles; Lambert, Paul F

2010-12-01

Human anal cancers are associated with high-risk human papillomaviruses (HPV) that cause other anogenital cancers and head and neck cancers. As with other cancers, HPV16 is the most common high-risk HPV in anal cancers. We describe the generation and characterization of a mouse model for human anal cancer. This model makes use of K14E6 and K14E7 transgenic mice in which the HPV16 E6 and E7 genes are directed in their expression to stratified squamous epithelia. HPV16 E6 and E7 possess oncogenic properties including, but not limited to, their capacity to inactivate the cellular tumor suppressors p53 and pRb, respectively. Both E6 and E7 were found to be functionally expressed in the anal epithelia of K14E6/K14E7 transgenic mice. To assess the susceptibility of these mice to anal cancer, mice were treated topically with dimethylbenz[a]anthracene (DMBA), a chemical carcinogen that is known to induce squamous cell carcinomas in other sites. Nearly 50% of DMBA-treated HPV16 E6/E7 transgenic mice showed overt signs of tumors, whereas none of the like-treated nontransgenic mice showed tumors. Histopathologic analyses confirmed that the HPV16 transgenic mice were increased in their susceptibility to anal cancers and precancerous lesions. Biomarker analyses demonstrated that these mouse anal cancers exhibit properties that are similar to those observed in HPV-positive precursors to human anal cancer. This is the first mouse model for investigating the contributions of viral and cellular factors in anal carcinogenesis, and should provide a platform for assessing new therapeutic modalities for treating and/or preventing this type of cancer. ©2010 AACR.

A Mouse Model for Human Anal Cancer

PubMed Central

Stelzer, Marie K.; Pitot, Henry C.; Liem, Amy; Schweizer, Johannes; Mahoney, Charles; Lambert, Paul F.

2010-01-01

Human anal cancers are associated with high-risk human papillomaviruses (HPVs) that cause other anogenital cancers and head and neck cancers. As with other cancers, HPV16 is the most common high-risk HPV in anal cancers. We describe the generation and characterization of a mouse model for human anal cancer. This model makes use of K14E6 and K14E7 transgenic mice in which the HPV16 E6 and E7 genes are directed in their expression to stratified squamous epithelia. HPV16 E6 and E7 possess oncogenic properties including but not limited to their capacity to inactivate the cellular tumor suppressors p53 and pRb, respectively. Both E6 and E7 were found to be functionally expressed in the anal epithelia of K14E6/K14E7 transgenic mice. To assess the susceptibility of these mice to anal cancer, mice were treated topically with dimethylbenz[a]anthracene (DMBA), a chemical carcinogen that is known to induce squamous cell carcinomas in other sites. Nearly 50% of DMBA-treated HPV16 E6/E7 transgenic mice showed overt signs of tumors; whereas, none of the like treated non-transgenic mice showed tumors. Histopathological analyses confirmed that the HPV16 transgenic mice were increased in their susceptibility to anal cancers and precancerous lesions. Biomarker analyses demonstrated that these mouse anal cancers exhibit properties that are similar to those observed in HPV-positive precursors to human anal cancer. This is the first mouse model for investigating the contributions of viral and cellular factors in anal carcinogenesis, and should provide a platform for assessing new therapeutic modalities for treating and/or preventing this type of cancer. PMID:20947489

Prolonged running, not fluoxetine treatment, increases neurogenesis, but does not alter neuropathology, in the 3xTg mouse model of Alzheimer's disease.

PubMed

Marlatt, Michael W; Potter, Michelle C; Bayer, Thomas A; van Praag, Henriette; Lucassen, Paul J

2013-01-01

Reductions in adult neurogenesis have been documented in the original 3xTg mouse model of Alzheimer's disease (AD), notably occurring at the same age when spatial memory deficits and amyloid plaque pathology appeared. As this suggested reduced neurogenesis was associated with behavioral deficits, we tested whether activity and pharmacological stimulation could prevent memory deficits and modify neurogenesis and/or neuropathology in the 3xTg model backcrossed to the C57Bl/6 strain. We chronically administered the antidepressant fluoxetine to one group of mice, allowed access to a running wheel in another, and combined both treatments in a third cohort. All treatments lasted for 11 months. The female 3xTg mice failed to exhibit any deficits in spatial learning and memory as measured in the Morris water maze, indicating that when backcrossed to the C57Bl/6 strain, the 3xTg mice lost the behavioral phenotype that was present in the original 3xTg mouse maintained on a hybrid background. Despite this, the backcrossed 3xTg mice expressed prominent intraneuronal amyloid beta (Aβ) levels in the cortex and amygdala, with lower levels in the CA1 area of the hippocampus. In the combined cohort, fluoxetine treatment interfered with exercise and reduced the total distance run. The extent of Aβ neuropathology, the tau accumulations, or BDNF levels, were not altered by prolonged exercise. Thus, neuropathology was present but not paralleled by spatial memory deficits in the backcrossed 3xTg mouse model of AD. Prolonged exercise for 11 months did improve the long-term survival of newborn neurons generated during middle-age, whereas fluoxetine had no effect. We further review and discuss the relevant literature in this respect.

System parameters for erythropoiesis control model: Comparison of normal values in human and mouse model

NASA Technical Reports Server (NTRS)

1979-01-01

The computer model for erythropoietic control was adapted to the mouse system by altering system parameters originally given for the human to those which more realistically represent the mouse. Parameter values were obtained from a variety of literature sources. Using the mouse model, the mouse was studied as a potential experimental model for spaceflight. Simulation studies of dehydration and hypoxia were performed. A comparison of system parameters for the mouse and human models is presented. Aside from the obvious differences expected in fluid volumes, blood flows and metabolic rates, larger differences were observed in the following: erythrocyte life span, erythropoietin half-life, and normal arterial pO2.

A predictive model of asymmetric morphogenesis from 3D reconstructions of mouse heart looping dynamics

PubMed Central

Le Garrec, Jean-François; Ivanovitch, Kenzo D; Raphaël, Etienne; Bangham, J Andrew; Torres, Miguel; Coen, Enrico; Mohun, Timothy J

2017-01-01

How left-right patterning drives asymmetric morphogenesis is unclear. Here, we have quantified shape changes during mouse heart looping, from 3D reconstructions by HREM. In combination with cell labelling and computer simulations, we propose a novel model of heart looping. Buckling, when the cardiac tube grows between fixed poles, is modulated by the progressive breakdown of the dorsal mesocardium. We have identified sequential left-right asymmetries at the poles, which bias the buckling in opposite directions, thus leading to a helical shape. Our predictive model is useful to explore the parameter space generating shape variations. The role of the dorsal mesocardium was validated in Shh-/- mutants, which recapitulate heart shape changes expected from a persistent dorsal mesocardium. Our computer and quantitative tools provide novel insight into the mechanism of heart looping and the contribution of different factors, beyond the simple description of looping direction. This is relevant to congenital heart defects. PMID:29179813

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

PubMed

Potter, Paul K; Bowl, Michael R; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E; Simon, Michelle M; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V; Law, Gemma; MacLaren, Robert E; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H; Foster, Russell G; Jackson, Ian J; Peirson, Stuart N; Thakker, Rajesh V; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D M

2016-08-18

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease

PubMed Central

Potter, Paul K.; Bowl, Michael R.; Jeyarajan, Prashanthini; Wisby, Laura; Blease, Andrew; Goldsworthy, Michelle E.; Simon, Michelle M.; Greenaway, Simon; Michel, Vincent; Barnard, Alun; Aguilar, Carlos; Agnew, Thomas; Banks, Gareth; Blake, Andrew; Chessum, Lauren; Dorning, Joanne; Falcone, Sara; Goosey, Laurence; Harris, Shelley; Haynes, Andy; Heise, Ines; Hillier, Rosie; Hough, Tertius; Hoslin, Angela; Hutchison, Marie; King, Ruairidh; Kumar, Saumya; Lad, Heena V.; Law, Gemma; MacLaren, Robert E.; Morse, Susan; Nicol, Thomas; Parker, Andrew; Pickford, Karen; Sethi, Siddharth; Starbuck, Becky; Stelma, Femke; Cheeseman, Michael; Cross, Sally H.; Foster, Russell G.; Jackson, Ian J.; Peirson, Stuart N.; Thakker, Rajesh V.; Vincent, Tonia; Scudamore, Cheryl; Wells, Sara; El-Amraoui, Aziz; Petit, Christine; Acevedo-Arozena, Abraham; Nolan, Patrick M.; Cox, Roger; Mallon, Anne-Marie; Brown, Steve D. M.

2016-01-01

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss. PMID:27534441

Nuclear receptor TLX stimulates hippocampal neurogenesis and enhances learning and memory in a transgenic mouse model.

PubMed

Murai, Kiyohito; Qu, Qiuhao; Sun, GuoQiang; Ye, Peng; Li, Wendong; Asuelime, Grace; Sun, Emily; Tsai, Guochuan E; Shi, Yanhong

2014-06-24

The role of the nuclear receptor TLX in hippocampal neurogenesis and cognition has just begun to be explored. In this study, we generated a transgenic mouse model that expresses TLX under the control of the promoter of nestin, a neural precursor marker. Transgenic TLX expression led to mice with enlarged brains with an elongated hippocampal dentate gyrus and increased numbers of newborn neurons. Specific expression of TLX in adult hippocampal dentate gyrus via lentiviral transduction increased the numbers of BrdU(+) cells and BrdU(+)NeuN(+) neurons. Furthermore, the neural precursor-specific expression of the TLX transgene substantially rescued the neurogenic defects of TLX-null mice. Consistent with increased neurogenesis in the hippocampus, the TLX transgenic mice exhibited enhanced cognition with increased learning and memory. These results suggest a strong association between hippocampal neurogenesis and cognition, as well as significant contributions of TLX to hippocampal neurogenesis, learning, and memory.

Nuclear Receptors in Neurodegenerative Diseases

PubMed Central

Skerrett, Rebecca; Malm, Tarja; Landreth, Gary

2014-01-01

Nuclear receptors have generated substantial interest in the past decade as potential therapeutic targets for the treatment of neurodegenerative disorders. Despite years of effort, effective treatments for progressive neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and ALS remain elusive, making non-classical drug targets such as nuclear receptors an attractive alternative. A substantial literature in mouse models of disease and several clinical trials have investigated the role of nuclear receptors in various neurodegenerative disorders, most prominently AD. These studies have met with mixed results, yet the majority of studies in mouse models report positive outcomes. The mechanisms by which nuclear receptor agonists affect disease pathology remain unclear. Deciphering the complex signaling underlying nuclear receptor action in neurodegenerative diseases is essential for understanding this variability in preclinical studies, and for the successful translation of nuclear receptor agonists into clinical therapies. PMID:24874548

Effects of photoacoustic imaging and photothermal ablation therapy mediated by targeted hollow gold nanospheres in an orthotopic mouse xenograft model of glioma

PubMed Central

Lu, Wei; Melancon, Marites P.; Xiong, Chiyi; Huang, Qian; Elliott, Andrew; Song, Shaoli; Zhang, Rui; Flores, Leo G.; Gelovani, Juri G.; Wang, Lihong V.; Ku, Geng; Stafford, R. Jason; Li, Chun

2011-01-01

Advancements in nanotechnology have made it possible to create multifunctional nanostructures that can be used simultaneously to image and treat cancers. For example, hollow gold nanospheres (HAuNS) have been shown to generate intense photoacoustic signals and induce efficient photothermal ablation (PTA) therapy. In this study, we used photoacoustic tomography (PAT), a hybrid imaging modality, to assess the intravenous delivery of HAuNS targeted to integrins that are overexpressed in both glioma and angiogenic blood vessels in a mouse model of glioma. Mice were then treated with near-infrared laser, which elevated tumor temperature by 20.7 °C. We found that PTA treatment significantly prolonged the survival of tumor-bearing mice. Taken together, these results demonstrate the feasibility of using a single nanostructure for image-guided local tumor PTA therapy using photoacoustic molecular imaging. PMID:21856744

Transgenic mouse models enabling photolabeling of individual neurons in vivo.

PubMed

Peter, Manuel; Bathellier, Brice; Fontinha, Bruno; Pliota, Pinelopi; Haubensak, Wulf; Rumpel, Simon

2013-01-01

One of the biggest tasks in neuroscience is to explain activity patterns of individual neurons during behavior by their cellular characteristics and their connectivity within the neuronal network. To greatly facilitate linking in vivo experiments with a more detailed molecular or physiological analysis in vitro, we have generated and characterized genetically modified mice expressing photoactivatable GFP (PA-GFP) that allow conditional photolabeling of individual neurons. Repeated photolabeling at the soma reveals basic morphological features due to diffusion of activated PA-GFP into the dendrites. Neurons photolabeled in vivo can be re-identified in acute brain slices and targeted for electrophysiological recordings. We demonstrate the advantages of PA-GFP expressing mice by the correlation of in vivo firing rates of individual neurons with their expression levels of the immediate early gene c-fos. Generally, the mouse models described in this study enable the combination of various analytical approaches to characterize living cells, also beyond the neurosciences.

Behavioral Characterization of a Mouse Model Overexpressing DSCR1/ RCAN1

PubMed Central

Dierssen, Mara; Arqué, Gloria; McDonald, Jerome; Andreu, Nuria; Martínez-Cué, Carmen; Flórez, Jesús; Fillat, Cristina

2011-01-01

DSCR1/ RCAN1 is a chromosome 21 gene found to be overexpressed in the brains of Down syndrome (DS) and postulated as a good candidate to contribute to mental disability. However, even though Rcan1 knockout mice have pronounced spatial learning and memory deficits, the possible deleterious effects of its overexpression in DS are not well understood. We have generated a transgenic mouse model overexpressing DSCR1/RCAN1 in the brain and analyzed the effect of RCAN1 overexpression on cognitive function. TgRCAN1 mice present a marked disruption of the learning process in a visuo-spatial learning task. However, no significant differences were observed in the performance of the memory phase of the test (removal session) nor in a step-down passive avoidance task, thus suggesting that once learning has been established, the animals are able to consolidate the information in the longer term. PMID:21364922

Behavioral Analysis of Genetically Modified Mice Indicates Essential Roles of Neurosteroidal Estrogen

PubMed Central

Honda, Shin-Ichiro; Wakatsuki, Toru; Harada, Nobuhiro

2011-01-01

Aromatase in the mouse brain is expressed only in the nerve cells of specific brain regions with a transient peak during the neonatal period when sexual behaviors become organized. The aromatase-knockout (ArKO) mouse, generated to shed light on the physiological functions of estrogen in the brain, exhibited various abnormal behaviors, concomitant with undetectable estrogen and increased androgen in the blood. To further elucidate the effects of neurosteroidal estrogens on behavioral phenotypes, we first prepared an brain-specific aromatase transgenic (bsArTG) mouse by introduction of a human aromatase transgene controlled under a −6.5 kb upstream region of the brain-specific promoter of the mouse aromatase gene into fertilized mouse eggs, because the −6.5 kb promoter region was previously shown to contain the minimal essential element responsible for brain-specific spatiotemporal expression. Then, an ArKO mouse expressing the human aromatase only in the brain was generated by crossing the bsArTG mouse with the ArKO mouse. The resulting mice (ArKO/bsArTG mice) nearly recovered from abnormal sexual, aggressive, and locomotive (exploratory) behaviors, in spite of having almost the same serum levels of estrogen and androgen as the adult ArKO mouse. These results suggest that estrogens locally synthesized in the specific neurons of the perinatal mouse brain directly act on the neurons and play crucial roles in the organization of neuronal networks participating in the control of sexual, aggressive, and locomotive (exploratory) behaviors. PMID:22654807

Murine tissue-engineered stomach demonstrates epithelial differentiation.

PubMed

Speer, Allison L; Sala, Frederic G; Matthews, Jamil A; Grikscheit, Tracy C

2011-11-01

Gastric cancer remains the second largest cause of cancer-related mortality worldwide. Postgastrectomy morbidity is considerable and quality of life is poor. Tissue-engineered stomach is a potential replacement solution to restore adequate food reservoir and gastric physiology. In this study, we performed a detailed investigation of the development of tissue-engineered stomach in a mouse model, specifically evaluating epithelial differentiation, proliferation, and the presence of putative stem cell markers. Organoid units were isolated from <3 wk-old mouse glandular stomach and seeded onto biodegradable scaffolds. The constructs were implanted into the omentum of adult mice. Implants were harvested at designated time points and analyzed with histology and immunohistochemistry. Tissue-engineered stomach grows as an expanding sphere with a simple columnar epithelium organized into gastric glands and an adjacent muscularis. The regenerated gastric epithelium demonstrates differentiation of all four cell types: mucous, enteroendocrine, chief, and parietal cells. Tissue-engineered stomach epithelium proliferates at a rate comparable to native glandular stomach and expresses two putative stem cell markers: DCAMKL-1 and Lgr5. This study demonstrates the successful generation of tissue-engineered stomach in a mouse model for the first time. Regenerated gastric epithelium is able to appropriately proliferate and differentiate. The generation of murine tissue-engineered stomach is a necessary advance as it provides the transgenic tools required to investigate the molecular and cellular mechanisms of this regenerative process. Delineating the mechanism of how tissue-engineered stomach develops in vivo is an important precursor to its use as a human stomach replacement therapy. Copyright © 2011 Elsevier Inc. All rights reserved.

Generation of transgenic mice expressing EGFP protein fused to NP68 MHC class I epitope using lentivirus vectors.

PubMed

Tomkowiak, Martine; Ghittoni, Raffaella; Teixeira, Marie; Blanquier, Bariza; Szécsi, Judit; Nègre, Didier; Aubert, Denise; Coupet, Charles-Antoine; Brunner, Molly; Verhoeyen, Els; Thoumas, Jean-Louis; Cosset, François-Loïc; Leverrier, Yann; Marvel, Jacqueline

2013-03-01

Immune tolerance to self-antigens is a complex process that utilizes multiple mechanisms working in concert to maintain homeostasis and prevent autoimmunity. Considerable progress in deciphering the mechanisms controlling the activation or deletion of T cells has been made by using T cell receptor (TCR) transgenic mice. One such model is the F5 model in which CD8 T cells express a TCR specific for an epitope derived from the influenza NP68 protein. Our aim was to create transgenic mouse models expressing constitutively the NP68 epitope fused to enhanced green fluorescent protein (EGFP) in order to assess unambiguously the relative levels of NP68 epitope expressed by single cells. We used a lentiviral-based approach to generate two independent transgenic mouse strains expressing the fusion protein EGFP-NP68 under the control of CAG (CMV immediate early enhancer and the chicken β-actin promoter) or spleen focus-forming virus (SFFV) promoters. Analysis of the pattern of EGFP expression in the hematopoietic compartment showed that CAG and SFFV promoters are differentially regulated during T cell development. However, both promoters drove high EGFP-NP68 expression in dendritic cells (pDCs, CD8α(+) cDCs, and CD8α(-) cDCs) from spleen or generated in vitro following differentiation from bone-marrow progenitors. NP68 epitope was properly processed and successfully presented by dendritic cells (DCs) by direct presentation and cross-presentation to F5 CD8 T cells. The models presented here are valuable tools to investigate the priming of F5 CD8 T cells by different subsets of DCs. Copyright © 2013 Wiley Periodicals, Inc.

Induction of a specific strong polyantigenic cellular immune response after short-term chemotherapy controls bacillary reactivation in murine and guinea pig experimental models of tuberculosis.

PubMed

Guirado, Evelyn; Gil, Olga; Cáceres, Neus; Singh, Mahavir; Vilaplana, Cristina; Cardona, Pere-Joan

2008-08-01

RUTI is a therapeutic vaccine that is generated from detoxified and liposomed Mycobacterium tuberculosis cell fragments that has demonstrated its efficacy in the control of bacillus reactivation after short-term chemotherapy. The aim of this study was to characterize the cellular immune response generated after the therapeutic administration of RUTI and to corroborate the lack of toxicity of the vaccine. Mouse and guinea pig experimental models were infected with a low-dose M. tuberculosis aerosol. RUTI-treated animals showed the lowest bacillary load in both experimental models. RUTI also decreased the percentage of pulmonary granulomatous infiltration in the mouse and guinea pig models. This was not the case after Mycobacterium bovis BCG treatment. Cellular immunity was studied through the characterization of the intracellular gamma interferon (IFN-gamma)-producing cells after the splenocytes' stimulation with M. tuberculosis-specific structural and growth-related antigens. Our data show that the difference between the therapeutic administration of BCG and RUTI resides mainly in the stronger activation of IFN-gamma(+) CD4(+) cells and CD8(+) cells against tuberculin purified protein derivative, ESAT-6, and Ag85B that RUTI generates. Both vaccines also triggered a specific immune response against the M. tuberculosis structural antigens Ag16kDa and Ag38kDa and a marked mRNA expression of IFN-gamma, tumor necrosis factor, interleukin-12, inducible nitric oxide synthase, and RANTES in the lung. The results show that RUTI's therapeutic effect is linked not only to the induction of a Th1 response but also to the stimulation of a quicker and stronger specific immunity against structural and growth-related antigens that reduces both the bacillary load and the pulmonary pathology.

Induction of a Specific Strong Polyantigenic Cellular Immune Response after Short-Term Chemotherapy Controls Bacillary Reactivation in Murine and Guinea Pig Experimental Models of Tuberculosis▿

PubMed Central

Guirado, Evelyn; Gil, Olga; Cáceres, Neus; Singh, Mahavir; Vilaplana, Cristina; Cardona, Pere-Joan

2008-01-01

RUTI is a therapeutic vaccine that is generated from detoxified and liposomed Mycobacterium tuberculosis cell fragments that has demonstrated its efficacy in the control of bacillus reactivation after short-term chemotherapy. The aim of this study was to characterize the cellular immune response generated after the therapeutic administration of RUTI and to corroborate the lack of toxicity of the vaccine. Mouse and guinea pig experimental models were infected with a low-dose M. tuberculosis aerosol. RUTI-treated animals showed the lowest bacillary load in both experimental models. RUTI also decreased the percentage of pulmonary granulomatous infiltration in the mouse and guinea pig models. This was not the case after Mycobacterium bovis BCG treatment. Cellular immunity was studied through the characterization of the intracellular gamma interferon (IFN-γ)-producing cells after the splenocytes' stimulation with M. tuberculosis-specific structural and growth-related antigens. Our data show that the difference between the therapeutic administration of BCG and RUTI resides mainly in the stronger activation of IFN-γ+ CD4+ cells and CD8+ cells against tuberculin purified protein derivative, ESAT-6, and Ag85B that RUTI generates. Both vaccines also triggered a specific immune response against the M. tuberculosis structural antigens Ag16kDa and Ag38kDa and a marked mRNA expression of IFN-γ, tumor necrosis factor, interleukin-12, inducible nitric oxide synthase, and RANTES in the lung. The results show that RUTI's therapeutic effect is linked not only to the induction of a Th1 response but also to the stimulation of a quicker and stronger specific immunity against structural and growth-related antigens that reduces both the bacillary load and the pulmonary pathology. PMID:18524883

Mouse model of proximal colon-specific tumorigenesis driven by microsatellite instability-induced Cre-mediated inactivation of Apc and activation of Kras.

PubMed

Kawaguchi, Yasuo; Hinoi, Takao; Saito, Yasufumi; Adachi, Tomohiro; Miguchi, Masashi; Niitsu, Hiroaki; Sasada, Tatsunari; Shimomura, Manabu; Egi, Hiroyuki; Oka, Shiro; Tanaka, Shinji; Chayama, Kazuaki; Sentani, Kazuhiro; Oue, Naohide; Yasui, Wataru; Ohdan, Hideki

2016-05-01

KRAS gene mutations are found in 40-50% of colorectal cancer cases, but their functional contribution is not fully understood. To address this issue, we generated genetically engineered mice with colon tumors expressing an oncogenic Kras(G12D) allele in the context of the Adenomatous polyposis coli (Apc) deficiency to compare them to tumors harboring Apc deficiency alone. CDX2P9.5-G22Cre (referred to as G22Cre) mice showing inducible Cre recombinase transgene expression in the proximal colon controlled under the CDX2 gene promoter were intercrossed with Apc (flox/flox) mice and LSL-Kras (G12D) mice carrying loxP-flanked Apc and Lox-Stop-Lox oncogenic Kras(G12D) alleles, respectively, to generate G22Cre; Apc(flox/flox); Kras(G12D) and G22Cre; Apc(flox/flox); KrasWT mice. Gene expression profiles of the tumors were analyzed using high-density oligonucleotide arrays. Morphologically, minimal difference in proximal colon tumor was observed between the two mouse models. Consistent with previous findings in vitro, Glut1 transcript and protein expression was up-regulated in the tumors of G22Cre;Apc (flox/flox) ; Kras(G12D) mice. Immunohistochemical staining analysis revealed that GLUT1 protein expression correlated with KRAS mutations in human colorectal cancer. Microarray analysis identified 11 candidate genes upregulated more than fivefold and quantitative PCR analysis confirmed that Aqp8, Ttr, Qpct, and Slc26a3 genes were upregulated 3.7- to 30.2-fold in tumors with mutant Kras. These results demonstrated the validity of the G22Cre; Apc(flox/flox) ;Kras (G12D) mice as a new mouse model with oncogenic Kras activation. We believe that this model can facilitate efforts to define novel factors that contribute to the pathogenesis of human colorectal cancer with KRAS mutations.

Human mammary microenvironment better regulates the biology of human breast cancer in humanized mouse model.

PubMed

Zheng, Ming-Jie; Wang, Jue; Xu, Lu; Zha, Xiao-Ming; Zhao, Yi; Ling, Li-Jun; Wang, Shui

2015-02-01

During the past decades, many efforts have been made in mimicking the clinical progress of human cancer in mouse models. Previously, we developed a human breast tissue-derived (HB) mouse model. Theoretically, it may mimic the interactions between "species-specific" mammary microenvironment of human origin and human breast cancer cells. However, detailed evidences are absent. The present study (in vivo, cellular, and molecular experiments) was designed to explore the regulatory role of human mammary microenvironment in the progress of human breast cancer cells. Subcutaneous (SUB), mammary fat pad (MFP), and HB mouse models were developed for in vivo comparisons. Then, the orthotopic tumor masses from three different mouse models were collected for primary culture. Finally, the biology of primary cultured human breast cancer cells was compared by cellular and molecular experiments. Results of in vivo mouse models indicated that human breast cancer cells grew better in human mammary microenvironment. Cellular and molecular experiments confirmed that primary cultured human breast cancer cells from HB mouse model showed a better proliferative and anti-apoptotic biology than those from SUB to MFP mouse models. Meanwhile, primary cultured human breast cancer cells from HB mouse model also obtained the migratory and invasive biology for "species-specific" tissue metastasis to human tissues. Comprehensive analyses suggest that "species-specific" mammary microenvironment of human origin better regulates the biology of human breast cancer cells in our humanized mouse model of breast cancer, which is more consistent with the clinical progress of human breast cancer.

The use of urinary proteomics in the assessment of suitability of mouse models for ageing.

PubMed

Nkuipou-Kenfack, Esther; Schanstra, Joost P; Bajwa, Seerat; Pejchinovski, Martin; Vinel, Claire; Dray, Cédric; Valet, Philippe; Bascands, Jean-Loup; Vlahou, Antonia; Koeck, Thomas; Borries, Melanie; Busch, Hauke; Bechtel-Walz, Wibke; Huber, Tobias B; Rudolph, Karl L; Pich, Andreas; Mischak, Harald; Zürbig, Petra

2017-01-01

Ageing is a complex process characterised by a systemic and progressive deterioration of biological functions. As ageing is associated with an increased prevalence of age-related chronic disorders, understanding its underlying molecular mechanisms can pave the way for therapeutic interventions and managing complications. Animal models such as mice are commonly used in ageing research as they have a shorter lifespan in comparison to humans and are also genetically close to humans. To assess the translatability of mouse ageing to human ageing, the urinary proteome in 89 wild-type (C57BL/6) mice aged between 8-96 weeks was investigated using capillary electrophoresis coupled to mass spectrometry (CE-MS). Using age as a continuous variable, 295 peptides significantly correlated with age in mice were identified. To investigate the relevance of using mouse models in human ageing studies, a comparison was performed with a previous correlation analysis using 1227 healthy subjects. In mice and humans, a decrease in urinary excretion of fibrillar collagens and an increase of uromodulin fragments was observed with advanced age. Of the 295 peptides correlating with age, 49 had a strong homology to the respective human age-related peptides. These ortholog peptides including several collagen (N = 44) and uromodulin (N = 5) fragments were used to generate an ageing classifier that was able to discriminate the age among both wild-type mice and healthy subjects. Additionally, the ageing classifier depicted that telomerase knock-out mice were older than their chronological age. Hence, with a focus on ortholog urinary peptides mouse ageing can be translated to human ageing.

Uncovering the mystery of opposite circadian rhythms between mouse and human leukocytes in humanized mice.

PubMed

Zhao, Yue; Liu, Min; Chan, Xue Ying; Tan, Sue Yee; Subramaniam, Sharrada; Fan, Yong; Loh, Eva; Chang, Kenneth Tou En; Tan, Thiam Chye; Chen, Qingfeng

2017-11-02

Many immune parameters show circadian rhythms during the 24-hour day in mammals. The most striking circadian oscillation is the number of circulating immune cells that display an opposite rhythm between humans and mice. The physiological roles and mechanisms of circadian variations in mouse leukocytes are well studied, whereas for humans they remain unclear because of the lack of a proper model. In this study, we found that consistent with their natural host species, mouse and human circulating leukocytes exhibited opposite circadian oscillations in humanized mice. This cyclic pattern of trafficking correlated well with the diurnal expression levels of C-X-C chemokine receptor 4, which were controlled by the intracellular hypoxia-inducible factor 1α/aryl hydrocarbon receptor nuclear translocator-like heterodimer. Furthermore, we also discovered that p38 mitogen-activated protein kinases/mitogen-activated 2 had opposite effects between mice and humans in generating intracellular reactive oxygen species, which subsequently regulated HIF-1α expression. In conclusion, we propose humanized mice as a robust model for human circadian studies and reveal insights on a novel molecular clock network in the human circadian rhythm. © 2017 by The American Society of Hematology.

Humanized mouse lines and their application for prediction of human drug metabolism and toxicological risk assessment

PubMed Central

Cheung, Connie; Gonzalez, Frank J

2008-01-01

Cytochrome P450s (P450s) are important enzymes involved in the metabolism of xenobiotics, particularly clinically used drugs, and are also responsible for metabolic activation of chemical carcinogens and toxins. Many xenobiotics can activate nuclear receptors that in turn induce the expression of genes encoding xenobiotic metabolizing enzymes and drug transporters. Marked species differences in the expression and regulation of cytochromes P450 and xenobiotic nuclear receptors exist. Thus obtaining reliable rodent models to accurately reflect human drug and carcinogen metabolism is severely limited. Humanized transgenic mice were developed in an effort to create more reliable in vivo systems to study and predict human responses to xenobiotics. Human P450s or human xenobiotic-activated nuclear receptors were introduced directly or replaced the corresponding mouse gene, thus creating “humanized” transgenic mice. Mice expressing human CYP1A1/CYP1A2, CYP2E1, CYP2D6, CYP3A4, CY3A7, PXR, PPARα were generated and characterized. These humanized mouse models offers a broad utility in the evaluation and prediction of toxicological risk that may aid in the development of safer drugs. PMID:18682571

Identifying mechanisms for superdiffusive dynamics in cell trajectories

NASA Astrophysics Data System (ADS)

Passucci, Giuseppe; Brasch, Megan; Henderson, James; Manning, M. Lisa

Self-propelled particle (SPP) models have been used to explore features of active matter such as motility-induced phase separation, jamming, and flocking, and are often used to model biological cells. However, many cells exhibit super-diffusive trajectories, where displacements scale faster than t 1 / 2 in all directions, and these are not captured by traditional SPP models. We extract cell trajectories from image stacks of mouse fibroblast cells moving on 2D substrates and find super-diffusive mean-squared displacements in all directions across varying densities. Two SPP model modifications have been proposed to capture super-diffusive dynamics: Levy walks and heterogeneous motility parameters. In mouse fibroblast cells displacement probability distributions collapse when time is rescaled by a power greater than 1/2, which is consistent with Levy walks. We show that a simple SPP model with heterogeneous rotational noise can also generate a similar collapse. Furthermore, a close examination of statistics extracted directly from cell trajectories is consistent with a heterogeneous mobility SPP model and inconsistent with a Levy walk model. Our work demonstrates that a simple set of analyses can distinguish between mechanisms for anomalous diffusion in active matter.

Animal models of pituitary neoplasia

PubMed Central

Lines, K.E.; Stevenson, M.; Thakker, R.V.

2016-01-01

Pituitary neoplasias can occur as part of a complex inherited disorder, or more commonly as sporadic (non-familial) disease. Studies of the molecular and genetic mechanisms causing such pituitary tumours have identified dysregulation of >35 genes, with many revealed by studies in mice, rats and zebrafish. Strategies used to generate these animal models have included gene knockout, gene knockin and transgenic over-expression, as well as chemical mutagenesis and drug induction. These animal models provide an important resource for investigation of tissue-specific tumourigenic mechanisms, and evaluations of novel therapies, illustrated by studies into multiple endocrine neoplasia type 1 (MEN1), a hereditary syndrome in which ∼30% of patients develop pituitary adenomas. This review describes animal models of pituitary neoplasia that have been generated, together with some recent advances in gene editing technologies, and an illustration of the use of the Men1 mouse as a pre clinical model for evaluating novel therapies. PMID:26320859

Generation and characterization of a human-mouse chimeric antibody against the extracellular domain of claudin-1 for cancer therapy using a mouse model.

PubMed

Hashimoto, Yosuke; Tada, Minoru; Iida, Manami; Nagase, Shotaro; Hata, Tomoyuki; Watari, Akihiro; Okada, Yoshiaki; Doi, Takefumi; Fukasawa, Masayoshi; Yagi, Kiyohito; Kondoh, Masuo

2016-08-12

Claudin-1 (CLDN-1), an integral transmembrane protein, is an attractive target for drug absorption, prevention of infection, and cancer therapy. Previously, we generated mouse anti-CLDN-1 monoclonal antibodies (mAbs) and found that they enhanced epidermal absorption of a drug and prevented hepatitis C virus infection in human hepatocytes. Here, we investigated anti-tumor activity of a human-mouse chimeric IgG1, xi-3A2, from one of the anti-CLDN-1 mAbs, clone 3A2. Xi-3A2 accumulated in the tumor tissues in mice bearing with human CLDN-1-expressing tumor cells. Xi-3A2 activated Fcγ receptor IIIa-expressing reporter cells in the presence of human CLDN-1-expressing cells, suggesting xi-3A2 has a potential to exhibit antibody-dependent cellular cytotoxicity against CLDN-1 expressing tumor cells. We also constructed a mutant xi-3A2 antibody with Gly, Ser, and Ile substituted with Ala, Asp, and Arg at positions 236, 239, and 332 of the Fc domain. This mutant antibody showed greater activation of Fcγ receptor IIIa and in vivo anti-tumor activity in mice bearing human CLDN-1-expressing tumors than xi-3A2 did. These findings indicate that the G236A/S239D/I332E mutant of xi-3A2 might be a promising lead for tumor therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

The Mouse Tumor Biology Database: A Comprehensive Resource for Mouse Models of Human Cancer.

PubMed

Krupke, Debra M; Begley, Dale A; Sundberg, John P; Richardson, Joel E; Neuhauser, Steven B; Bult, Carol J

2017-11-01

Research using laboratory mice has led to fundamental insights into the molecular genetic processes that govern cancer initiation, progression, and treatment response. Although thousands of scientific articles have been published about mouse models of human cancer, collating information and data for a specific model is hampered by the fact that many authors do not adhere to existing annotation standards when describing models. The interpretation of experimental results in mouse models can also be confounded when researchers do not factor in the effect of genetic background on tumor biology. The Mouse Tumor Biology (MTB) database is an expertly curated, comprehensive compendium of mouse models of human cancer. Through the enforcement of nomenclature and related annotation standards, MTB supports aggregation of data about a cancer model from diverse sources and assessment of how genetic background of a mouse strain influences the biological properties of a specific tumor type and model utility. Cancer Res; 77(21); e67-70. ©2017 AACR . ©2017 American Association for Cancer Research.

Differential Plasma Glycoproteome of p19 Skin Cancer Mouse Model Using the Corra Label-Free LC-MS Proteomics Platform.

PubMed

Letarte, Simon; Brusniak, Mi-Youn; Campbell, David; Eddes, James; Kemp, Christopher J; Lau, Hollis; Mueller, Lukas; Schmidt, Alexander; Shannon, Paul; Kelly-Spratt, Karen S; Vitek, Olga; Zhang, Hui; Aebersold, Ruedi; Watts, Julian D

2008-12-01

A proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented here, using a mouse model for skin cancer as an example. Blood plasma was collected from 10 control mice, and 10 mice having a mutation in the p19(ARF) gene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the modified tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists. We next assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localisation, trasport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application. These results thus show that an LC-MS-based workflow can be a useful tool for the generation of candidate proteins of interest as part of a disease biomarker discovery effort.

Differential Plasma Glycoproteome of p19ARF Skin Cancer Mouse Model Using the Corra Label-Free LC-MS Proteomics Platform

PubMed Central

Letarte, Simon; Brusniak, Mi-Youn; Campbell, David; Eddes, James; Kemp, Christopher J.; Lau, Hollis; Mueller, Lukas; Schmidt, Alexander; Shannon, Paul; Kelly-Spratt, Karen S.; Vitek, Olga; Zhang, Hui; Aebersold, Ruedi; Watts, Julian D.

2010-01-01

A proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented here, using a mouse model for skin cancer as an example. Blood plasma was collected from 10 control mice, and 10 mice having a mutation in the p19ARF gene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the modified tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists. We next assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localisation, trasport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application. These results thus show that an LC-MS-based workflow can be a useful tool for the generation of candidate proteins of interest as part of a disease biomarker discovery effort. PMID:20157627

Automated segmentation of mouse OCT volumes (ASiMOV): Validation & clinical study of a light damage model.

PubMed

Antony, Bhavna Josephine; Kim, Byung-Jin; Lang, Andrew; Carass, Aaron; Prince, Jerry L; Zack, Donald J

2017-01-01

The use of spectral-domain optical coherence tomography (SD-OCT) is becoming commonplace for the in vivo longitudinal study of murine models of ophthalmic disease. Longitudinal studies, however, generate large quantities of data, the manual analysis of which is very challenging due to the time-consuming nature of generating delineations. Thus, it is of importance that automated algorithms be developed to facilitate accurate and timely analysis of these large datasets. Furthermore, as the models target a variety of diseases, the associated structural changes can also be extremely disparate. For instance, in the light damage (LD) model, which is frequently used to study photoreceptor degeneration, the outer retina appears dramatically different from the normal retina. To address these concerns, we have developed a flexible graph-based algorithm for the automated segmentation of mouse OCT volumes (ASiMOV). This approach incorporates a machine-learning component that can be easily trained for different disease models. To validate ASiMOV, the automated results were compared to manual delineations obtained from three raters on healthy and BALB/cJ mice post LD. It was also used to study a longitudinal LD model, where five control and five LD mice were imaged at four timepoints post LD. The total retinal thickness and the outer retina (comprising the outer nuclear layer, and inner and outer segments of the photoreceptors) were unchanged the day after the LD, but subsequently thinned significantly (p < 0.01). The retinal nerve fiber-ganglion cell complex and the inner plexiform layers, however, remained unchanged for the duration of the study.

Automated segmentation of mouse OCT volumes (ASiMOV): Validation & clinical study of a light damage model

PubMed Central

Lang, Andrew; Carass, Aaron; Prince, Jerry L.; Zack, Donald J.

2017-01-01

The use of spectral-domain optical coherence tomography (SD-OCT) is becoming commonplace for the in vivo longitudinal study of murine models of ophthalmic disease. Longitudinal studies, however, generate large quantities of data, the manual analysis of which is very challenging due to the time-consuming nature of generating delineations. Thus, it is of importance that automated algorithms be developed to facilitate accurate and timely analysis of these large datasets. Furthermore, as the models target a variety of diseases, the associated structural changes can also be extremely disparate. For instance, in the light damage (LD) model, which is frequently used to study photoreceptor degeneration, the outer retina appears dramatically different from the normal retina. To address these concerns, we have developed a flexible graph-based algorithm for the automated segmentation of mouse OCT volumes (ASiMOV). This approach incorporates a machine-learning component that can be easily trained for different disease models. To validate ASiMOV, the automated results were compared to manual delineations obtained from three raters on healthy and BALB/cJ mice post LD. It was also used to study a longitudinal LD model, where five control and five LD mice were imaged at four timepoints post LD. The total retinal thickness and the outer retina (comprising the outer nuclear layer, and inner and outer segments of the photoreceptors) were unchanged the day after the LD, but subsequently thinned significantly (p < 0.01). The retinal nerve fiber-ganglion cell complex and the inner plexiform layers, however, remained unchanged for the duration of the study. PMID:28817571

Using dried blood spot sampling to improve data quality and reduce animal use in mouse pharmacokinetic studies.

PubMed

Wickremsinhe, Enaksha R; Perkins, Everett J

2015-03-01

Traditional pharmacokinetic analysis in nonclinical studies is based on the concentration of a test compound in plasma and requires approximately 100 to 200 μL blood collected per time point. However, the total blood volume of mice limits the number of samples that can be collected from an individual animal-often to a single collection per mouse-thus necessitating dosing multiple mice to generate a pharmacokinetic profile in a sparse-sampling design. Compared with traditional methods, dried blood spot (DBS) analysis requires smaller volumes of blood (15 to 20 μL), thus supporting serial blood sampling and the generation of a complete pharmacokinetic profile from a single mouse. Here we compare plasma-derived data with DBS-derived data, explain how to adopt DBS sampling to support discovery mouse studies, and describe how to generate pharmacokinetic and pharmacodynamic data from a single mouse. Executing novel study designs that use DBS enhances the ability to identify and streamline better drug candidates during drug discovery. Implementing DBS sampling can reduce the number of mice needed in a drug discovery program. In addition, the simplicity of DBS sampling and the smaller numbers of mice needed translate to decreased study costs. Overall, DBS sampling is consistent with 3Rs principles by achieving reductions in the number of animals used, decreased restraint-associated stress, improved data quality, direct comparison of interanimal variability, and the generation of multiple endpoints from a single study.

Development and proof-of-concept of three-dimensional lung histology volumes

NASA Astrophysics Data System (ADS)

Mathew, Lindsay; Alabousi, Mostafa; Wheatley, Andrew; Aladl, Usaf; Slipetz, Deborah; Hogg, James C.; Fenster, Aaron; Parraga, Grace

2012-03-01

Most medical imaging is inherently three-dimensional (3D) but for validation of pathological findings, histopathology is commonly used and typically histopathology images are acquired as twodimensional slices with quantitative analysis performed in a single dimension. Histopathology is invasive, labour-intensive, and the analysis cannot be performed in real time, yet it remains the gold standard for the pathological diagnosis and validation of clinical or radiological diagnoses of disease. A major goal worldwide is to improve medical imaging resolution, sensitivity and specificity to better guide therapy and biopsy and to one day delay or replace biopsy. A key limitation however is the lack of tools to directly compare 3D macroscopic imaging acquired in patients with histopathology findings, typically provided in a single dimension (1D) or in two dimensions (2D). To directly address this, we developed methods for 2D histology slice visualization/registration to generate 3D volumes and quantified tissue components in the 3D volume for direct comparison to volumetric micro-CT and clinical CT. We used the elastase-instilled mouse emphysema lung model to evaluate our methods with murine lungs sectioned (5 μm thickness/10 μm gap) and digitized with 2μm in-plane resolution. 3D volumes were generated for wildtype and elastase mouse lung sections after semi-automated registration of all tissue slices. The 1D mean linear intercept (Lm) for wildtype (WT) (47.1 μm +/- 9.8 μm) and elastase mouse lung (64.5 μm +/- 14.0 μm) was significantly different (p<.001). We also generated 3D measurements based on tissue and airspace morphometry from the 3D volumes and all of these were significantly different (p<.0001) when comparing elastase and WT mouse lung. The ratio of the airspace-to-lung volume for the entire lung volume was also significantly and strongly correlated with Lm.

Mucosal-associated invariant T cell-rich congenic mouse strain allows functional evaluation.

PubMed

Cui, Yue; Franciszkiewicz, Katarzyna; Mburu, Yvonne K; Mondot, Stanislas; Le Bourhis, Lionel; Premel, Virginie; Martin, Emmanuel; Kachaner, Alexandra; Duban, Livine; Ingersoll, Molly A; Rabot, Sylvie; Jaubert, Jean; De Villartay, Jean-Pierre; Soudais, Claire; Lantz, Olivier

2015-11-02

Mucosal-associated invariant T cells (MAITs) have potent antimicrobial activity and are abundant in humans (5%-10% in blood). Despite strong evolutionary conservation of the invariant TCR-α chain and restricting molecule MR1, this population is rare in laboratory mouse strains (≈0.1% in lymphoid organs), and lack of an appropriate mouse model has hampered the study of MAIT biology. Herein, we show that MAITs are 20 times more frequent in clean wild-derived inbred CAST/EiJ mice than in C57BL/6J mice. Increased MAIT frequency was linked to one CAST genetic trait that mapped to the TCR-α locus and led to higher usage of the distal Vα segments, including Vα19. We generated a MAIThi congenic strain that was then crossed to a transgenic Rorcgt-GFP reporter strain. Using this tool, we characterized polyclonal mouse MAITs as memory (CD44+) CD4-CD8lo/neg T cells with tissue-homing properties (CCR6+CCR7-). Similar to human MAITs, mouse MAITs expressed the cytokine receptors IL-7R, IL-18Rα, and IL-12Rβ and the transcription factors promyelocytic leukemia zinc finger (PLZF) and RAR-related orphan receptor γ (RORγt). Mouse MAITs produced Th1/2/17 cytokines upon TCR stimulation and recognized a bacterial compound in an MR1-dependent manner. During experimental urinary tract infection, MAITs migrated to the bladder and decreased bacterial load. Our study demonstrates that the MAIThi congenic strain allows phenotypic and functional characterization of naturally occurring mouse MAITs in health and disease.

Mucosal-associated invariant T cell–rich congenic mouse strain allows functional evaluation

PubMed Central

Cui, Yue; Franciszkiewicz, Katarzyna; Mburu, Yvonne K.; Mondot, Stanislas; Le Bourhis, Lionel; Premel, Virginie; Martin, Emmanuel; Kachaner, Alexandra; Duban, Livine; Ingersoll, Molly A.; Rabot, Sylvie; Jaubert, Jean; De Villartay, Jean-Pierre; Soudais, Claire; Lantz, Olivier

2015-01-01

Mucosal-associated invariant T cells (MAITs) have potent antimicrobial activity and are abundant in humans (5%–10% in blood). Despite strong evolutionary conservation of the invariant TCR-α chain and restricting molecule MR1, this population is rare in laboratory mouse strains (≈0.1% in lymphoid organs), and lack of an appropriate mouse model has hampered the study of MAIT biology. Herein, we show that MAITs are 20 times more frequent in clean wild-derived inbred CAST/EiJ mice than in C57BL/6J mice. Increased MAIT frequency was linked to one CAST genetic trait that mapped to the TCR-α locus and led to higher usage of the distal Vα segments, including Vα19. We generated a MAIThi congenic strain that was then crossed to a transgenic Rorcgt-GFP reporter strain. Using this tool, we characterized polyclonal mouse MAITs as memory (CD44+) CD4–CD8lo/neg T cells with tissue-homing properties (CCR6+CCR7–). Similar to human MAITs, mouse MAITs expressed the cytokine receptors IL-7R, IL-18Rα, and IL-12Rβ and the transcription factors promyelocytic leukemia zinc finger (PLZF) and RAR-related orphan receptor γ (RORγt). Mouse MAITs produced Th1/2/17 cytokines upon TCR stimulation and recognized a bacterial compound in an MR1-dependent manner. During experimental urinary tract infection, MAITs migrated to the bladder and decreased bacterial load. Our study demonstrates that the MAIThi congenic strain allows phenotypic and functional characterization of naturally occurring mouse MAITs in health and disease. PMID:26524590

SU-F-T-668: Irradiating Mouse Brain with a Clinical Linear Accelerator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perez-Torres, C

Purpose: To design and construct a “mouse jig” device that would allow for irradiation of the mouse brain with a clinical Varian 6 MeV Linear Accelerator. This device must serve as a head immobilizer, gaseous anesthesia delivery, and radiation bolus concurrently. Methods: The mouse jig was machined out of nylon given that it is inexpensive, easy to machine, and has similar electron density to water. A cylindrical opening with diameter of 16 mm and 40 mm depth was drilled into a nylon block sized 56×56×50 mm (width, length, depth). Additional slots were included in the block for ear bars andmore » a tooth bar to serve as a three-point immobilization device as well as for anesthesia delivery and scavenging. For ease of access when loading the mouse into the holder, there is a removable piece at the top of the block that is 15 mm in depth. This serves a dual purpose, as with the proper extra shielding, the mouse jig could be used with lower linear energy transfer photons with this piece removed. A baseplate was then constructed with five square slots where the mouse jig can securely be inserted plus additional slots that would allow the baseplate to be mounted on a standard lock bar in the treatment couch. This maximizes the reproducibility of placement between imaging and treatment and between treatment sessions. Results: CT imaging and radiation treatment planning was performed that showed acceptable coverage and uniformity of radiation dose in the mouse brain while sparing the throat and eyes. Conclusion: We have designed and manufactured a device that fulfills our criteria allowing us to selectively irradiate the mouse brain with a clinical linear accelerator. This setup will be used for generating mouse models of radiation-induced brain injury.« less

Generation of Novel Traj18-Deficient Mice Lacking Vα14 Natural Killer T Cells with an Undisturbed T Cell Receptor α-Chain Repertoire.

PubMed

Dashtsoodol, Nyambayar; Shigeura, Tomokuni; Ozawa, Ritsuko; Harada, Michishige; Kojo, Satoshi; Watanabe, Takashi; Koseki, Haruhiko; Nakayama, Manabu; Ohara, Osamu; Taniguchi, Masaru

2016-01-01

Invariant Vα14 natural killer T (NKT) cells, characterized by the expression of a single invariant T cell receptor (TCR) α chain encoded by rearranged Trav11 (Vα14)-Traj18 (Jα18) gene segments in mice, and TRAV10 (Vα24)-TRAJ18 (Jα18) in humans, mediate adjuvant effects to activate various effector cell types in both innate and adaptive immune systems that facilitates the potent antitumor effects. It was recently reported that the Jα18-deficient mouse described by our group in 1997 harbors perturbed TCRα repertoire, which raised concerns regarding the validity of some of the experimental conclusions that have been made using this mouse line. To resolve this concern, we generated a novel Traj18-deficient mouse line by specifically targeting the Traj18 gene segment using Cre-Lox approach. Here we showed the newly generated Traj18-deficient mouse has, apart from the absence of Traj18, an undisturbed TCRα chain repertoire by using next generation sequencing and by detecting normal generation of Vα19Jα33 expressing mucosal associated invariant T cells, whose development was abrogated in the originally described Jα18-KO mice. We also demonstrated here the definitive requirement for NKT cells in the protection against tumors and their potent adjuvant effects on antigen-specific CD8 T cells.

Deleterious effects of neuronal accumulation of glycogen in flies and mice.

PubMed

Duran, Jordi; Tevy, María Florencia; Garcia-Rocha, Mar; Calbó, Joaquim; Milán, Marco; Guinovart, Joan J

2012-08-01

Under physiological conditions, most neurons keep glycogen synthase (GS) in an inactive form and do not show detectable levels of glycogen. Nevertheless, aberrant glycogen accumulation in neurons is a hallmark of patients suffering from Lafora disease or other polyglucosan disorders. Although these diseases are associated with mutations in genes involved in glycogen metabolism, the role of glycogen accumulation remains elusive. Here, we generated mouse and fly models expressing an active form of GS to force neuronal accumulation of glycogen. We present evidence that the progressive accumulation of glycogen in mouse and Drosophila neurons leads to neuronal loss, locomotion defects and reduced lifespan. Our results highlight glycogen accumulation in neurons as a direct cause of neurodegeneration. Copyright © 2012 EMBO Molecular Medicine.

Deleterious effects of neuronal accumulation of glycogen in flies and mice

PubMed Central

Duran, Jordi; Tevy, María Florencia; Garcia-Rocha, Mar; Calbó, Joaquim; Milán, Marco; Guinovart, Joan J

2012-01-01

Under physiological conditions, most neurons keep glycogen synthase (GS) in an inactive form and do not show detectable levels of glycogen. Nevertheless, aberrant glycogen accumulation in neurons is a hallmark of patients suffering from Lafora disease or other polyglucosan disorders. Although these diseases are associated with mutations in genes involved in glycogen metabolism, the role of glycogen accumulation remains elusive. Here, we generated mouse and fly models expressing an active form of GS to force neuronal accumulation of glycogen. We present evidence that the progressive accumulation of glycogen in mouse and Drosophila neurons leads to neuronal loss, locomotion defects and reduced lifespan. Our results highlight glycogen accumulation in neurons as a direct cause of neurodegeneration. PMID:22549942

Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning

PubMed Central

Morgani, Sophie M; Metzger, Jakob J; Nichols, Jennifer

2018-01-01

During gastrulation epiblast cells exit pluripotency as they specify and spatially arrange the three germ layers of the embryo. Similarly, human pluripotent stem cells (PSCs) undergo spatially organized fate specification on micropatterned surfaces. Since in vivo validation is not possible for the human, we developed a mouse PSC micropattern system and, with direct comparisons to mouse embryos, reveal the robust specification of distinct regional identities. BMP, WNT, ACTIVIN and FGF directed mouse epiblast-like cells to undergo an epithelial-to-mesenchymal transition and radially pattern posterior mesoderm fates. Conversely, WNT, ACTIVIN and FGF patterned anterior identities, including definitive endoderm. By contrast, epiblast stem cells, a developmentally advanced state, only specified anterior identities, but without patterning. The mouse micropattern system offers a robust scalable method to generate regionalized cell types present in vivo, resolve how signals promote distinct identities and generate patterns, and compare mechanisms operating in vivo and in vitro and across species. PMID:29412136

Generation and characterization of Lhx9 – GFPCreERT2 knock-in mouse line

PubMed Central

Xie, Xiaoling; Deng, Min; Gan, Lin

2014-01-01

Summary LHX9 is a LIM-homeodomain transcription factor essential for the development of gonads, spinal cord interneurons, and thalamic neurons to name a few. We recently reported the expression of LHX9 in retinal amacrine cells during development. In this study, we generated an Lhx9 - GFPCreERT2 (GCE) knock-in mouse line by knocking-in a GCE cassette at the Lhx9 locus, thus inactivating endogenous Lhx9. Lhx9GCE/+ mice were viable, fertile, and displayed no overt phenotypical characteristics. Lhx9GCE/GCE mice were all phenotypically female, smaller in size, viable, but infertile. The specificity and efficacy of the Lhx9-GCE mouse line was verified by crossing it to a Rosa26 - tdTomato reporter mouse line, which reveals the Cre recombinase activities in retinal amacrine cells, developing limbs, testis, hippocampal neurons, thalamic neurons, and cerebellar neurons. Taken together, the Lhx9-GCE mouse line could serve as a beneficial tool for lineage tracing and gene manipulation experiments. PMID:25112520

Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells.

PubMed

Brown, Rachel C; Morris, Andrew P; O'Neil, Roger G

2007-01-26

Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions.

TIGHT JUNCTION PROTEIN EXPRESSION AND BARRIER PROPERTIES OF IMMORTALIZED MOUSE BRAIN MICROVESSEL ENDOTHELIAL CELLS

PubMed Central

Brown, Rachel C.; Morris, Andrew P.; O’Neil, Roger G.

2007-01-01

Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions. PMID:17169347

Interspecies chimera between primate embryonic stem cells and mouse embryos: monkey ESCs engraft into mouse embryos, but not post-implantation fetuses.

PubMed

Simerly, Calvin; McFarland, Dave; Castro, Carlos; Lin, Chih-Cheng; Redinger, Carrie; Jacoby, Ethan; Mich-Basso, Jocelyn; Orwig, Kyle; Mills, Parker; Ahrens, Eric; Navara, Chris; Schatten, Gerald

2011-07-01

Unequivocal evidence for pluripotency in which embryonic stem cells contribute to chimeric offspring has yet to be demonstrated in human or nonhuman primates (NHPs). Here, rhesus and baboons ESCs were investigated in interspecific mouse chimera generated by aggregation or blastocyst injection. Aggregation chimera produced mouse blastocysts with GFP-nhpESCs at the inner cell mass (ICM), and embryo transfers (ETs) generated dimly-fluorescencing abnormal fetuses. Direct injection of GFP-nhpESCs into blastocysts produced normal non-GFP-fluorescencing fetuses. Injected chimera showed >70% loss of GFP-nhpESCs after 21 h culture. Outgrowths of all chimeric blastocysts established distinct but separate mouse- and NHP-ESC colonies. Extensive endogenous autofluorescence compromised anti-GFP detection and PCR analysis did not detect nhpESCs in fetuses. NhpESCs localize to the ICM in chimera and generate pregnancies. Because primate ESCs do not engraft post-implantation, and also because endogenous autofluorescence results in misleading positive signals, interspecific chimera assays for pluripotency with primate stem cells is unreliable with the currently available ESCs. Testing primate ESCs reprogrammed into even more naïve states in these inter-specific chimera assays will be an important future endeavor. Copyright © 2011 Elsevier B.V. All rights reserved.

Strategies and Considerations for Distributing and Recovering Mouse Lines

PubMed Central

Du, Yubin; Xie, Wen; Liu, Chengyu

2012-01-01

As more and more genetically modified mouse lines are being generated, it becomes increasingly common to share animal models among different research institutions. Live mice are routinely transferred between animal facilities. Due to various issues concerning animal welfare, intellectual property rights, colony health status and biohazard, significant paperwork and coordination are required before any animal travel can take place. Shipping fresh or frozen preimplantation embryos, gametes, or reproductive organs can bypass some of the issues associated with live animal transfer, but it requires the receiving facilities to be able to perform delicate and sometimes intricate procedures such as embryo transfer, in vitro fertilization (IVF), or ovary transplantation. Here, we summarize the general requirements for live animal transport and review some of the assisted reproductive technologies (ART) that can be applied to shipping and reviving mouse lines. Intended users of these methods should consult their institution’s responsible official to find out whether each specific method is legal or appropriate in their own animal facilities. PMID:20691859

Molecular determinants of nucleosome retention at CpG-rich sequences in mouse spermatozoa.

PubMed

Erkek, Serap; Hisano, Mizue; Liang, Ching-Yeu; Gill, Mark; Murr, Rabih; Dieker, Jürgen; Schübeler, Dirk; van der Vlag, Johan; Stadler, Michael B; Peters, Antoine H F M

2013-07-01

In mammalian spermatozoa, most but not all of the genome is densely packaged by protamines. Here we reveal the molecular logic underlying the retention of nucleosomes in mouse spermatozoa, which contain only 1% residual histones. We observe high enrichment throughout the genome of nucleosomes at CpG-rich sequences that lack DNA methylation. Residual nucleosomes are largely composed of the histone H3.3 variant and are trimethylated at Lys4 of histone H3 (H3K4me3). Canonical H3.1 and H3.2 histones are also enriched at CpG-rich promoters marked by Polycomb-mediated H3K27me3, a modification predictive of gene repression in preimplantation embryos. Histone variant-specific nucleosome retention in sperm is strongly associated with nucleosome turnover in round spermatids. Our data show evolutionary conservation of the basic principles of nucleosome retention in mouse and human sperm, supporting a model of epigenetic inheritance by nucleosomes between generations.

Prevention of the degeneration of human dopaminergic neurons in an astrocyte co-culture system allowing endogenous drug metabolism

PubMed Central

Efremova, Liudmila; Schildknecht, Stefan; Adam, Martina; Pape, Regina; Gutbier, Simon; Hanf, Benjamin; Bürkle, Alexander; Leist, Marcel

2015-01-01

Background and Purpose Few neuropharmacological model systems use human neurons. Moreover, available test systems rarely reflect functional roles of co-cultured glial cells. There is no human in vitro counterpart of the widely used 1-methyl-4-phenyl-tetrahydropyridine (MPTP) mouse model of Parkinson's disease Experimental Approach We generated such a model by growing an intricate network of human dopaminergic neurons on a dense layer of astrocytes. In these co-cultures, MPTP was metabolized to 1-methyl-4-phenyl-pyridinium (MPP+) by the glial cells, and the toxic metabolite was taken up through the dopamine transporter into neurons. Cell viability was measured biochemically and by quantitative neurite imaging, siRNA techniques were also used. Key Results We initially characterized the activation of PARP. As in mouse models, MPTP exposure induced (poly-ADP-ribose) synthesis and neurodegeneration was blocked by PARP inhibitors. Several different putative neuroprotectants were then compared in mono-cultures and co-cultures. Rho kinase inhibitors worked in both models; CEP1347, ascorbic acid or a caspase inhibitor protected mono-cultures from MPP+ toxicity, but did not protect co-cultures, when used alone or in combination. Application of GSSG prevented degeneration in co-cultures, but not in mono-cultures. The surprisingly different pharmacological profiles of the models suggest that the presence of glial cells, and the in situ generation of the toxic metabolite MPP+ within the layered cultures played an important role in neuroprotection. Conclusions and Implications Our new model system is a closer model of human brain tissue than conventional cultures. Its use for screening of candidate neuroprotectants may increase the predictiveness of a test battery. PMID:25989025

In Vivo Pharmacokinetics/Pharmacodynamics of Cefquinome in an Experimental Mouse Model of Staphylococcus Aureus Mastitis following Intramammary Infusion

PubMed Central

Yu, Yang; Zhou, Yu-Feng; Chen, Mei-Ren; Li, Xiao; Qiao, Gui-Lin; Sun, Jian; Liao, Xiao-Ping; Liu, Ya-Hong

2016-01-01

Staphylococcus aureus remains the major cause of morbidity of bovine mastitis worldwide leading to massive economic losses. Cefquinome is a fourth generation cephalosporin, which preserves susceptibility and antibacterial activity against S. aureus. This work aims to study the pharmacokinetic (PK) and pharmacodynamic (PD) modeling following intramammary administration of cefquinome against S. aureus mastitis. The mouse model of S. aureus mastitis was developed for the PK/PD experiments. The plasma PK characteristics after intramammary injection of cefquinome at various single doses of 25, 50, 100, 200, 400 μg per gland (both fourth pairs of glands: L4 and R4) were calculated using one-compartment and first-order absorption model. PD study was investigated based on twenty-one intermittent dosing regimens, of which total daily dose ranged from 25 to 4800 μg per mouse and dosage intervals included 8, 12 or 24 h. The sigmoid Emax model of inhibitory effect was employed for PK/PD modeling. The results of PK/PD integration of cefquinome against S. aureus suggested that the percentage of duration that drug concentration exceeded the minimal inhibitory concentration (%T>MIC) and the ratio of area under time-concentration curve over MIC (AUC/MIC) are important indexes to evaluate the antibacterial activity. The PK/PD parameters of %T>MIC and AUC0-24/MIC were 35.98% and 137.43 h to obtain a 1.8 logCFU/gland reduction of bacterial colony counts in vivo, against S. aureus strains with cefquinome MIC of 0.5μg/ml. PMID:27218674

In Vivo Pharmacokinetics/Pharmacodynamics of Cefquinome in an Experimental Mouse Model of Staphylococcus Aureus Mastitis following Intramammary Infusion.

PubMed

Yu, Yang; Zhou, Yu-Feng; Chen, Mei-Ren; Li, Xiao; Qiao, Gui-Lin; Sun, Jian; Liao, Xiao-Ping; Liu, Ya-Hong

2016-01-01

Staphylococcus aureus remains the major cause of morbidity of bovine mastitis worldwide leading to massive economic losses. Cefquinome is a fourth generation cephalosporin, which preserves susceptibility and antibacterial activity against S. aureus. This work aims to study the pharmacokinetic (PK) and pharmacodynamic (PD) modeling following intramammary administration of cefquinome against S. aureus mastitis. The mouse model of S. aureus mastitis was developed for the PK/PD experiments. The plasma PK characteristics after intramammary injection of cefquinome at various single doses of 25, 50, 100, 200, 400 μg per gland (both fourth pairs of glands: L4 and R4) were calculated using one-compartment and first-order absorption model. PD study was investigated based on twenty-one intermittent dosing regimens, of which total daily dose ranged from 25 to 4800 μg per mouse and dosage intervals included 8, 12 or 24 h. The sigmoid Emax model of inhibitory effect was employed for PK/PD modeling. The results of PK/PD integration of cefquinome against S. aureus suggested that the percentage of duration that drug concentration exceeded the minimal inhibitory concentration (%T>MIC) and the ratio of area under time-concentration curve over MIC (AUC/MIC) are important indexes to evaluate the antibacterial activity. The PK/PD parameters of %T>MIC and AUC0-24/MIC were 35.98% and 137.43 h to obtain a 1.8 logCFU/gland reduction of bacterial colony counts in vivo, against S. aureus strains with cefquinome MIC of 0.5μg/ml.

Differentiation and Characterization of Myeloid Cells

PubMed Central

Gupta, Dipti; Shah, Hetavi Parag; Malu, Krishnakumar; Berliner, Nancy; Gaines, Peter

2015-01-01

Recent molecular studies of myeloid differentiation have utilized several in vitro models of myelopoiesis, generated from either ex vivo differentiated bone marrow progenitors or induced immortalized myeloid cell lines. Ex vivo differentiation begins with an enriched population of bone marrow-derived hematopoietic stem cells generated by lineage depletion and/or positive selection for CD34+ antigen (human) or Sca-1+ (mouse) cells, which are then expanded and subsequently induced in vitro in a process that recapitulates normal myeloid development. Myeloid cell lines include two human leukemic cell lines, NB-4 and HL-60, which have been demonstrated to undergo retinoic acid–induced myeloid development, however, both cell lines exhibit defects in the upregulation of late-expressed neutrophil-specific genes. Multiple murine factor–dependent cell models of myelopoiesis are also available that express the full range of neutrophil maturation markers, including: 32Dcl3 cells, which undergo G-CSF-induced myeloid maturation, EML/EPRO cells, which develop into mature neutrophils in response to cytokines and retinoic acid, and ER-Hoxb8 cells, which undergo myeloid maturation upon removal of estradial in the maintenance medium. In this unit, the induction of myeloid maturation in each of these model systems is described, including their differentiation to either neutrophils or macrophages, if applicable. Commonly used techniques to test for myeloid characteristics of developing cells are also described, including flow cytometry and real time RT-PCR. Together, these assays provide a solid foundation for in vitro investigations of myeloid development with either human or mouse models. PMID:24510620

Genetically engineered mouse models of human B-cell precursor leukemias.

PubMed

Hauer, Julia; Borkhardt, Arndt; Sánchez-García, Isidro; Cobaleda, César

2014-01-01

B-cell precursor acute lymphoblastic leukemias (pB-ALLs) are the most frequent type of malignancies of the childhood, and also affect an important proportion of adult patients. In spite of their apparent homogeneity, pB-ALL comprises a group of diseases very different both clinically and pathologically, and with very diverse outcomes as a consequence of their biology, and underlying molecular alterations. Their understanding (as a prerequisite for their cure) will require a sustained multidisciplinary effort from professionals coming from many different fields. Among all the available tools for pB-ALL research, the use of animal models stands, as of today, as the most powerful approach, not only for the understanding of the origin and evolution of the disease, but also for the development of new therapies. In this review we go over the most relevant (historically, technically or biologically) genetically engineered mouse models (GEMMs) of human pB-ALLs that have been generated over the last 20 years. Our final aim is to outline the most relevant guidelines that should be followed to generate an "ideal" animal model that could become a standard for the study of human pB-ALL leukemia, and which could be shared among research groups and drug development companies in order to unify criteria for studies like drug testing, analysis of the influence of environmental risk factors, or studying the role of both low-penetrance mutations and cancer susceptibility alterations.

Comparative Evaluation of Two Vaccine Candidates against Experimental Leishmaniasis Due to Leishmania major Infection in Four Inbred Mouse Strains▿

PubMed Central

Benhnini, Fouad; Chenik, Mehdi; Laouini, Dhafer; Louzir, Hechmi; Cazenave, Pierre André; Dellagi, Koussay

2009-01-01

Experimental leishmaniasis in BALB/c and C57BL/6 mice are the most investigated murine models that were used for the preclinical evaluation of Leishmania vaccine candidates. We have previously described two new inbred mouse strains named PWK and MAI issued from feral founders that also support the development of experimental leishmaniasis due to L. major. In this study, we sought to determine whether different mouse inbred strains generate concordant or discordant results when used to evaluate the potential of Leishmania proteins to protect against experimental leishmaniasis. To this end, two Leishmania proteins, namely, LACK (for Leishmania homolog of receptor for activated C kinase) and LmPDI (for L. major protein disulfide isomerase) were compared for their capacity to protect against experimental leishmaniasis in PWK, MAI, BALB/c, and C57BL/6 inbred mouse strains. Our data show that the capacity of Leishmania proteins to confer protection depends on the mouse strain used, stressing the important role played by the genetic background in shaping the immune response against the pathogen. These results may have important implications for the preclinical evaluation of candidate Leishmania vaccines: rather than using a single mouse strain, a panel of different inbred strains of various genetic backgrounds should be tested in parallel. The antigen that confers protection in the larger range of inbred strains may have better chances to be also protective in outbred human populations and should be selected for clinical trials. PMID:19726616

Comparative evaluation of two vaccine candidates against experimental leishmaniasis due to Leishmania major infection in four inbred mouse strains.

PubMed

Benhnini, Fouad; Chenik, Mehdi; Laouini, Dhafer; Louzir, Hechmi; Cazenave, Pierre André; Dellagi, Koussay

2009-11-01

Experimental leishmaniasis in BALB/c and C57BL/6 mice are the most investigated murine models that were used for the preclinical evaluation of Leishmania vaccine candidates. We have previously described two new inbred mouse strains named PWK and MAI issued from feral founders that also support the development of experimental leishmaniasis due to L. major. In this study, we sought to determine whether different mouse inbred strains generate concordant or discordant results when used to evaluate the potential of Leishmania proteins to protect against experimental leishmaniasis. To this end, two Leishmania proteins, namely, LACK (for Leishmania homolog of receptor for activated C kinase) and LmPDI (for L. major protein disulfide isomerase) were compared for their capacity to protect against experimental leishmaniasis in PWK, MAI, BALB/c, and C57BL/6 inbred mouse strains. Our data show that the capacity of Leishmania proteins to confer protection depends on the mouse strain used, stressing the important role played by the genetic background in shaping the immune response against the pathogen. These results may have important implications for the preclinical evaluation of candidate Leishmania vaccines: rather than using a single mouse strain, a panel of different inbred strains of various genetic backgrounds should be tested in parallel. The antigen that confers protection in the larger range of inbred strains may have better chances to be also protective in outbred human populations and should be selected for clinical trials.

Multi-tissue DNA methylation age predictor in mouse.

PubMed

Stubbs, Thomas M; Bonder, Marc Jan; Stark, Anne-Katrien; Krueger, Felix; von Meyenn, Ferdinand; Stegle, Oliver; Reik, Wolf

2017-04-11

DNA methylation changes at a discrete set of sites in the human genome are predictive of chronological and biological age. However, it is not known whether these changes are causative or a consequence of an underlying ageing process. It has also not been shown whether this epigenetic clock is unique to humans or conserved in the more experimentally tractable mouse. We have generated a comprehensive set of genome-scale base-resolution methylation maps from multiple mouse tissues spanning a wide range of ages. Many CpG sites show significant tissue-independent correlations with age which allowed us to develop a multi-tissue predictor of age in the mouse. Our model, which estimates age based on DNA methylation at 329 unique CpG sites, has a median absolute error of 3.33 weeks and has similar properties to the recently described human epigenetic clock. Using publicly available datasets, we find that the mouse clock is accurate enough to measure effects on biological age, including in the context of interventions. While females and males show no significant differences in predicted DNA methylation age, ovariectomy results in significant age acceleration in females. Furthermore, we identify significant differences in age-acceleration dependent on the lipid content of the diet. Here we identify and characterise an epigenetic predictor of age in mice, the mouse epigenetic clock. This clock will be instrumental for understanding the biology of ageing and will allow modulation of its ticking rate and resetting the clock in vivo to study the impact on biological age.

A critical assessment of Mus musculus gene function prediction using integrated genomic evidence

PubMed Central

Peña-Castillo, Lourdes; Tasan, Murat; Myers, Chad L; Lee, Hyunju; Joshi, Trupti; Zhang, Chao; Guan, Yuanfang; Leone, Michele; Pagnani, Andrea; Kim, Wan Kyu; Krumpelman, Chase; Tian, Weidong; Obozinski, Guillaume; Qi, Yanjun; Mostafavi, Sara; Lin, Guan Ning; Berriz, Gabriel F; Gibbons, Francis D; Lanckriet, Gert; Qiu, Jian; Grant, Charles; Barutcuoglu, Zafer; Hill, David P; Warde-Farley, David; Grouios, Chris; Ray, Debajyoti; Blake, Judith A; Deng, Minghua; Jordan, Michael I; Noble, William S; Morris, Quaid; Klein-Seetharaman, Judith; Bar-Joseph, Ziv; Chen, Ting; Sun, Fengzhu; Troyanskaya, Olga G; Marcotte, Edward M; Xu, Dong; Hughes, Timothy R; Roth, Frederick P

2008-01-01

Background: Several years after sequencing the human genome and the mouse genome, much remains to be discovered about the functions of most human and mouse genes. Computational prediction of gene function promises to help focus limited experimental resources on the most likely hypotheses. Several algorithms using diverse genomic data have been applied to this task in model organisms; however, the performance of such approaches in mammals has not yet been evaluated. Results: In this study, a standardized collection of mouse functional genomic data was assembled; nine bioinformatics teams used this data set to independently train classifiers and generate predictions of function, as defined by Gene Ontology (GO) terms, for 21,603 mouse genes; and the best performing submissions were combined in a single set of predictions. We identified strengths and weaknesses of current functional genomic data sets and compared the performance of function prediction algorithms. This analysis inferred functions for 76% of mouse genes, including 5,000 currently uncharacterized genes. At a recall rate of 20%, a unified set of predictions averaged 41% precision, with 26% of GO terms achieving a precision better than 90%. Conclusion: We performed a systematic evaluation of diverse, independently developed computational approaches for predicting gene function from heterogeneous data sources in mammals. The results show that currently available data for mammals allows predictions with both breadth and accuracy. Importantly, many highly novel predictions emerge for the 38% of mouse genes that remain uncharacterized. PMID:18613946

Mesenchymal stem cells generate a CD4+CD25+Foxp3+ regulatory T cell population during the differentiation process of Th1 and Th17 cells.

PubMed

Luz-Crawford, Patricia; Kurte, Monica; Bravo-Alegría, Javiera; Contreras, Rafael; Nova-Lamperti, Estefania; Tejedor, Gautier; Noël, Danièle; Jorgensen, Christian; Figueroa, Fernando; Djouad, Farida; Carrión, Flavio

2013-06-04

Mesenchymal stem cells (MSCs) are adult, multipotent, stem cells with immunomodulatory properties. The mechanisms involved in the capacity of MSCs to inhibit the proliferation of proinflammatory T lymphocytes, which appear responsible for causing autoimmune disease, have yet to be fully elucidated. One of the underlying mechanisms studied recently is the ability of MSCs to generate T regulatory (Treg) cells in vitro and in vivo from activated peripheral blood mononuclear cells (PBMC), T-CD4+ and also T-CD8(+) cells. In the present work we investigated the capacity of MSCs to generate Treg cells using T-CD4(+) cells induced to differentiate toward the proinflammatory Th1 and Th17 lineages. MSCs were obtained from mouse bone marrow and characterized according to their surface antigen expression and their multilineage differentiation potential. CD4(+) T cells isolated from mouse spleens were induced to differentiate into Th1 or Th17 cells and co-cultured with MSCs added at day 0, 2 or 4 of the differentiation processes. After six days, CD25, Foxp3, IL-17 and IFN-γ expression was assessed by flow cytometry and helios and neuropilin 1 mRNA levels were assessed by RT-qPCR. For the functional assays, the 'conditioned' subpopulation generated in the presence of MSCs was cultured with concanavalin A-activated CD4(+) T cells labeled with carboxyfluorescein succinimidyl ester. Finally, we used the encephalomyelitis autoimmune diseases (EAE) mouse model, in which mice were injected with MSCs at day 18 and 30 after immunization. At day 50, the mice were euthanized and draining lymph nodes were extracted for Th1, Th17 and Treg detection by flow cytometry. MSCs were able to suppress the proliferation, activation and differentiation of CD4(+) T cells induced to differentiate into Th1 and Th17 cells. This substantial suppressive effect was associated with an increase of the percentage of functional induced CD4(+)CD25(+)Foxp3(+) regulatory T cells and IL-10 secretion. However, using mature Th1 or Th17 cells our results demonstrated that while MSCs suppress the proliferation and phenotype of mature Th1 and Th17 cells they did not generate Treg cells. Finally, we showed that the beneficial effect observed following MSC injection in an EAE mouse model was associated with the suppression of Th17 cells and an increase in the percentage of CD4(+)CD25(+)Foxp3(+) T lymphocytes when administrated at early stages of the disease. This study demonstrated that MSCs contribute to the generation of an immunosuppressive environment via the inhibition of proinflammatory T cells and the induction of T cells with a regulatory phenotype. Together, these results might have important clinical implications for inflammatory and autoimmune diseases.

A versatile strategy for gene trapping and trap conversion in emerging model organisms.

PubMed

Kontarakis, Zacharias; Pavlopoulos, Anastasios; Kiupakis, Alexandros; Konstantinides, Nikolaos; Douris, Vassilis; Averof, Michalis

2011-06-01

Genetic model organisms such as Drosophila, C. elegans and the mouse provide formidable tools for studying mechanisms of development, physiology and behaviour. Established models alone, however, allow us to survey only a tiny fraction of the morphological and functional diversity present in the animal kingdom. Here, we present iTRAC, a versatile gene-trapping approach that combines the implementation of unbiased genetic screens with the generation of sophisticated genetic tools both in established and emerging model organisms. The approach utilises an exon-trapping transposon vector that carries an integrase docking site, allowing the targeted integration of new constructs into trapped loci. We provide proof of principle for iTRAC in the emerging model crustacean Parhyale hawaiensis: we generate traps that allow specific developmental and physiological processes to be visualised in unparalleled detail, we show that trapped genes can be easily cloned from an unsequenced genome, and we demonstrate targeting of new constructs into a trapped locus. Using this approach, gene traps can serve as platforms for generating diverse reporters, drivers for tissue-specific expression, gene knockdown and other genetic tools not yet imagined.

A knock-in mouse line conditionally expressing the tumor suppressor WTX/AMER1.

PubMed

Boutet, Agnès; Comai, Glenda; Charlet, Aurélie; Jian Motamedi, Fariba; Dhib, Haroun; Bandiera, Roberto; Schedl, Andreas

2017-11-01

WTX/AMER1 is an important developmental regulator, mutations in which have been identified in a proportion of patients suffering from the renal neoplasm Wilms' tumor and in the bone malformation syndrome Osteopathia Striata with Cranial Sclerosis (OSCS). Its cellular functions appear complex and the protein can be found at the membrane, within the cytoplasm and the nucleus. To understand its developmental and cellular function an allelic series for Wtx in the mouse is crucial. Whereas mice carrying a conditional knock out allele for Wtx have been previously reported, a gain-of-function mouse model that would allow studying the molecular, cellular and developmental role of Wtx is still missing. Here we describe the generation of a novel mouse strain that permits the conditional activation of WTX expression. Wtx fused to GFP was introduced downstream a stop cassette flanked by loxP sites into the Rosa26 locus by gene targeting. Ectopic WTX expression is reported after crosses with several Cre transgenic mice in different embryonic tissues. Further, functionality of the fusion protein was demonstrated in the context of a Wtx null allele. © 2017 Wiley Periodicals, Inc.

Proteomic analysis of mouse islets after multiple low-dose streptozotocin injection.

PubMed

Xie, Xiaolei; Li, Shuai; Liu, Siyu; Lu, Yan; Shen, Pingping; Ji, Jianguo

2008-02-01

The islets of Langerhans are scattered throughout the pancreas and play a major role in the control of metabolic fuel homeostasis. To get a better understanding of the mechanisms underlying type 1 diabetes mellitus, we have generated a mouse model by injections of multiple low-dose streptozotocin. The islets in the mouse pancreas were handpicked and proteins from the islets were then isolated and separated by two-dimensional gel electrophoresis. Seven proteins were found to be altered significantly at expression level. Among the seven proteins, four were up-regulated and three were down-regulated in diabetic mice as compared with controls. These proteins were successfully identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and the changes of selected protein expression were further validated by quantitative real time PCR and Western blotting. Voltage-dependent anion-selective channel protein 1 and peroxiredoxin-4 were found for the first time to be associated with type 1 diabetes mellitus in mouse islets in the current study. These results suggest that glucose transport, beta cell proliferation/death, and oxidative stress play important roles in maintaining the balance of glucose level. Our study also provides novel insight into the mechanism of type 1 diabetes mellitus.

Adaptive evolution influences the infectious dose of MERS-CoV necessary to achieve severe respiratory disease.

PubMed

Douglas, Madeline G; Kocher, Jacob F; Scobey, Trevor; Baric, Ralph S; Cockrell, Adam S

2018-04-01

We recently established a mouse model (288-330 +/+ ) that developed acute respiratory disease resembling human pathology following infection with a high dose (5 × 10 6 PFU) of mouse-adapted MERS-CoV (icMERSma1). Although this high dose conferred fatal respiratory disease in mice, achieving similar pathology at lower viral doses may more closely reflect naturally acquired infections. Through continued adaptive evolution of icMERSma1 we generated a novel mouse-adapted MERS-CoV (maM35c4) capable of achieving severe respiratory disease at doses between 10 3 and 10 5 PFU. Novel mutations were identified in the maM35c4 genome that may be responsible for eliciting etiologies of acute respiratory distress syndrome at 10-1000 fold lower viral doses. Importantly, comparative genetics of the two mouse-adapted MERS strains allowed us to identify specific mutations that remained fixed through an additional 20 cycles of adaptive evolution. Our data indicate that the extent of MERS-CoV adaptation determines the minimal infectious dose required to achieve severe respiratory disease. Copyright © 2017 Elsevier Inc. All rights reserved.

Drug discovery in prostate cancer mouse models.

PubMed

Valkenburg, Kenneth C; Pienta, Kenneth J

2015-01-01

The mouse is an important, though imperfect, organism with which to model human disease and to discover and test novel drugs in a preclinical setting. Many experimental strategies have been used to discover new biological and molecular targets in the mouse, with the hopes of translating these discoveries into novel drugs to treat prostate cancer in humans. Modeling prostate cancer in the mouse, however, has been challenging, and often drugs that work in mice have failed in human trials. The authors discuss the similarities and differences between mice and men; the types of mouse models that exist to model prostate cancer; practical questions one must ask when using a mouse as a model; and potential reasons that drugs do not often translate to humans. They also discuss the current value in using mouse models for drug discovery to treat prostate cancer and what needs are still unmet in field. With proper planning and following practical guidelines by the researcher, the mouse is a powerful experimental tool. The field lacks genetically engineered metastatic models, and xenograft models do not allow for the study of the immune system during the metastatic process. There remain several important limitations to discovering and testing novel drugs in mice for eventual human use, but these can often be overcome. Overall, mouse modeling is an essential part of prostate cancer research and drug discovery. Emerging technologies and better and ever-increasing forms of communication are moving the field in a hopeful direction.

Identification of HMX1 target genes: A predictive promoter model approach

PubMed Central

Boulling, Arnaud; Wicht, Linda

2013-01-01

Purpose A homozygous mutation in the H6 family homeobox 1 (HMX1) gene is responsible for a new oculoauricular defect leading to eye and auricular developmental abnormalities as well as early retinal degeneration (MIM 612109). However, the HMX1 pathway remains poorly understood, and in the first approach to better understand the pathway’s function, we sought to identify the target genes. Methods We developed a predictive promoter model (PPM) approach using a comparative transcriptomic analysis in the retina at P15 of a mouse model lacking functional Hmx1 (dmbo mouse) and its respective wild-type. This PPM was based on the hypothesis that HMX1 binding site (HMX1-BS) clusters should be more represented in promoters of HMX1 target genes. The most differentially expressed genes in the microarray experiment that contained HMX1-BS clusters were used to generate the PPM, which was then statistically validated. Finally, we developed two genome-wide target prediction methods: one that focused on conserving PPM features in human and mouse and one that was based on the co-occurrence of HMX1-BS pairs fitting the PPM, in human or in mouse, independently. Results The PPM construction revealed that sarcoglycan, gamma (35kDa dystrophin-associated glycoprotein) (Sgcg), teashirt zinc finger homeobox 2 (Tshz2), and solute carrier family 6 (neurotransmitter transporter, glycine) (Slc6a9) genes represented Hmx1 targets in the mouse retina at P15. Moreover, the genome-wide target prediction revealed that mouse genes belonging to the retinal axon guidance pathway were targeted by Hmx1. Expression of these three genes was experimentally validated using a quantitative reverse transcription PCR approach. The inhibitory activity of Hmx1 on Sgcg, as well as protein tyrosine phosphatase, receptor type, O (Ptpro) and Sema3f, two targets identified by the PPM, were validated with luciferase assay. Conclusions Gene expression analysis between wild-type and dmbo mice allowed us to develop a PPM that identified the first target genes of Hmx1. PMID:23946633

Orthology for comparative genomics in the mouse genome database.

PubMed

Dolan, Mary E; Baldarelli, Richard M; Bello, Susan M; Ni, Li; McAndrews, Monica S; Bult, Carol J; Kadin, James A; Richardson, Joel E; Ringwald, Martin; Eppig, Janan T; Blake, Judith A

2015-08-01

The mouse genome database (MGD) is the model organism database component of the mouse genome informatics system at The Jackson Laboratory. MGD is the international data resource for the laboratory mouse and facilitates the use of mice in the study of human health and disease. Since its beginnings, MGD has included comparative genomics data with a particular focus on human-mouse orthology, an essential component of the use of mouse as a model organism. Over the past 25 years, novel algorithms and addition of orthologs from other model organisms have enriched comparative genomics in MGD data, extending the use of orthology data to support the laboratory mouse as a model of human biology. Here, we describe current comparative data in MGD and review the history and refinement of orthology representation in this resource.

Transgenic overexpression of NanogP8 in the mouse prostate is insufficient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model.

PubMed

Liu, Bigang; Gong, Shuai; Li, Qiuhui; Chen, Xin; Moore, John; Suraneni, Mahipal V; Badeaux, Mark D; Jeter, Collene R; Shen, Jianjun; Mehmood, Rashid; Fan, Qingxia; Tang, Dean G

2017-08-08

This project was undertaken to address a critical cancer biology question: Is overexpression of the pluripotency molecule Nanog sufficient to initiate tumor development in a somatic tissue? Nanog1 is critical for the self-renewal and pluripotency of ES cells, and its retrotransposed homolog, NanogP8 is preferentially expressed in somatic cancer cells. Our work has shown that shRNA-mediated knockdown of NanogP8 in prostate, breast, and colon cancer cells inhibits tumor regeneration whereas inducible overexpression of NanogP8 promotes cancer stem cell phenotypes and properties. To address the key unanswered question whether tissue-specific overexpression of NanogP8 is sufficient to promote tumor development in vivo , we generated a NanogP8 transgenic mouse model, in which the ARR 2 PB promoter was used to drive NanogP8 cDNA. Surprisingly, the ARR 2 PB-NanogP8 transgenic mice were viable, developed normally, and did not form spontaneous tumors in >2 years. Also, both wild type and ARR 2 PB-NanogP8 transgenic mice responded similarly to castration and regeneration and castrated ARR 2 PB-NanogP8 transgenic mice also did not develop tumors. By crossing the ARR 2 PB-NanogP8 transgenic mice with ARR 2 PB-Myc (i.e., Hi-Myc) mice, we found that the double transgenic (i.e., ARR 2 PB-NanogP8; Hi-Myc) mice showed similar tumor incidence and histology to the Hi-Myc mice. Interestingly, however, we observed white dots in the ventral lobes of the double transgenic prostates, which were characterized as overgrown ductules/buds featured by crowded atypical Nanog-expressing luminal cells. Taken together, our present work demonstrates that transgenic overexpression of NanogP8 in the mouse prostate is insufficient to initiate tumorigenesis but weakly promotes tumor development in the Hi-Myc mouse model.

CIC-DUX4 Induces Small Round Cell Sarcomas Distinct from Ewing Sarcoma.

PubMed

Yoshimoto, Toyoki; Tanaka, Miwa; Homme, Mizuki; Yamazaki, Yukari; Takazawa, Yutaka; Antonescu, Cristina R; Nakamura, Takuro

2017-06-01

CIC-DUX4 sarcoma (CDS) or CIC-rearranged sarcoma is a subcategory of small round cell sarcoma resembling the morphological phenotypes of Ewing sarcoma (ES). However, recent clinicopathologic and molecular genetic analyses indicate that CDS is an independent disease entity from ES. Few ancillary markers have been used in the differential diagnosis of CDS, and additional CDS-specific biomarkers are needed for more definitive classification. Here, we report the generation of an ex vivo mouse model for CDS by transducing embryonic mesenchymal cells (eMC) with human CIC-DUX4 cDNA. Recipient mice transplanted with eMC-expressing CIC-DUX4 rapidly developed an aggressive, undifferentiated sarcoma composed of small round to short spindle cells. Gene-expression profiles of CDS and eMC revealed upregulation of CIC-DUX4 downstream genes such as PEA3 family genes, Ccnd2, Crh , and Zic1 IHC analyses for both mouse and human tumors showed that CCND2 and MUC5AC are reliable biomarkers to distinguish CDS from ES. Gene silencing of CIC-DUX4 as well as Ccnd2, Ret , and Bcl2 effectively inhibited CDS tumor growth in vitro The CDK4/6 inhibitor palbociclib and the soft tissue sarcoma drug trabectedin also blocked the growth of mouse CDS. In summary, our mouse model provides important biological information about CDS and provides a useful platform to explore biomarkers and therapeutic agents for CDS. Cancer Res; 77(11); 2927-37. ©2017 AACR . ©2017 American Association for Cancer Research.

A novel mouse model carrying a human cytoplasmic dynein mutation shows motor behavior deficits consistent with Charcot-Marie-Tooth type 2O disease.

PubMed

Sabblah, Thywill T; Nandini, Swaran; Ledray, Aaron P; Pasos, Julio; Calderon, Jami L Conley; Love, Rachal; King, Linda E; King, Stephen J

2018-01-29

Charcot-Marie-Tooth disease (CMT) is a peripheral neuromuscular disorder in which axonal degeneration causes progressive loss of motor and sensory nerve function. The loss of motor nerve function leads to distal muscle weakness and atrophy, resulting in gait problems and difficulties with walking, running, and balance. A mutation in the cytoplasmic dynein heavy chain (DHC) gene was discovered to cause an autosomal dominant form of the disease designated Charcot-Marie-Tooth type 2 O disease (CMT2O) in 2011. The mutation is a single amino acid change of histidine into arginine at amino acid 306 (H306R) in DHC. In order to understand the onset and progression of CMT2, we generated a knock-in mouse carrying the corresponding CMT2O mutation (H304R/+). We examined H304R/+ mouse cohorts in a 12-month longitudinal study of grip strength, tail suspension, and rotarod assays. H304R/+ mice displayed distal muscle weakness and loss of motor coordination phenotypes consistent with those of individuals with CMT2. Analysis of the gastrocnemius of H304R/+ male mice showed prominent defects in neuromuscular junction (NMJ) morphology including reduced size, branching, and complexity. Based on these results, the H304R/+ mouse will be an important model for uncovering functions of dynein in complex organisms, especially related to CMT onset and progression.

Obesity genetics in mouse and human: back and forth, and back again

PubMed Central

Yazdi, Fereshteh T.; Clee, Susanne M.

2015-01-01

Obesity is a major public health concern. This condition results from a constant and complex interplay between predisposing genes and environmental stimuli. Current attempts to manage obesity have been moderately effective and a better understanding of the etiology of obesity is required for the development of more successful and personalized prevention and treatment options. To that effect, mouse models have been an essential tool in expanding our understanding of obesity, due to the availability of their complete genome sequence, genetically identified and defined strains, various tools for genetic manipulation and the accessibility of target tissues for obesity that are not easily attainable from humans. Our knowledge of monogenic obesity in humans greatly benefited from the mouse obesity genetics field. Genes underlying highly penetrant forms of monogenic obesity are part of the leptin-melanocortin pathway in the hypothalamus. Recently, hypothesis-generating genome-wide association studies for polygenic obesity traits in humans have led to the identification of 119 common gene variants with modest effect, most of them having an unknown function. These discoveries have led to novel animal models and have illuminated new biologic pathways. Integrated mouse-human genetic approaches have firmly established new obesity candidate genes. Innovative strategies recently developed by scientists are described in this review to accelerate the identification of causal genes and deepen our understanding of obesity etiology. An exhaustive dissection of the molecular roots of obesity may ultimately help to tackle the growing obesity epidemic worldwide. PMID:25825681

UV-induced somatic mutations elicit a functional T cell response in the YUMMER1.7 mouse melanoma model.

PubMed

Wang, Jake; Perry, Curtis J; Meeth, Katrina; Thakral, Durga; Damsky, William; Micevic, Goran; Kaech, Susan; Blenman, Kim; Bosenberg, Marcus

2017-07-01

Human melanomas exhibit relatively high somatic mutation burden compared to other malignancies. These somatic mutations may produce neoantigens that are recognized by the immune system, leading to an antitumor response. By irradiating a parental mouse melanoma cell line carrying three driver mutations with UVB and expanding a single-cell clone, we generated a mutagenized model that exhibits high somatic mutation burden. When inoculated at low cell numbers in immunocompetent C57BL/6J mice, YUMMER1.7 (Yale University Mouse Melanoma Exposed to Radiation) regresses after a brief period of growth. This regression phenotype is dependent on T cells as YUMMER1.7 tumors grow significantly faster in immunodeficient Rag1 -/- mice and C57BL/6J mice depleted of CD4 and CD8 T cells. Interestingly, regression can be overcome by injecting higher cell numbers of YUMMER1.7, which results in tumors that grow without effective rejection. Mice that have previously rejected YUMMER1.7 tumors develop immunity against higher doses of YUMMER1.7 tumor challenge. In addition, escaping YUMMER1.7 tumors are sensitive to anti-CTLA-4 and anti-PD-1 therapy, establishing a new model for the evaluation of immune checkpoint inhibition and antitumor immune responses. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Copper chelator induced efficient episodic memory recovery in a non-transgenic Alzheimer's mouse model.

PubMed

Ceccom, Johnatan; Coslédan, Frédéric; Halley, Hélène; Francès, Bernard; Lassalle, Jean Michel; Meunier, Bernard

2012-01-01

Alzheimer's disease (AD) is a neurodegenerative syndrom involving many different biological parameters, including the accumulation of copper metal ions in Aβ amyloid peptides due to a perturbation of copper circulation and homeostasis within the brain. Copper-containing amyloids activated by endogenous reductants are able to generate an oxidative stress that is involved in the toxicity of abnormal amyloids and contribute to the progressive loss of neurons in AD. Since only few drugs are currently available for the treatment of AD, we decided to design small molecules able to interact with copper and we evaluated these drug-candidates with non-transgenic mice, since AD is mainly an aging disease, not related to genetic disorders. We created a memory deficit mouse model by a single icv injection of Aβ(1-42) peptide, in order to mimic the early stage of the disease and the key role of amyloid oligomers in AD. No memory deficit was observed in the control mice with the antisense Aβ(42-1) peptide. Here we report the capacity of a new copper-specific chelating agent, a bis-8-aminoquinoline PA1637, to fully reverse the deficit of episodic memory after three weeks of treatment by oral route on non-transgenic amyloid-impaired mice. Clioquinol and memantine have been used as comparators to validate this fast and efficient mouse model.

mNos2 deletion and human NOS2 replacement in Alzheimer disease models.

PubMed

Colton, Carol A; Wilson, Joan G; Everhart, Angela; Wilcock, Donna M; Puoliväli, Jukka; Heikkinen, Taneli; Oksman, Juho; Jääskeläinen, Olli; Lehtimäki, Kimmo; Laitinen, Teemu; Vartiainen, Nina; Vitek, Michael P

2014-08-01

Understanding the pathophysiologic mechanisms underlying Alzheimer disease relies on knowledge of disease onset and the sequence of development of brain pathologies. We present a comprehensive analysis of early and progressive changes in a mouse model that demonstrates a full spectrum of characteristic Alzheimer disease-like pathologies. This model demonstrates an altered immune redox state reminiscent of the human disease and capitalizes on data indicating critical differences between human and mouse immune responses, particularly in nitric oxide levels produced by immune activation of the NOS2 gene. Using the APPSwDI(+)/(+)mNos2(-/-) (CVN-AD) mouse strain, we show a sequence of pathologic events leading to neurodegeneration,which include pathologically hyperphosphorylated tau in the perforant pathway at 6 weeks of age progressing to insoluble tau, early appearance of β-amyloid peptides in perivascular deposits around blood vessels in brain regions known to be vulnerable to Alzheimer disease, and progression to damage and overt loss in select vulnerable neuronal populations in these regions. The role of species differences between hNOS2 and mNos2 was supported by generating mice in which the human NOS2 gene replaced mNos2. When crossed with CVN-AD mice, pathologic characteristics of this new strain (APPSwDI(+)/(-)/HuNOS2(tg+)/(+)/mNos2(-/-)) mimicked the pathologic phenotypes found in the CVN-AD strain.

Phenylquinoxalinone CFTR activator as potential prosecretory therapy for constipation

PubMed Central

CIL, ONUR; PHUAN, PUAY-WAH; SON, JUNG-HO; ZHU, JIE S.; KU, COLTON K.; TABIB, NILOUFAR AKHAVAN; TEUTHORN, ANDREW P.; FERRERA, LORETTA; ZACHOS, NICHOLAS C.; LIN, RUXIAN; GALIETTA, LUIS J. V.; DONOWITZ, MARK; KURTH, MARK J.; VERKMAN, ALAN S.

2017-01-01

Constipation is a common condition for which current treatments can have limited efficacy. By high-throughput screening, we recently identified a phenylquinoxalinone activator of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel that stimulated intestinal fluid secretion and normalized stool output in a mouse model of opioid-induced constipation. Here, we report phenylquinoxalinone structure-activity analysis, mechanism of action, animal efficacy data in acute and chronic models of constipation, and functional data in ex vivo primary cultured human enterocytes. Structure-activity analysis was done on 175 phenylquinoxalinone analogs, including 15 synthesized compounds. The most potent compound, CFTRact-J027, activated CFTR with EC50 ~ 200 nM, with patch-clamp analysis showing a linear CFTR current-voltage relationship with direct CFTR activation. CFTRact-J027 corrected reduced stool output and hydration in a mouse model of acute constipation produced by scopolamine and in a chronically constipated mouse strain (C3H/HeJ). Direct comparison with the approved prosecretory drugs lubiprostone and linaclotide showed substantially greater intestinal fluid secretion with CFTRact-J027, as well as greater efficacy in a constipation model. As evidence to support efficacy in human constipation, CFTRact-J027 increased transepithelial fluid transport in enteroids generated from normal human small intestine. Also, CFTRact-J027 was rapidly metabolized in vitro in human hepatic microsomes, suggesting minimal systemic exposure upon oral administration. These data establish structure-activity and mechanistic data for phenylquinoxalinone CFTR activators, and support their potential efficacy in human constipation. PMID:27815136

Gene signatures distinguish stage-specific prostate cancer stem cells isolated from transgenic adenocarcinoma of the mouse prostate lesions and predict the malignancy of human tumors.

PubMed

Mazzoleni, Stefania; Jachetti, Elena; Morosini, Sara; Grioni, Matteo; Piras, Ignazio Stefano; Pala, Mauro; Bulfone, Alessandro; Freschi, Massimo; Bellone, Matteo; Galli, Rossella

2013-09-01

The relevant social and economic impact of prostate adenocarcinoma, one of the leading causes of death in men, urges critical improvements in knowledge of the pathogenesis and cure of this disease. These can also be achieved by implementing in vitro and in vivo preclinical models by taking advantage of prostate cancer stem cells (PCSCs). The best-characterized mouse model of prostate cancer is the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. TRAMP mice develop a progressive lesion called prostatic intraepithelial neoplasia that evolves into adenocarcinoma (AD) between 24 and 30 weeks of age. ADs often metastasize to lymph nodes, lung, bones, and kidneys. Eventually, approximately 5% of the mice develop an androgen-independent neuroendocrine adenocarcinoma. Here we report the establishment of long-term self-renewing PCSC lines from the different stages of TRAMP progression by application of the neurosphere assay. Stage-specific prostate cell lines were endowed with the critical features expected from malignant bona fide cancer stem cells, namely, self-renewal, multipotency, and tumorigenicity. Notably, transcriptome analysis of stage-specific PCSCs resulted in the generation of well-defined, meaningful gene signatures, which identify distinct stages of human tumor progression. As such, TRAMP-derived PCSCs represent a novel and valuable preclinical model for elucidating the pathogenetic mechanisms leading to prostate adenocarcinoma and for the identification of molecular mediators to be pursued as therapeutic targets.

Distinct roles for motor neuron autophagy early and late in the SOD1G93A mouse model of ALS

PubMed Central

Rudnick, Noam D.; Griffey, Christopher J.; Guarnieri, Paolo; Gerbino, Valeria; Wang, Xueyong; Piersaint, Jason A.; Tapia, Juan Carlos; Rich, Mark M.; Maniatis, Tom

2017-01-01

Mutations in autophagy genes can cause familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of autophagy in ALS pathogenesis is poorly understood, in part due to the lack of cell type-specific manipulations of this pathway in animal models. Using a mouse model of ALS expressing mutant superoxide dismutase 1 (SOD1G93A), we show that motor neurons form large autophagosomes containing ubiquitinated aggregates early in disease progression. To investigate whether this response is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 was specifically disrupted in motor neurons (Atg7 cKO). Atg7 cKO mice were viable but exhibited structural and functional defects at a subset of vulnerable neuromuscular junctions. By crossing Atg7 cKO mice to the SOD1G93A mouse model, we found that autophagy inhibition accelerated early neuromuscular denervation of the tibialis anterior muscle and the onset of hindlimb tremor. Surprisingly, however, lifespan was extended in Atg7 cKO; SOD1G93A double-mutant mice. Autophagy inhibition did not prevent motor neuron cell death, but it reduced glial inflammation and blocked activation of the stress-related transcription factor c-Jun in spinal interneurons. We conclude that motor neuron autophagy is required to maintain neuromuscular innervation early in disease but eventually acts in a non–cell-autonomous manner to promote disease progression. PMID:28904095

Generation of Functional Thyroid Tissue Using 3D-Based Culture of Embryonic Stem Cells.

PubMed

Antonica, Francesco; Kasprzyk, Dominika Figini; Schiavo, Andrea Alex; Romitti, Mírian; Costagliola, Sabine

2017-01-01

During the last decade three-dimensional (3D) cultures of pluripotent stem cells have been intensively used to understand morphogenesis and molecular signaling important for the embryonic development of many tissues. In addition, pluripotent stem cells have been shown to be a valid tool for the in vitro modeling of several congenital or chronic human diseases, opening new possibilities to study their physiopathology without using animal models. Even more interestingly, 3D culture has proved to be a powerful and versatile tool to successfully generate functional tissues ex vivo. Using similar approaches, we here describe a protocol for the generation of functional thyroid tissue using mouse embryonic stem cells and give all the details and references for its characterization and analysis both in vitro and in vivo. This model is a valid approach to study the expression and the function of genes involved in the correct morphogenesis of thyroid gland, to elucidate the mechanisms of production and secretion of thyroid hormones and to test anti-thyroid drugs.

Genetically Engineered Mouse Models for Studying Inflammatory Bowel Disease

PubMed Central

Mizoguchi, Atsushi; Takeuchi, Takahito; Himuro, Hidetomo; Okada, Toshiyuki; Mizoguchi, Emiko

2015-01-01

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition that is mediated by very complex mechanisms controlled by genetic, immune, and environmental factors. More than 74 kinds of genetically engineered mouse strains have been established since 1993 for studying IBD. Although mouse models cannot fully reflect human IBD, they have provided significant contributions for not only understanding the mechanism, but also developing new therapeutic means for IBD. Indeed, 20 kinds of genetically engineered mouse models carry the susceptibility genes identified in human IBD, and the functions of some other IBD susceptibility genes have also been dissected out using mouse models. Cutting-edge technologies such as cell-specific and inducible knockout systems, which were recently employed to mouse IBD models, have further enhanced the ability of investigators to provide important and unexpected rationales for developing new therapeutic strategies for IBD. In this review article, we briefly introduce 74 kinds of genetically engineered mouse models that spontaneously develop intestinal inflammation. PMID:26387641

Highly efficient methods to obtain homogeneous dorsal neural progenitor cells from human and mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Zhang, Meixiang; Ngo, Justine; Pirozzi, Filomena; Sun, Ying-Pu; Wynshaw-Boris, Anthony

2018-03-15

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used to generate cellular models harboring specific disease-related genotypes. Of particular importance are ESC and iPSC applications capable of producing dorsal telencephalic neural progenitor cells (NPCs) that are representative of the cerebral cortex and overcome the challenges of maintaining a homogeneous population of cortical progenitors over several passages in vitro. While previous studies were able to derive NPCs from pluripotent cell types, the fraction of dorsal NPCs in this population is small and decreases over several passages. Here, we present three protocols that are highly efficient in differentiating mouse and human ESCs, as well as human iPSCs, into a homogeneous and stable population of dorsal NPCs. These protocols will be useful for modeling cerebral cortical neurological and neurodegenerative disorders in both mouse and human as well as for high-throughput drug screening for therapeutic development. We optimized three different strategies for generating dorsal telencephalic NPCs from mouse and human pluripotent cell types through single or double inhibition of bone morphogenetic protein (BMP) and/or SMAD pathways. Mouse and human pluripotent cells were aggregated to form embryoid bodies in suspension and were treated with dorsomorphin alone (BMP inhibition) or combined with SB431542 (double BMP/SMAD inhibition) during neural induction. Neural rosettes were then selected from plated embryoid bodies to purify the population of dorsal NPCs. We tested the expression of key dorsal NPC markers as well as nonectodermal markers to confirm the efficiency of our three methods in comparison to published and commercial protocols. Single and double inhibition of BMP and/or SMAD during neural induction led to the efficient differentiation of dorsal NPCs, based on the high percentage of PAX6-positive cells and the NPC gene expression profile. There were no statistically significant differences in the variation of PAX6 and SOX1-positive NPCs between the two human pluripotent cell-derived methods; therefore, both methods are suitable for producing stable dorsal NPCs. When further differentiated into mature neurons, NPCs gave rise to a population of almost exclusively forebrain cortical neurons, confirming the dorsal fate commitment of the progenitors. The methods described in this study show improvements over previously published studies and are highly efficient at differentiating human and mouse pluripotent cell types into dorsal PAX6-positive NPCs and eventually into forebrain cortical neurons.

Establishment of a Mouse Model with Misregulated Chromosome Condensation due to Defective Mcph1 Function

PubMed Central

Walther, Diego J.; Dopatka, Monika; Dutrannoy, Véronique; Busche, Andreas; Meyer, Franziska; Nowak, Stefanie; Nowak, Jean; Zabel, Claus; Klose, Joachim; Esquitino, Veronica; Garshasbi, Masoud; Kuss, Andreas W.; Ropers, Hans-Hilger; Mueller, Susanne; Poehlmann, Charlotte; Gavvovidis, Ioannis; Schindler, Detlev; Sperling, Karl; Neitzel, Heidemarie

2010-01-01

Mutations in the human gene MCPH1 cause primary microcephaly associated with a unique cellular phenotype with premature chromosome condensation (PCC) in early G2 phase and delayed decondensation post-mitosis (PCC syndrome). The gene encodes the BRCT-domain containing protein microcephalin/BRIT1. Apart from its role in the regulation of chromosome condensation, the protein is involved in the cellular response to DNA damage. We report here on the first mouse model of impaired Mcph1-function. The model was established based on an embryonic stem cell line from BayGenomics (RR0608) containing a gene trap in intron 12 of the Mcph1 gene deleting the C-terminal BRCT-domain of the protein. Although residual wild type allele can be detected by quantitative real-time PCR cell cultures generated from mouse tissues bearing the homozygous gene trap mutation display the cellular phenotype of misregulated chromosome condensation that is characteristic for the human disorder, confirming defective Mcph1 function due to the gene trap mutation. While surprisingly the DNA damage response (formation of repair foci, chromosomal breakage, and G2/M checkpoint function after irradiation) appears to be largely normal in cell cultures derived from Mcph1gt/gt mice, the overall survival rates of the Mcph1gt/gt animals are significantly reduced compared to wild type and heterozygous mice. However, we could not detect clear signs of premature malignant disease development due to the perturbed Mcph1 function. Moreover, the animals show no obvious physical phenotype and no reduced fertility. Body and brain size are within the range of wild type controls. Gene expression on RNA and protein level did not reveal any specific pattern of differentially regulated genes. To the best of our knowledge this represents the first mammalian transgenic model displaying a defect in mitotic chromosome condensation and is also the first mouse model for impaired Mcph1-function. PMID:20169082

Establishment of a mouse model with misregulated chromosome condensation due to defective Mcph1 function.

PubMed

Trimborn, Marc; Ghani, Mahdi; Walther, Diego J; Dopatka, Monika; Dutrannoy, Véronique; Busche, Andreas; Meyer, Franziska; Nowak, Stefanie; Nowak, Jean; Zabel, Claus; Klose, Joachim; Esquitino, Veronica; Garshasbi, Masoud; Kuss, Andreas W; Ropers, Hans-Hilger; Mueller, Susanne; Poehlmann, Charlotte; Gavvovidis, Ioannis; Schindler, Detlev; Sperling, Karl; Neitzel, Heidemarie

2010-02-16

Mutations in the human gene MCPH1 cause primary microcephaly associated with a unique cellular phenotype with premature chromosome condensation (PCC) in early G2 phase and delayed decondensation post-mitosis (PCC syndrome). The gene encodes the BRCT-domain containing protein microcephalin/BRIT1. Apart from its role in the regulation of chromosome condensation, the protein is involved in the cellular response to DNA damage. We report here on the first mouse model of impaired Mcph1-function. The model was established based on an embryonic stem cell line from BayGenomics (RR0608) containing a gene trap in intron 12 of the Mcph1 gene deleting the C-terminal BRCT-domain of the protein. Although residual wild type allele can be detected by quantitative real-time PCR cell cultures generated from mouse tissues bearing the homozygous gene trap mutation display the cellular phenotype of misregulated chromosome condensation that is characteristic for the human disorder, confirming defective Mcph1 function due to the gene trap mutation. While surprisingly the DNA damage response (formation of repair foci, chromosomal breakage, and G2/M checkpoint function after irradiation) appears to be largely normal in cell cultures derived from Mcph1(gt/gt) mice, the overall survival rates of the Mcph1(gt/gt) animals are significantly reduced compared to wild type and heterozygous mice. However, we could not detect clear signs of premature malignant disease development due to the perturbed Mcph1 function. Moreover, the animals show no obvious physical phenotype and no reduced fertility. Body and brain size are within the range of wild type controls. Gene expression on RNA and protein level did not reveal any specific pattern of differentially regulated genes. To the best of our knowledge this represents the first mammalian transgenic model displaying a defect in mitotic chromosome condensation and is also the first mouse model for impaired Mcph1-function.

Developing a Reliable Mouse Model for Cancer Therapy-Induced Cardiovascular Toxicity in Cancer Patients and Survivors.

PubMed

Ko, Kyung Ae; Wang, Yin; Kotla, Sivareddy; Fujii, Yuka; Vu, Hang Thi; Venkatesulu, Bhanu P; Thomas, Tamlyn N; Medina, Jan L; Gi, Young Jin; Hada, Megumi; Grande-Allen, Jane; Patel, Zarana S; Milgrom, Sarah A; Krishnan, Sunil; Fujiwara, Keigi; Abe, Jun-Ichi

2018-01-01

The high incidence of cardiovascular events in cancer survivors has long been noted, but the mechanistic insights of cardiovascular toxicity of cancer treatments, especially for vessel diseases, remain unclear. It is well known that atherosclerotic plaque formation begins in the area exposed to disturbed blood flow, but the relationship between cancer therapy and disturbed flow in regulating plaque formation has not been well studied. Therefore, we had two goals for this study; (1) Generate an affordable, reliable, and reproducible mouse model to recapitulate the cancer therapy-induced cardiovascular events in cancer survivors, and (2) Establish a mouse model to investigate the interplay between disturbed flow and various cancer therapies in the process of atherosclerotic plaque formation. We examined the effects of two cancer drugs and ionizing radiation (IR) on disturbed blood flow-induced plaque formation using a mouse carotid artery partial ligation (PCL) model of atherosclerosis. We found that doxorubicin and cisplatin, which are commonly used anti-cancer drugs, had no effect on plaque formation in partially ligated carotid arteries. Similarly, PCL-induced plaque formation was not affected in mice that received IR (2 Gy) and PCL surgery performed one week later. In contrast, when PCL surgery was performed 26 days after IR treatment, not only the atherosclerotic plaque formation but also the necrotic core formation was significantly enhanced. Lastly, we found a significant increase in p90RSK phosphorylation in the plaques from the IR-treated group compared to those from the non-IR treated group. Our results demonstrate that IR not only increases atherosclerotic events but also vulnerable plaque formation. These increases were a somewhat delayed effect of IR as they were observed in mice with PCL surgery performed 26 days, but not 10 days, after IR exposure. A proper animal model must be developed to study how to minimize the cardiovascular toxicity due to cancer treatment.

Genetically Engineered Mouse Model of Diffuse Intrinsic Pontine Glioma as a Preclinical Tool

DTIC Science & Technology

2012-09-01

Hydrocephalus mice were excluded from this calculation. With this particular experiment the hydrocephalus rate is 57% (due to the formation of...is completed. We have generated 10 tumors by injecting 14 mice and an example of one is described in the figure below. Hydrocephalus mice were...excluded from the 4 analysis. The hydrocephalus rate was 51% for this experiment due to the formation of leptomeningeal tumor

Analysis of second-harmonic-generation microscopy in a mouse model of ovarian carcinoma

NASA Astrophysics Data System (ADS)

Watson, Jennifer M.; Rice, Photini F.; Marion, Samuel L.; Brewer, Molly A.; Davis, John R.; Rodriguez, Jeffrey J.; Utzinger, Urs; Hoyer, Patricia B.; Barton, Jennifer K.

2012-07-01

Second-harmonic-generation (SHG) imaging of mouse ovaries ex vivo was used to detect collagen structure changes accompanying ovarian cancer development. Dosing with 4-vinylcyclohexene diepoxide and 7,12-dimethylbenz[a]anthracene resulted in histologically confirmed cases of normal, benign abnormality, dysplasia, and carcinoma. Parameters for each SHG image were calculated using the Fourier transform matrix and gray-level co-occurrence matrix (GLCM). Cancer versus normal and cancer versus all other diagnoses showed the greatest separation using the parameters derived from power in the highest-frequency region and GLCM energy. Mixed effects models showed that these parameters were significantly different between cancer and normal (P<0.008). Images were classified with a support vector machine, using 25% of the data for training and 75% for testing. Utilizing all images with signal greater than the noise level, cancer versus not-cancer specimens were classified with 81.2% sensitivity and 80.0% specificity, and cancer versus normal specimens were classified with 77.8% sensitivity and 79.3% specificity. Utilizing only images with greater than of 75% of the field of view containing signal improved sensitivity and specificity for cancer versus normal to 81.5% and 81.1%. These results suggest that using SHG to visualize collagen structure in ovaries could help with early cancer detection.

Examination of diagnostic features in multiphoton microscopy and optical coherence tomography images of ovarian tumorigenesis in a mouse model

NASA Astrophysics Data System (ADS)

Watson, Jennifer M.

Ovarian cancer is a deadly disease owing to the non-specific symptoms and suspected rapid progression, leading to frequent late stage detection and poor prognosis. Medical imaging methods such as CT, MRI and ultrasound as well as serum testing for cancer markers have had extremely poor performance for early disease detection. Due to the poor performance of available screening methods, and the impracticality and ineffectiveness of taking tissue biopsies from the ovary, women at high risk for developing ovarian cancer are often advised to undergo prophylactic salpingo-oophorectomy. This surgery results in many side effects and is most often unnecessary since only a fraction of high risk women go on to develop ovarian cancer. Better understanding of the early development of ovarian cancer and characterization of morphological changes associated with early disease could lead to the development of an effective screening test for women at high risk. Optical imaging methods including optical coherence tomography (OCT) and multiphoton microscopy (MPM) are excellent tools for studying disease progression owing to the high resolution and depth sectioning capabilities. Further, these techniques are excellent for optical biopsy because they can image in situ non-destructively. In the studies described in this dissertation OCT and MPM are used to identify cellular and tissue morphological changes associated with early tumor development in a mouse model of ovarian cancer. This work is organized into three specific aims. The first aim is to use the images from the MPM phenomenon of second harmonic generation to quantitatively examine the morphological differences in collagen structure in normal mouse ovarian tissue and mouse ovarian tumors. The second aim is to examine the differences in endogenous two-photon excited fluorescence in normal mouse ovarian tissue and mouse ovarian tumors. The third and final aim is to identify changes in ovarian microstructure resulting from early disease development by imaging animals in vivo at three time points during a long-term survival study.

Metabolic characterization of a mouse deficient in all known leptin receptor isoforms.

PubMed

Osborn, Olivia; Sanchez-Alavez, Manuel; Brownell, Sara E; Ross, Brendon; Klaus, Joe; Dubins, Jeffrey; Beutler, Bruce; Conti, Bruno; Bartfai, Tamas

2010-01-01

We have characterized a newly generated mouse model of obesity, a mouse strain deficient in all five previously described leptin receptor isoforms. These transgenic mice, named the db (333)/db (333) mice, were identified from an ENU mutagenesis screen and carry a point mutation in the seventh exon of the db gene encoding the leptin receptor, resulting in a premature stop codon (Y(333)Stop) and gene product that lacks STAT signaling domains. db (333)/db (333) mice have a morbidly obese phenotype, with body weights diverging from wild type as early as 4 weeks of age (P < 0.05). To determine the contribution of the short isoforms of the leptin receptor in this metabolic phenotype, we performed an extensive metabolic characterization of the db (333)/db (333) mouse in relation to the well-characterized db/db mouse lacking only the long form of the leptin receptor. db (333)/db (333) mice have similar endocrine and metabolic parameters as previously described in other leptin receptor transgenic mice including db/db mice that lack only the long isoform of the leptin receptor. However, db (333)/db (333) mice show a subtle trend toward higher body weight and insulin levels, lower oxygen, carbon dioxide production, respiratory exchange ratio (RER), and temperature than db/db mice suggesting the short isoforms may play an additional role in energy homeostasis.

Craniomandibular form and body size variation of first generation mouse hybrids: A model for hominin hybridization.

PubMed

Warren, Kerryn A; Ritzman, Terrence B; Humphreys, Robyn A; Percival, Christopher J; Hallgrímsson, Benedikt; Ackermann, Rebecca Rogers

2018-03-01

Hybridization occurs in a number of mammalian lineages, including among primate taxa. Analyses of ancient genomes have shown that hybridization between our lineage and other archaic hominins in Eurasia occurred numerous times in the past. However, we still have limited empirical data on what a hybrid skeleton looks like, or how to spot patterns of hybridization among fossils for which there are no genetic data. Here we use experimental mouse models to supplement previous studies of primates. We characterize size and shape variation in the cranium and mandible of three wild-derived inbred mouse strains and their first generation (F 1 ) hybrids. The three parent taxa in our analysis represent lineages that diverged over approximately the same period as the human/Neanderthal/Denisovan lineages and their hybrids are variably successful in the wild. Comparisons of body size, as quantified by long bone measurements, are also presented to determine whether the identified phenotypic effects of hybridization are localized to the cranium or represent overall body size changes. The results indicate that hybrid cranial and mandibular sizes, as well as limb length, exceed that of the parent taxa in all cases. All three F 1 hybrid crosses display similar patterns of size and form variation. These results are generally consistent with earlier studies on primates and other mammals, suggesting that the effects of hybridization may be similar across very different scenarios of hybridization, including different levels of hybrid fitness. This paper serves to supplement previous studies aimed at identifying F 1 hybrids in the fossil record and to introduce further research that will explore hybrid morphologies using mice as a proxy for better understanding hybridization in the hominin fossil record. Copyright © 2017 Elsevier Ltd. All rights reserved.

Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

PubMed

Shuda, Masahiro; Guastafierro, Anna; Geng, Xuehui; Shuda, Yoko; Ostrowski, Stephen M; Lukianov, Stefan; Jenkins, Frank J; Honda, Kord; Maricich, Stephen M; Moore, Patrick S; Chang, Yuan

2015-01-01

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

Production of cloned mice and ES cells from adult somatic cells by nuclear transfer: how to improve cloning efficiency?

PubMed

Wakayama, Teruhiko

2007-02-01

Although it has now been 10 years since the first cloned mammals were generated from somatic cells using nuclear transfer (NT), most cloned embryos usually undergo developmental arrest prior to or soon after implantation, and the success rate for producing live offspring by cloning remains below 5%. The low success rate is believed to be associated with epigenetic errors, including abnormal DNA hypermethylation, but the mechanism of "reprogramming" is unclear. We have been able to develop a stable NT method in the mouse in which donor nuclei are directly injected into the oocyte using a piezo-actuated micromanipulator. Especially in the mouse, only a few laboratories can make clones from adult somatic cells, and cloned mice are never successfully produced from most mouse strains. However, this technique promises to be an important tool for future research in basic biology. For example, NT can be used to generate embryonic stem (NT-ES) cell lines from a patient's own somatic cells. We have shown that NT-ES cells are equivalent to ES cells derived from fertilized embryos and that they can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. In general, NT-ES cell techniques are expected to be applied to regenerative medicine; however, this technique can also be applied to the preservation of genetic resources of mouse strain instead of embryos, oocytes and spermatozoa. This review describes how to improve cloning efficiency and NT-ES cell establishment and further applications.

A humanoid mouse model of autism.

PubMed

Takumi, Toru

2010-10-01

Even now fruit of the human genome project is available, we have difficulties to approach neuropsychiatric disorders at the molecular level. Autism is a complex psychiatric illness but has received considerable attention as a developmental brain disorder not only from basic researchers but also from society. Substantial evidence suggests that chromosomal abnormalities contribute to autism risk. The duplication of human chromosome 15q11-13 is known to be the most frequent cytogenetic abnormality in autism. We succeeded to generate mice with a 6.3-Mb-wide interstitial duplication in mouse chromosome 7c that is highly syntenic to human 15q11-13 by using a Cre-loxP-based chromosome-engineering technique. The only paternally duplicated mice display autistic behavioral features such as poor social interaction and stereotypical behavior, and exhibit a developmental abnormality in ultrasonic vocalizations as well as anxiety. The detailed analysis focusing on a non-coding small nucleolar RNA, MBII52, within the duplicated region, revealed that the paternally duplicated mice alter the editing ratio of serotonin (5-HT) 2c receptor pre-mRNA and intracellular calcium responses by a 5-HT2c receptor specific agonist are changed in neurons. This result may explain one of molecular mechanisms of abnormal behaviors in the paternal duplicated mice. The first chromosome-engineered mouse model for human chromosome 15q11-13 duplication fulfills not only face validity of human autistic phenotypes but also construct validity based on human chromosome abnormality. This model will be a founder mouse for forward genetics of autistic disease and an invaluable tool for its therapeutic development. Copyright © 2010 Elsevier B.V. All rights reserved.

A new mouse model of ARX dup24 recapitulates the patients' behavioral and fine motor alterations.

PubMed

Dubos, Aline; Meziane, Hamid; Iacono, Giovanni; Curie, Aurore; Riet, Fabrice; Martin, Christelle; Loaëc, Nadège; Birling, Marie-Christine; Selloum, Mohammed; Normand, Elisabeth; Pavlovic, Guillaume; Sorg, Tania; Stunnenberg, Henk G; Chelly, Jamel; Humeau, Yann; Friocourt, Gaëlle; Hérault, Yann

2018-06-15

The aristaless-related homeobox (ARX) transcription factor is involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopmental disorders in humans, among which the most frequent, a 24 bp duplication in the polyalanine tract 2 (c.428_451dup24), gives rise to intellectual disability, fine motor defects with or without epilepsy. To understand the functional consequences of this mutation, we generated a partially humanized mouse model carrying the c.428_451dup24 duplication (Arxdup24/0) that we characterized at the behavior, neurological and molecular level. Arxdup24/0 males presented with hyperactivity, enhanced stereotypies and altered contextual fear memory. In addition, Arxdup24/0 males had fine motor defects with alteration of reaching and grasping abilities. Transcriptome analysis of Arxdup24/0 forebrains at E15.5 showed a down-regulation of genes specific to interneurons and an up-regulation of genes normally not expressed in this cell type, suggesting abnormal interneuron development. Accordingly, interneuron migration was altered in the cortex and striatum between E15.5 and P0 with consequences in adults, illustrated by the defect in the inhibitory/excitatory balance in Arxdup24/0 basolateral amygdala. Altogether, we showed that the c.428_451dup24 mutation disrupts Arx function with a direct consequence on interneuron development, leading to hyperactivity and defects in precise motor movement control and associative memory. Interestingly, we highlighted striking similarities between the mouse phenotype and a cohort of 33 male patients with ARX c.428_451dup24, suggesting that this new mutant mouse line is a good model for understanding the pathophysiology and evaluation of treatment.

R6/2 Huntington's disease mice develop early and progressive abnormal brain metabolism and seizures.

PubMed

Cepeda-Prado, Efrain; Popp, Susanna; Khan, Usman; Stefanov, Dimitre; Rodríguez, Jorge; Menalled, Liliana B; Dow-Edwards, Diana; Small, Scott A; Moreno, Herman

2012-05-09

A hallmark feature of Huntington's disease pathology is the atrophy of brain regions including, but not limited to, the striatum. Though MRI studies have identified structural CNS changes in several Huntington's disease (HD) mouse models, the functional consequences of HD pathology during the progression of the disease have yet to be investigated using in vivo functional MRI (fMRI). To address this issue, we first established the structural and functional MRI phenotype of juvenile HD mouse model R6/2 at early and advanced stages of disease. Significantly higher fMRI signals [relative cerebral blood volumes (rCBVs)] and atrophy were observed in both age groups in specific brain regions. Next, fMRI results were correlated with electrophysiological analysis, which showed abnormal increases in neuronal activity in affected brain regions, thus identifying a mechanism accounting for the abnormal fMRI findings. [(14)C] 2-deoxyglucose maps to investigate patterns of glucose utilization were also generated. An interesting mismatch between increases in rCBV and decreases in glucose uptake was observed. Finally, we evaluated the sensitivity of this mouse line to audiogenic seizures early in the disease course. We found that R6/2 mice had an increased susceptibility to develop seizures. Together, these findings identified seizure activity in R6/2 mice and show that neuroimaging measures sensitive to oxygen metabolism can be used as in vivo biomarkers, preceding the onset of an overt behavioral phenotype. Since fMRI-rCBV can also be obtained in patients, we propose that it may serve as a translational tool to evaluate therapeutic responses in humans and HD mouse models.

Characterization of a new, inducible transgenic mouse model with GFP expression in melanocytes and their precursors.

PubMed

Joshi, Sandeep S; Tandukar, Bishal; Castaneda, Maira; Jiang, Shunlin; Diwakar, Ganesh; Hertzano, Ronna P; Hornyak, Thomas J

2018-01-01

Melanocytes are neural crest-derived cells that are responsible for mammalian hair follicle (HF) pigmentation. The Dct-LacZ transgenic mouse is extensively used to study melanocyte biology but lacks conditionally-inducible labelling and fluorescent labelling, enabling specific, viable isolation of melanocytes using fluorescence-activated cell sorting (FACS). Here, we have generated a Tet-off bitransgenic mouse model, Dct-H2BGFP, containing Dct-tTA and TRE-H2BGFP transgenes. Characterization of Dct-H2BGFP mice confirmed a pattern of Dct-H2BGFP expression in melanoblasts, melanocyte stem cells (McSCs), and terminally differentiated melanocytes similar to the expression pattern of previously published mouse models Dct-LacZ and iDct-GFP. GFP expression is regulated by doxycycline. GFP is shown to co-localize with melanocyte label-retaining cells (LRCs) identified through BrdU retention. The GFP-expressing cells identified in vivo in the bulge and the secondary hair germ of telogen HFs of Dct-H2BGFP mice express the melanocyte and melanocyte stem cell markers Dct and Kit. Using Dct-H2BGFP mice, we separated GFP-expressing cells from the telogen HF based on FACS and showed that GFP-expressing cells express high levels of Kit and Dct, and lower levels of HF epithelial keratin genes. We also show that GFP-expressing cells express high levels of the melanocyte differentiation genes Tyr, Tyrp1, and Pmel17, further substantiating their identity within the melanocyte lineage. Thus, Dct-H2BGFP mice are not only useful for the in vivo identification of melanocytic cells, but also for isolating them viably and studying their molecular and biological properties. Published by Elsevier B.V.

Berberine blocks the relapse of Clostridium difficile infection in C57BL/6 mice after standard vancomycin treatment.

PubMed

Lv, Zhi; Peng, Guoli; Liu, Weihua; Xu, Hufeng; Su, JianRong

2015-07-01

Vancomycin is a preferred antibiotic for treating Clostridium difficile infection (CDI) and has been associated with a rate of recurrence of CDI of as high as 20% in treated patients. Recent studies have suggested that berberine, an alternative medical therapy for gastroenteritis and diarrhea, exhibits several beneficial effects, including induction of anti-inflammatory responses and restoration of the intestinal barrier function. This study investigated the therapeutic effects of berberine on preventing CDI relapse and restoring the gut microbiota in a mouse model. Berberine was administered through gavage to C57BL/6 mice with established CDI-induced intestinal injury and colitis. The disease activity index (DAI), mean relative weight, histopathology scores, and levels of toxins A and B in fecal samples were measured. An Illumina sequencing-based analysis of 16S rRNA genes was used to determine the overall structural change in the microbiota in the mouse ileocecum. Berberine administration significantly promoted the restoration of the intestinal microbiota by inhibiting the expansion of members of the family Enterobacteriaceae and counteracting the side effects of vancomycin treatment. Therapy consisting of vancomycin and berberine combined prevented weight loss, improved the DAI and the histopathology scores, and effectively decreased the mortality rate. Berberine prevented CDIs from relapsing and significantly improved survival in the mouse model of CDI. Our data indicate that a combination of berberine and vancomycin is more effective than vancomycin alone for treating CDI. One of the possible mechanisms by which berberine prevents a CDI relapse is through modulation of the gut microbiota. Although this conclusion was generated in the case of the mouse model, use of the combination of vancomycin and berberine and represent a novel therapeutic approach targeting CDI. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A new mouse model of ARX dup24 recapitulates the patients’ behavioral and fine motor alterations

PubMed Central

Dubos, Aline; Meziane, Hamid; Iacono, Giovanni; Curie, Aurore; Riet, Fabrice; Martin, Christelle; Loaëc, Nadège; Birling, Marie-Christine; Selloum, Mohammed; Normand, Elisabeth; Pavlovic, Guillaume; Sorg, Tania; Stunnenberg, Henk G; Chelly, Jamel; Humeau, Yann; Friocourt, Gaëlle; Hérault, Yann

2018-01-01

Abstract The aristaless-related homeobox (ARX) transcription factor is involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopmental disorders in humans, among which the most frequent, a 24 bp duplication in the polyalanine tract 2 (c.428_451dup24), gives rise to intellectual disability, fine motor defects with or without epilepsy. To understand the functional consequences of this mutation, we generated a partially humanized mouse model carrying the c.428_451dup24 duplication (Arxdup24/0) that we characterized at the behavior, neurological and molecular level. Arxdup24/0 males presented with hyperactivity, enhanced stereotypies and altered contextual fear memory. In addition, Arxdup24/0 males had fine motor defects with alteration of reaching and grasping abilities. Transcriptome analysis of Arxdup24/0 forebrains at E15.5 showed a down-regulation of genes specific to interneurons and an up-regulation of genes normally not expressed in this cell type, suggesting abnormal interneuron development. Accordingly, interneuron migration was altered in the cortex and striatum between E15.5 and P0 with consequences in adults, illustrated by the defect in the inhibitory/excitatory balance in Arxdup24/0 basolateral amygdala. Altogether, we showed that the c.428_451dup24 mutation disrupts Arx function with a direct consequence on interneuron development, leading to hyperactivity and defects in precise motor movement control and associative memory. Interestingly, we highlighted striking similarities between the mouse phenotype and a cohort of 33 male patients with ARX c.428_451dup24, suggesting that this new mutant mouse line is a good model for understanding the pathophysiology and evaluation of treatment. PMID:29659809

Poliomyelitis in transgenic mice expressing CD155 under the control of the Tage4 promoter after oral and parenteral poliovirus inoculation

PubMed Central

Khan, Shaukat; Toyoda, Hidemi; Linehan, Melissa; Iwasaki, Akiko; Nomoto, Akio; Bernhardt, Günter; Wimmer, Eckard

2014-01-01

An important step in poliovirus (PV) infection by the oral route in humans is replication of the virus in lymphatic tissues of the gastrointestinal (GI) tract, thought to be mainly in the Peyer’s patches of the small intestine. No immunocompetent transgenic (tg) mice that express human PV receptor (CD155) under the control of different promoters can be infected orally. The mouse orthologue of human CD155 is Tage4, a protein expressed at the surface of enterocytes and in the Peyer’s patches. We describe here the generation of a tg mouse model in which the Tage4 promoter was used to drive expression of the human PV receptor-coding region (Tage4-CD155tg mice). In this model, CD155 expression was observed by immunostaining in different regions in the Peyer’s patches but not in their germinal centres. Although a similar pattern of staining was observed between 3- and 6-week-old Tage4-CD155tg mice, poliomyelitis was only seen in the younger mice after PV infection by the oral route. When compared with TgPVR21 mice that expressed CD155 driven by its human promoter, 3-week-old Tage4-CD155tg mice were more susceptible to gut infection and paralysis following feeding with PV. Also, Tage4-CD155tg mice exhibited higher susceptibility to poliomyelitis after parenteral inoculation of PV. Remarkably, the LD50 after intracerebral inoculation of PV was similar in both CD155 tg mouse strains. The CD155 tg mouse model reported here, although moderately susceptible to oral infection, may be suitable to study mechanisms of PV replication in the gastrointestinal tract and to dissect important aspects of PV neuroinvasiveness. PMID:24784416

Poliomyelitis in transgenic mice expressing CD155 under the control of the Tage4 promoter after oral and parenteral poliovirus inoculation.

PubMed

Khan, Shaukat; Toyoda, Hidemi; Linehan, Melissa; Iwasaki, Akiko; Nomoto, Akio; Bernhardt, Günter; Cello, Jeronimo; Wimmer, Eckard

2014-08-01

An important step in poliovirus (PV) infection by the oral route in humans is replication of the virus in lymphatic tissues of the gastrointestinal (GI) tract, thought to be mainly in the Peyer's patches of the small intestine. No immunocompetent transgenic (tg) mice that express human PV receptor (CD155) under the control of different promoters can be infected orally. The mouse orthologue of human CD155 is Tage4, a protein expressed at the surface of enterocytes and in the Peyer's patches. We describe here the generation of a tg mouse model in which the Tage4 promoter was used to drive expression of the human PV receptor-coding region (Tage4-CD155tg mice). In this model, CD155 expression was observed by immunostaining in different regions in the Peyer's patches but not in their germinal centres. Although a similar pattern of staining was observed between 3- and 6-week-old Tage4-CD155tg mice, poliomyelitis was only seen in the younger mice after PV infection by the oral route. When compared with TgPVR21 mice that expressed CD155 driven by its human promoter, 3-week-old Tage4-CD155tg mice were more susceptible to gut infection and paralysis following feeding with PV. Also, Tage4-CD155tg mice exhibited higher susceptibility to poliomyelitis after parenteral inoculation of PV. Remarkably, the LD50 after intracerebral inoculation of PV was similar in both CD155 tg mouse strains. The CD155 tg mouse model reported here, although moderately susceptible to oral infection, may be suitable to study mechanisms of PV replication in the gastrointestinal tract and to dissect important aspects of PV neuroinvasiveness. © 2014 The Authors.

XGAP: a uniform and extensible data model and software platform for genotype and phenotype experiments

PubMed Central

2010-01-01

We present an extensible software model for the genotype and phenotype community, XGAP. Readers can download a standard XGAP (http://www.xgap.org) or auto-generate a custom version using MOLGENIS with programming interfaces to R-software and web-services or user interfaces for biologists. XGAP has simple load formats for any type of genotype, epigenotype, transcript, protein, metabolite or other phenotype data. Current functionality includes tools ranging from eQTL analysis in mouse to genome-wide association studies in humans. PMID:20214801

XGAP: a uniform and extensible data model and software platform for genotype and phenotype experiments.

PubMed

Swertz, Morris A; Velde, K Joeri van der; Tesson, Bruno M; Scheltema, Richard A; Arends, Danny; Vera, Gonzalo; Alberts, Rudi; Dijkstra, Martijn; Schofield, Paul; Schughart, Klaus; Hancock, John M; Smedley, Damian; Wolstencroft, Katy; Goble, Carole; de Brock, Engbert O; Jones, Andrew R; Parkinson, Helen E; Jansen, Ritsert C

2010-01-01

We present an extensible software model for the genotype and phenotype community, XGAP. Readers can download a standard XGAP (http://www.xgap.org) or auto-generate a custom version using MOLGENIS with programming interfaces to R-software and web-services or user interfaces for biologists. XGAP has simple load formats for any type of genotype, epigenotype, transcript, protein, metabolite or other phenotype data. Current functionality includes tools ranging from eQTL analysis in mouse to genome-wide association studies in humans.

Genome-wide expression profiling of five mouse models identifies similarities and differences with human psoriasis.

PubMed

Swindell, William R; Johnston, Andrew; Carbajal, Steve; Han, Gangwen; Wohn, Christian; Lu, Jun; Xing, Xianying; Nair, Rajan P; Voorhees, John J; Elder, James T; Wang, Xiao-Jing; Sano, Shigetoshi; Prens, Errol P; DiGiovanni, John; Pittelkow, Mark R; Ward, Nicole L; Gudjonsson, Johann E

2011-04-04

Development of a suitable mouse model would facilitate the investigation of pathomechanisms underlying human psoriasis and would also assist in development of therapeutic treatments. However, while many psoriasis mouse models have been proposed, no single model recapitulates all features of the human disease, and standardized validation criteria for psoriasis mouse models have not been widely applied. In this study, whole-genome transcriptional profiling is used to compare gene expression patterns manifested by human psoriatic skin lesions with those that occur in five psoriasis mouse models (K5-Tie2, imiquimod, K14-AREG, K5-Stat3C and K5-TGFbeta1). While the cutaneous gene expression profiles associated with each mouse phenotype exhibited statistically significant similarity to the expression profile of psoriasis in humans, each model displayed distinctive sets of similarities and differences in comparison to human psoriasis. For all five models, correspondence to the human disease was strong with respect to genes involved in epidermal development and keratinization. Immune and inflammation-associated gene expression, in contrast, was more variable between models as compared to the human disease. These findings support the value of all five models as research tools, each with identifiable areas of convergence to and divergence from the human disease. Additionally, the approach used in this paper provides an objective and quantitative method for evaluation of proposed mouse models of psoriasis, which can be strategically applied in future studies to score strengths of mouse phenotypes relative to specific aspects of human psoriasis.

Integration of Oncogenes via Sleeping Beauty as a Mouse Model of HPV16+ Oral Tumors and Immunologic Control.

PubMed

Lin, Yi-Hsin; Yang, Ming-Chieh; Tseng, Ssu-Hsueh; Jiang, Rosie; Yang, Andrew; Farmer, Emily; Peng, Shiwen; Henkle, Talia; Chang, Yung-Nien; Hung, Chien-Fu; Wu, T-C

2018-01-23

Human papillomavirus type 16 (HPV16) is the etiologic factor for cervical cancer and a subset of oropharyngeal cancers. Although several prophylactic HPV vaccines are available, no effective therapeutic strategies to control active HPV diseases exist. Tumor implantation models are traditionally used to study HPV-associated buccal tumors. However, they fail to address precancerous phases of disease progression and display tumor microenvironments distinct from those observed in patients. Previously, K14-E6/E7 transgenic mouse models have been used to generate spontaneous tumors. However, the rate of tumor formation is inconsistent, and the host often develops immune tolerance to the viral oncoproteins. We developed a preclinical, spontaneous, HPV16 + buccal tumor model using submucosal injection of oncogenic plasmids expressing HPV16-E6/E7, NRas G12V , luciferase, and sleeping beauty (SB) transposase, followed by electroporation in the buccal mucosa. We evaluated responses to immunization with a pNGVL4a-CRT/E7(detox) therapeutic HPV DNA vaccine and tumor cell migration to distant locations. Mice transfected with plasmids encoding HPV16-E6/E7, NRas G12V , luciferase, and SB transposase developed tumors within 3 weeks. We also found transient anti-CD3 administration is required to generate tumors in immunocompetent mice. Bioluminescence signals from luciferase correlated strongly with tumor growth, and tumors expressed HPV16-associated markers. We showed that pNGVL4a-CRT/E7(detox) administration resulted in antitumor immunity in tumor-bearing mice. Lastly, we demonstrated that the generated tumor could migrate to tumor-draining lymph nodes. Our model provides an efficient method to induce spontaneous HPV + tumor formation, which can be used to identify effective therapeutic interventions, analyze tumor migration, and conduct tumor biology research. Cancer Immunol Res; 6(3); 1-15. ©2018 AACR. ©2018 American Association for Cancer Research.

Epigenetic transgenerational inheritance of vinclozolin induced mouse adult onset disease and associated sperm epigenome biomarkers.

PubMed

Guerrero-Bosagna, Carlos; Covert, Trevor R; Haque, Md M; Settles, Matthew; Nilsson, Eric E; Anway, Matthew D; Skinner, Michael K

2012-12-01

The endocrine disruptor vinclozolin has previously been shown to promote epigenetic transgenerational inheritance of adult onset disease in the rat. The current study was designed to investigate the transgenerational actions of vinclozolin on the mouse. Transient exposure of the F0 generation gestating female during gonadal sex determination promoted transgenerational adult onset disease in F3 generation male and female mice, including spermatogenic cell defects, testicular abnormalities, prostate abnormalities, kidney abnormalities and polycystic ovarian disease. Pathology analysis demonstrated 75% of the vinclozolin lineage animals developed disease with 34% having two or more different disease states. Interestingly, the vinclozolin induced transgenerational disease was observed in the outbred CD-1 strain, but not the inbred 129 mouse strain. Analysis of the F3 generation sperm epigenome identified differential DNA methylation regions that can potentially be utilized as epigenetic biomarkers for transgenerational exposure and disease. Copyright © 2012 Elsevier Inc. All rights reserved.

Epigenetic Transgenerational Inheritance of Vinclozolin Induced Mouse Adult Onset Disease and Associated Sperm Epigenome Biomarkers

PubMed Central

Guerrero-Bosagna, Carlos; Covert, Trevor R.; Haque, Md. M.; Settles, Matthew; Nilsson, Eric E.; Anway, Matthew D.; Skinner, Michael K.

2012-01-01

The endocrine disruptor vinclozolin has previously been shown to promote epigenetic transgenerational inheritance of adult onset disease in the rat. The current study was designed to investigate the transgenerational actions of vinclozolin on the mouse. Transient exposure of the F0 generation gestating female during gonadal sex determination promoted transgenerational adult onset disease in F3 generation male and female mice, including spermatogenic cell defects, testicular abnormalities, prostate abnormalities, kidney abnormalities and polycystic ovarian disease. Pathology analysis demonstrated 75% of the vinclozolin lineage animals developed disease with 34% having two or more different disease states. Interestingly, the vinclozolin induced transgenerational disease was observed in the outbred CD-1 strain, but not the inbred 129 mouse strain. Analysis of the F3 generation sperm epigenome identified differential DNA methylation regions that can potentially be utilized as epigenetic biomarkers for transgenerational exposure and disease. PMID:23041264

Generation and characterization of PDGFRα-GFPCreERT2 knock-In mouse line.

PubMed

Miwa, Hiroyuki; Era, Takumi

2015-05-01

Platelet-derived growth factor (PDGF) and its receptor play an important role in embryogenesis. PDGF receptor α (PDGFRα) is expressed specifically in the embryonic day 7.5 (E7.5) mesoderm and in the E9.5 neural crest among other tissues. PDGFRα-expressing cells and their descendants are involved in the formation of various tissues. To trace PDGFRα-expressing cells in vivo, we generated a knock-in mouse line that expressed a fusion protein of green fluorescent protein (GFP), Cre recombinase (Cre), and mutated estrogen receptor ligand-binding domain (ERT2) under the control of the PDGFRα promoter. In these mice, Cre activity in PDGFRα-expressing cells could be induced by tamoxifen treatment. Taken together, our results suggest that the knock-in mouse line generated here could be useful for studying PDGFRα-expressing cells and their descendants in vivo at various stages of development. © 2015 Wiley Periodicals, Inc.

Third-generation oncolytic herpes simplex virus inhibits the growth of liver tumors in mice.

PubMed

Nakatake, Richi; Kaibori, Masaki; Nakamura, Yusuke; Tanaka, Yoshito; Matushima, Hideyuki; Okumura, Tadayoshi; Murakami, Takashi; Ino, Yasushi; Todo, Tomoki; Kon, Masanori

2018-03-01

Multimodality therapies are used to manage patients with hepatocellular carcinoma (HCC), although advanced HCC is incurable. Oncolytic virus therapy is probably the next major breakthrough in cancer treatment. The third-generation oncolytic herpes simplex virus type 1 (HSV-1) T-01 kills tumor cells without damaging the surrounding normal tissues. Here we investigated the antitumor effects of T-01 on HCC and the host's immune response to HCC cells. The cytopathic activities of T-01 were tested in 14 human and 1 murine hepatoma cell line in vitro. In various mouse xenograft models, HuH-7, KYN-2, PLC/PRF/5 and HepG2 human cells and Hepa1-6 murine cells were used to investigate the in vivo efficacy of T-01. T-01 was cytotoxic to 13 cell lines (in vitro). In mouse xenograft models of subcutaneous, orthotopic and peritoneal tumor metastasis in athymic mice (BALB/c nu/nu), the growth of tumors formed by the human HCC cell lines and hepatoblastoma cell line was inhibited by T-01 compared with that of mock-inoculated tumors. In a bilateral Hepa1-6 subcutaneous tumor model in C57BL/6 mice, the growth of tumors inoculated with T-01 was inhibited, as was the case for contralateral tumors. T-01 also significantly reduced tumor growth. T-01 infection significantly enhanced antitumor efficacy via T cell-mediated immune responses. Results demonstrate that a third-generation oncolytic HSV-1 may serve as a novel treatment for patients with HCC. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Two waves of anisotropic growth generate enlarged follicles in the spiny mouse

PubMed Central

2014-01-01

Background Mammals exhibit a remarkable variety of phenotypes and comparative studies using novel model species are needed to uncover the evolutionary developmental mechanisms generating this diversity. Here, we undertake a developmental biology and numerical modeling approach to investigate the development of skin appendages in the spiny mouse, Acomys dimidiatus. Results We demonstrate that Acomys spines, possibly involved in display and protection, are enlarged awl hairs with a concave morphology. The Acomys spines originate from enlarged placodes that are characterized by a rapid downwards growth which results in voluminous follicles. The dermal condensation (dermal papilla) at the core of the follicle is very large and exhibits a curved geometry. Given its off-centered position, the dermal papilla generates two waves of anisotropic proliferation, first of the posterior matrix, then of the anterior inner root sheath (IRS). Higher in the follicle, the posterior and anterior cortex cross-section areas substantially decrease due to cortex cell elongation and accumulation of keratin intermediate filaments. Milder keratinization in the medulla gives rise to a foamy material that eventually collapses under the combined compression of the anterior IRS and elongation of the cortex cells. Simulations, using linear elasticity theory and the finite-element method, indicate that these processes are sufficient to replicate the time evolution of the Acomys spine layers and the final shape of the emerging spine shaft. Conclusions Our analyses reveal how hair follicle morphogenesis has been altered during the evolution of the Acomys lineage, resulting in a shift from ancestral awl follicles to enlarged asymmetrical spines. This study contributes to a better understanding of the evolutionary developmental mechanisms that generated the great diversity of skin appendage phenotypes observed in mammals. PMID:25705371

Evidence of Highly Conserved β-Crystallin Disulfidome that Can be Mimicked by In Vitro Oxidation in Age-related Human Cataract and Glutathione Depleted Mouse Lens*

PubMed Central

Fan, Xingjun; Zhou, Sheng; Wang, Benlian; Hom, Grant; Guo, Minfei; Li, Binbin; Yang, Jing; Vaysburg, Dennis; Monnier, Vincent M

2015-01-01

Low glutathione levels are associated with crystallin oxidation in age-related nuclear cataract. To understand the role of cysteine residue oxidation, we used the novel approach of comparing human cataracts with glutathione-depleted LEGSKO mouse lenses for intra- versus intermolecular disulfide crosslinks using 2D-PAGE and proteomics, and then systematically identified in vivo and in vitro all disulfide forming sites using ICAT labeling method coupled with proteomics. Crystallins rich in intramolecular disulfides were abundant at young age in human and WT mouse lens but shifted to multimeric intermolecular disulfides at older age. The shift was ∼4x accelerated in LEGSKO lens. Most cysteine disulfides in β-crystallins (except βA4 in human) were highly conserved in mouse and human and could be generated by oxidation with H2O2, whereas γ-crystallin oxidation selectively affected γC23/42/79/80/154, γD42/33, and γS83/115/130 in human cataracts, and γB79/80/110, γD19/109, γF19/79, γE19, γS83/130, and γN26/128 in mouse. Analysis based on available crystal structure suggests that conformational changes are needed to expose Cys42, Cys79/80, Cys154 in γC; Cys42, Cys33 in γD, and Cys83, Cys115, and Cys130 in γS. In conclusion, the β-crystallin disulfidome is highly conserved in age-related nuclear cataract and LEGSKO mouse, and reproducible by in vitro oxidation, whereas some of the disulfide formation sites in γ-crystallins necessitate prior conformational changes. Overall, the LEGSKO mouse model is closely reminiscent of age-related nuclear cataract. PMID:26453637

EMG1 is essential for mouse pre-implantation embryo development.

PubMed

Wu, Xiaoli; Sandhu, Sumit; Patel, Nehal; Triggs-Raine, Barbara; Ding, Hao

2010-09-21

Essential for mitotic growth 1 (EMG1) is a highly conserved nucleolar protein identified in yeast to have a critical function in ribosome biogenesis. A mutation in the human EMG1 homolog causes Bowen-Conradi syndrome (BCS), a developmental disorder characterized by severe growth failure and psychomotor retardation leading to death in early childhood. To begin to understand the role of EMG1 in mammalian development, and how its deficiency could lead to Bowen-Conradi syndrome, we have used mouse as a model. The expression of Emg1 during mouse development was examined and mice carrying a null mutation for Emg1 were generated and characterized. Our studies indicated that Emg1 is broadly expressed during early mouse embryonic development. However, in late embryonic stages and during postnatal development, Emg1 exhibited specific expression patterns. To assess a developmental role for EMG1 in vivo, we exploited a mouse gene-targeting approach. Loss of EMG1 function in mice arrested embryonic development prior to the blastocyst stage. The arrested Emg1-/- embryos exhibited defects in early cell lineage-specification as well as in nucleologenesis. Further, loss of p53, which has been shown to rescue some phenotypes resulting from defects in ribosome biogenesis, failed to rescue the Emg1-/- pre-implantation lethality. Our data demonstrate that Emg1 is highly expressed during mouse embryonic development, and essential for mouse pre-implantation development. The absolute requirement for EMG1 in early embryonic development is consistent with its essential role in yeast. Further, our findings also lend support to the previous study that showed Bowen-Conradi syndrome results from a partial EMG1 deficiency. A complete deficiency would not be expected to be compatible with a live birth.

Neutral Sphingomyelinase-2 Deficiency Ameliorates Alzheimer's Disease Pathology and Improves Cognition in the 5XFAD Mouse.

PubMed

Dinkins, Michael B; Enasko, John; Hernandez, Caterina; Wang, Guanghu; Kong, Jina; Helwa, Inas; Liu, Yutao; Terry, Alvin V; Bieberich, Erhard

2016-08-17

Recent evidence implicates exosomes in the aggregation of Aβ and spreading of tau in Alzheimer's disease. In neural cells, exosome formation can be blocked by inhibition or silencing of neutral sphingomyelinase-2 (nSMase2). We generated genetically nSMase2-deficient 5XFAD mice (fro;5XFAD) to assess AD-related pathology in a mouse model with consistently reduced ceramide generation. We conducted in vitro assays to assess Aβ42 aggregation and glial clearance with and without exosomes isolated by ultracentrifugation and determined exosome-induced amyloid aggregation by particle counting. We analyzed brain exosome content, amyloid plaque formation, neuronal degeneration, sphingolipid, Aβ42 and phospho-tau levels, and memory-related behaviors in 5XFAD versus fro;5XFAD mice using contextual and cued fear conditioning. Astrocyte-derived exosomes accelerated aggregation of Aβ42 and blocked glial clearance of Aβ42 in vitro Aβ42 aggregates were colocalized with extracellular ceramide in vitro using a bifunctional ceramide analog preloaded into exosomes and in vivo using anticeramide IgG, implicating ceramide-enriched exosomes in plaque formation. Compared with 5XFAD mice, the fro;5XFAD mice had reduced brain exosomes, ceramide levels, serum anticeramide IgG, glial activation, total Aβ42 and plaque burden, tau phosphorylation, and improved cognition in a fear-conditioned learning task. Ceramide-enriched exosomes appear to exacerbate AD-related brain pathology by promoting the aggregation of Aβ. Reduction of exosome secretion by nSMase2 loss of function improves pathology and cognition in the 5XFAD mouse model. We present for the first time evidence, using Alzheimer's disease (AD) model mice deficient in neural exosome secretion due to lack of neutral sphingomyelinase-2 function, that ceramide-enriched exosomes exacerbate AD-related pathologies and cognitive deficits. Our results provide rationale to pursue a means of inhibiting exosome secretion as a potential therapy for individuals at risk for developing AD. Copyright © 2016 the authors 0270-6474/16/368653-15$15.00/0.

Knock-in reporter mice demonstrate that DNA repair by non-homologous end joining declines with age.

PubMed

Vaidya, Amita; Mao, Zhiyong; Tian, Xiao; Spencer, Brianna; Seluanov, Andrei; Gorbunova, Vera

2014-07-01

Accumulation of genome rearrangements is a characteristic of aged tissues. Since genome rearrangements result from faulty repair of DNA double strand breaks (DSBs), we hypothesized that DNA DSB repair becomes less efficient with age. The Non-Homologous End Joining (NHEJ) pathway repairs a majority of DSBs in vertebrates. To examine age-associated changes in NHEJ, we have generated an R26NHEJ mouse model in which a GFP-based NHEJ reporter cassette is knocked-in to the ROSA26 locus. In this model, NHEJ repair of DSBs generated by the site-specific endonuclease, I-SceI, reconstitutes a functional GFP gene. In this system NHEJ efficiency can be compared across tissues of the same mouse and in mice of different age. Using R26NHEJ mice, we found that NHEJ efficiency was higher in the skin, lung, and kidney fibroblasts, and lower in the heart fibroblasts and brain astrocytes. Furthermore, we observed that NHEJ efficiency declined with age. In the 24-month old animals compared to the 5-month old animals, NHEJ efficiency declined 1.8 to 3.8-fold, depending on the tissue, with the strongest decline observed in the skin fibroblasts. The sequence analysis of 300 independent NHEJ repair events showed that, regardless of age, mice utilize microhomology sequences at a significantly higher frequency than expected by chance. Furthermore, the frequency of microhomology-mediated end joining (MMEJ) events increased in the heart and lung fibroblasts of old mice, suggesting that NHEJ becomes more mutagenic with age. In summary, our study provides a versatile mouse model for the analysis of NHEJ in a wide range of tissues and demonstrates that DNA repair by NHEJ declines with age in mice, which could provide a mechanism for age-related genomic instability and increased cancer incidence with age.

Xenogeneic spermatogenesis following transplantation of hamster germ cells to mouse testes.

PubMed

Ogawa, T; Dobrinski, I; Avarbock, M R; Brinster, R L

1999-02-01

It was recently demonstrated that rat spermatogenesis can occur in the seminiferous tubules of an immunodeficient recipient mouse after transplantation of testis cells from a donor rat. In the present study, hamster donor testis cells were transplanted to mice to determine whether xenogeneic spermatogenesis would result. The hamster diverged at least 16 million years ago from the mouse and produces spermatozoa that are larger than, and have a shape distinctly different from, those of the mouse. In four separate experiments with a total of 13 recipient mice, hamster spermatogenesis was identified in the testes of each mouse. Approximately 6% of the tubules examined demonstrated xenogeneic spermatogenesis. In addition, cryopreserved hamster testis cells generated spermatogenesis in recipients. However, abnormalities were noted in hamster spermatids and acrosomes in seminiferous tubules of recipient mice. Hamster spermatozoa were also found in the epididymis of recipient animals, but these spermatozoa generally lacked acrosomes, and heads and tails were separated. Thus, defects in spermiogenesis occur in hamster spermatogenesis in the mouse, which may reflect a limited ability of endogenous mouse Sertoli cells to support fully the larger and evolutionarily distant hamster germ cell. The generation of spermatogenesis from frozen hamster cells now adds this species to the mouse and rat, in which spermatogonial stem cells also can be cryopreserved. This finding has immediate application to valuable animals of many species, because the cells could be stored until suitable recipients are identified or culture techniques devised to expand the stem cell population.

Methods for non-surgical cancer nano-theranostics of ocular tumors in the mouse eye (Conference Presentation)

NASA Astrophysics Data System (ADS)

Goswami, Mayank; Wang, Xinlei; Zhang, Pengfei; Xiao, Wenwu; Lam, Kit S.; Pugh, Edward N.; Zawadzki, Robert J.

2017-02-01

We will present our results of evaluating the feasibility of using the mouse eye as a window for non-invasive, long-term, optical investigation of xenograft models, using multimodal, cellular-resolution ocular imaging. As an "approachable part of the brain", the retina allows examination of such issues as drug delivery across the blood retinal barrier (BRB) and blood brain barrier (BBB). Our custom-built wide-field SLO/OCT provided repeatable in vivo imaging over many weeks, allowing quantitative tracking of tumor growth, the delivery of theranostic nanoparticles, and the measurement of tumor microenvironment responses. Additionally, we were able to specifically control the spatial extent of light activated photodynamic therapy (PDT) and photothermal therapy (PTT) via efficient free radical and heat generation at the tumor site, respectively.

Heterogeneous mechanics of the mouse pulmonary arterial network.

PubMed

Lee, Pilhwa; Carlson, Brian E; Chesler, Naomi; Olufsen, Mette S; Qureshi, M Umar; Smith, Nicolas P; Sochi, Taha; Beard, Daniel A

2016-10-01

Individualized modeling and simulation of blood flow mechanics find applications in both animal research and patient care. Individual animal or patient models for blood vessel mechanics are based on combining measured vascular geometry with a fluid structure model coupling formulations describing dynamics of the fluid and mechanics of the wall. For example, one-dimensional fluid flow modeling requires a constitutive law relating vessel cross-sectional deformation to pressure in the lumen. To investigate means of identifying appropriate constitutive relationships, an automated segmentation algorithm was applied to micro-computerized tomography images from a mouse lung obtained at four different static pressures to identify the static pressure-radius relationship for four generations of vessels in the pulmonary arterial network. A shape-fitting function was parameterized for each vessel in the network to characterize the nonlinear and heterogeneous nature of vessel distensibility in the pulmonary arteries. These data on morphometric and mechanical properties were used to simulate pressure and flow velocity propagation in the network using one-dimensional representations of fluid and vessel wall mechanics. Moreover, wave intensity analysis was used to study effects of wall mechanics on generation and propagation of pressure wave reflections. Simulations were conducted to investigate the role of linear versus nonlinear formulations of wall elasticity and homogeneous versus heterogeneous treatments of vessel wall properties. Accounting for heterogeneity, by parameterizing the pressure/distention equation of state individually for each vessel segment, was found to have little effect on the predicted pressure profiles and wave propagation compared to a homogeneous parameterization based on average behavior. However, substantially different results were obtained using a linear elastic thin-shell model than were obtained using a nonlinear model that has a more physiologically realistic pressure versus radius relationship.

A mouse model for Costello syndrome reveals an Ang II–mediated hypertensive condition

PubMed Central

Schuhmacher, Alberto J.; Guerra, Carmen; Sauzeau, Vincent; Cañamero, Marta; Bustelo, Xosé R.; Barbacid, Mariano

2008-01-01

Germline activation of H-RAS oncogenes is the primary cause of Costello syndrome (CS), a neuro-cardio-facio-cutaneous developmental syndrome. Here we describe the generation of a mouse model of CS by introduction of an oncogenic Gly12Val mutation in the mouse H-Ras locus using homologous recombination in ES cells. Germline expression of the endogenous H-RasG12V oncogene, even in homozygosis, resulted in hyperplasia of the mammary gland. However, development of tumors in these mice was rare. H-RasG12V mutant mice closely phenocopied some of the abnormalities observed in patients with CS, including facial dysmorphia and cardiomyopathies. These mice also displayed alterations in the homeostasis of the cardiovascular system, including development of systemic hypertension, extensive vascular remodeling, and fibrosis in both the heart and the kidneys. This phenotype was age dependent and was a consequence of the abnormal upregulation of the renin–Ang II system. Treatment with captopril, an inhibitor of Ang II biosynthesis, prevented development of the hypertension condition, vascular remodeling, and heart and kidney fibrosis. In addition, it partially alleviated the observed cardiomyopathies. These mice should help in elucidating the etiology of CS symptoms, identifying additional defects, and evaluating potential therapeutic strategies. PMID:18483625

Loss of the E3 ubiquitin ligase LRSAM1 sensitizes peripheral axons to degeneration in a mouse model of Charcot-Marie-Tooth disease.

PubMed

Bogdanik, Laurent P; Sleigh, James N; Tian, Cong; Samuels, Mark E; Bedard, Karen; Seburn, Kevin L; Burgess, Robert W

2013-05-01

Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous condition characterized by peripheral axon degeneration with subsequent motor and sensory deficits. Several CMT gene products function in endosomal sorting and trafficking to the lysosome, suggesting that defects in this cellular pathway might present a common pathogenic mechanism for these conditions. LRSAM1 is an E3 ubiquitin ligase that is implicated in this process, and mutations in LRSAM1 have recently been shown to cause CMT. We have generated mouse mutations in Lrsam1 to create an animal model of this form of CMT (CMT2P). Mouse Lrsam1 is abundantly expressed in the motor and sensory neurons of the peripheral nervous system. Both homozygous and heterozygous mice have largely normal neuromuscular performance and only a very mild neuropathy phenotype with age. However, Lrsam1 mutant mice are more sensitive to challenge with acrylamide, a neurotoxic agent that causes axon degeneration, indicating that the axons in the mutant mice are indeed compromised. In transfected cells, LRSAM1 primarily localizes in a perinuclear compartment immediately beyond the Golgi and shows little colocalization with components of the endosome to lysosome trafficking pathway, suggesting that other cellular mechanisms also merit consideration.

Experience with a mouse intranasal test for the predictive identification of respiratory sensitization potential of proteins.

PubMed

Blaikie, L; Basketter, D A

1999-08-01

The predictive identification of respiratory allergenic potential is an important primary step in the safety evaluation of (novel) proteins, such as the enzymes used in a range of consumer laundry products. In the past this has been achieved by assessing the relative ability of proteins to give rise to the formation of anaphylactic antibody in the guinea pig. Recently, an alternative model has been proposed which assesses the formation of specific IgG1 antibody in a mouse intranasal test (MINT), the assumption being that specific IgG1 antibody is a surrogate for anaphylactic antibody in the mouse. This procedure has undergone successful initial intralaboratory and interlaboratory assessment. In the present work, the MINT has been evaluated in a more thorough intralaboratory study using eight enzymes plus ovalbumin. While the data generated with a reference enzyme protein, Alcalase, showed good reproducibility, results with the remaining eight proteins led to estimates of their relative antigenic or sensitization potential several of which were at variance from those derived from the guinea pig/ human experience. In consequence, it is concluded that the MINT requires substantial further investigation before it can be adopted as a model for the assessment of the relative ability of proteins to behave as respiratory allergens.

Cell Migration in Tissues: Explant Culture and Live Imaging.

PubMed

Staneva, Ralitza; Barbazan, Jorge; Simon, Anthony; Vignjevic, Danijela Matic; Krndija, Denis

2018-01-01

Cell migration is a process that ensures correct cell localization and function in development and homeostasis. In disease such as cancer, cells acquire an upregulated migratory capacity that leads to their dissemination throughout the body. Live imaging of cell migration allows for better understanding of cell behaviors in development, adult tissue homeostasis and disease. We have optimized live imaging procedures to track cell migration in adult murine tissue explants derived from: (1) healthy gut; (2) primary intestinal carcinoma; and (3) the liver, a common metastatic site. To track epithelial cell migration in the gut, we generated an inducible fluorescent reporter mouse, enabling us to visualize and track individual cells in unperturbed gut epithelium. To image intratumoral cancer cells, we use a spontaneous intestinal cancer model based on the activation of Notch1 and deletion of p53 in the mouse intestinal epithelium, which gives rise to aggressive carcinoma. Interaction of cancer cells with a metastatic niche, the mouse liver, is addressed using a liver colonization model. In summary, we describe a method for long-term 3D imaging of tissue explants by two-photon excitation microscopy. Explant culturing and imaging can help understand dynamic behavior of cells in homeostasis and disease, and would be applicable to various tissues.

Targeting human Mas-related G protein-coupled receptor X1 to inhibit persistent pain

PubMed Central

Li, Zhe; Tseng, Pang-Yen; Tiwari, Vinod; Xu, Qian; He, Shao-Qiu; Wang, Yan; Zheng, Qin; Han, Liang; Wu, Zhiping; Blobaum, Anna L.; Cui, Yiyuan; Tiwari, Vineeta; Sun, Shuohao; Cheng, Yingying; Huang-Lionnet, Julie H. Y.; Geng, Yixun; Xiao, Bo; Peng, Junmin; Hopkins, Corey; Raja, Srinivasa N.; Guan, Yun; Dong, Xinzhong

2017-01-01

Human Mas-related G protein-coupled receptor X1 (MRGPRX1) is a promising target for pain inhibition, mainly because of its restricted expression in nociceptors within the peripheral nervous system. However, constrained by species differences across Mrgprs, drug candidates that activate MRGPRX1 do not activate rodent receptors, leaving no responsive animal model to test the effect on pain in vivo. Here, we generated a transgenic mouse line in which we replaced mouse Mrgprs with human MrgprX1. This humanized mouse allowed us to characterize an agonist [bovine adrenal medulla 8–22 (BAM8–22)] and a positive allosteric modulator (PAM), ML382, of MRGPRX1. Cellular studies suggested that ML382 enhances the ability of BAM8–22 to inhibit high-voltage-activated Ca2+ channels and attenuate spinal nociceptive transmission. Importantly, both BAM8–22 and ML382 effectively attenuated evoked, persistent, and spontaneous pain without causing obvious side effects. Notably, ML382 by itself attenuated both evoked pain hypersensitivity and spontaneous pain in MrgprX1 mice after nerve injury without acquiring coadministration of an exogenous agonist. Our findings suggest that humanized MrgprX1 mice provide a promising preclinical model and that activating MRGPRX1 is an effective way to treat persistent pain. PMID:28223516

Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2.

PubMed

Vidal, Ruben; Barbeito, Ana G; Miravalle, Leticia; Ghetti, Bernardino

2009-01-01

Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease clinically characterized by the presence of cataracts, hearing impairment, cerebellar ataxia and dementia. Neuropathologically, FDD is characterized by the presence of widespread cerebral amyloid angiopathy (CAA), parenchymal amyloid deposition and neurofibrillary tangles. FDD is caused by a 10-nucleotide duplication-insertion in the BRI(2) gene that generates a larger-than-normal precursor protein, of which the Danish amyloid subunit (ADan) comprises the last 34 amino acids. Here, we describe a transgenic mouse model for FDD (Tg-FDD) in which the mouse Prnp (prion protein) promoter drives the expression of the Danish mutant form of human BRI(2). The main neuropathological findings in Tg-FDD mice are the presence of widespread CAA and parenchymal deposition of ADan. In addition, we observe the presence of amyloid-associated gliosis, an inflammatory response and deposition of oligomeric ADan. As the animals aged, they showed abnormal grooming behavior, an arched back, and walked with a wide-based gait and shorter steps. This mouse model may give insights on the pathogenesis of FDD and will prove useful for the development of therapeutics. Moreover, the study of Tg-FDD mice may offer new insights into the role of amyloid in neurodegeneration in other disorders, including Alzheimer disease.

Raman spectroscopy detects deterioration in biomechanical properties of bone in a glucocorticoid-treated mouse model of rheumatoid arthritis

NASA Astrophysics Data System (ADS)

Maher, Jason R.; Takahata, Masahiko; Awad, Hani A.; Berger, Andrew J.

2011-08-01

Although glucocorticoids are frequently prescribed for the symptomatic management of inflammatory disorders such as rheumatoid arthritis, extended glucocorticoid exposure is the leading cause of physician-induced osteoporosis and leaves patients at a high risk of fracture. To study the biochemical effects of glucocorticoid exposure and how they might affect biomechanical properties of the bone, Raman spectra were acquired from ex vivo tibiae of glucocorticoid- and placebo-treated wild-type mice and a transgenic mouse model of rheumatoid arthritis. Statistically significant spectral differences were observed due to both treatment regimen and mouse genotype. These differences are attributed to changes in the overall bone mineral composition, as well as the degree of phosphate mineralization in tibial cortical bone. In addition, partial least squares regression was used to generate a Raman-based prediction of each tibia's biomechanical strength as quantified by a torsion test. The Raman-based predictions were as accurate as those produced by microcomputed tomography derived parameters, and more accurate than the clinically-used parameter of bone mineral density. These results suggest that Raman spectroscopy could be a valuable tool for monitoring bone biochemistry in studies of bone diseases such as osteoporosis, including tests of drugs being developed to combat these diseases.

Applications and Limitations of Mouse Models for Understanding Human Atherosclerosis

PubMed Central

von Scheidt, Moritz; Zhao, Yuqi; Kurt, Zeyneb; Pan, Calvin; Zeng, Lingyao; Yang, Xia; Schunkert, Heribert; Lusis, Aldons J.

2017-01-01

Most of the biological understanding of mechanisms underlying coronary artery disease (CAD) derives from studies of mouse models. The identification of multiple CAD loci and strong candidate genes in large human genome-wide association studies (GWAS) presented an opportunity to examine the relevance of mouse models for the human disease. We comprehensively reviewed the mouse literature, including 827 literature-derived genes, and compared it to human data. First, we observed striking concordance of risk factors for atherosclerosis in mice and humans. Second, there was highly significant overlap of mouse genes with human genes identified by GWAS. In particular, of the 46 genes with strong association signals in CAD-GWAS that were studied in mouse models all but one exhibited consistent effects on atherosclerosis-related phenotypes. Third, we compared 178 CAD-associated pathways derived from human GWAS with 263 from mouse studies and observed that over 50% were consistent between both species. PMID:27916529

Insights into mammalian biology from the wild house mouse Mus musculus

PubMed Central

Phifer-Rixey, Megan; Nachman, Michael W

2015-01-01

The house mouse, Mus musculus, was established in the early 1900s as one of the first genetic model organisms owing to its short generation time, comparatively large litters, ease of husbandry, and visible phenotypic variants. For these reasons and because they are mammals, house mice are well suited to serve as models for human phenotypes and disease. House mice in the wild consist of at least three distinct subspecies and harbor extensive genetic and phenotypic variation both within and between these subspecies. Wild mice have been used to study a wide range of biological processes, including immunity, cancer, male sterility, adaptive evolution, and non-Mendelian inheritance. Despite the extensive variation that exists among wild mice, classical laboratory strains are derived from a limited set of founders and thus contain only a small subset of this variation. Continued efforts to study wild house mice and to create new inbred strains from wild populations have the potential to strengthen house mice as a model system. DOI: http://dx.doi.org/10.7554/eLife.05959.001 PMID:25875302

Molecular dynamics in drug design: new generations of compstatin analogs.

PubMed

Tamamis, Phanourios; López de Victoria, Aliana; Gorham, Ronald D; Bellows-Peterson, Meghan L; Pierou, Panayiota; Floudas, Christodoulos A; Morikis, Dimitrios; Archontis, Georgios

2012-05-01

We report the computational and rational design of new generations of potential peptide-based inhibitors of the complement protein C3 from the compstatin family. The binding efficacy of the peptides is tested by extensive molecular dynamics-based structural and physicochemical analysis, using 32 atomic detail trajectories in explicit water for 22 peptides bound to human, rat or mouse target protein C3, with a total of 257 ns. The criteria for the new design are: (i) optimization for C3 affinity and for the balance between hydrophobicity and polarity to improve solubility compared to known compstatin analogs; and (ii) development of dual specificity, human-rat/mouse C3 inhibitors, which could be used in animal disease models. Three of the new analogs are analyzed in more detail as they possess strong and novel binding characteristics and are promising candidates for further optimization. This work paves the way for the development of an improved therapeutic for age-related macular degeneration, and other complement system-mediated diseases, compared to known compstatin variants. © 2012 John Wiley & Sons A/S.

Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres

PubMed Central

Remeseiro, Silvia; Cuadrado, Ana; Carretero, María; Martínez, Paula; Drosopoulos, William C; Cañamero, Marta; Schildkraut, Carl L; Blasco, María A; Losada, Ana

2012-01-01

Cohesin is a protein complex originally identified for its role in sister chromatid cohesion, although increasing evidence portrays it also as a major organizer of interphase chromatin. Vertebrate cohesin consists of Smc1, Smc3, Rad21/Scc1 and either stromal antigen 1 (SA1) or SA2. To explore the functional specificity of these two versions of cohesin and their relevance for embryonic development and cancer, we generated a mouse model deficient for SA1. Complete ablation of SA1 results in embryonic lethality, while heterozygous animals have shorter lifespan and earlier onset of tumourigenesis. SA1-null mouse embryonic fibroblasts show decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromeric cohesion, which depends on cohesin-SA2. Instead, they arise from defective telomere replication, which requires cohesion mediated specifically by cohesin-SA1. We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis. PMID:22415365

HSV-1 interaction to 3-O-sulfated heparan sulfate in mouse-derived DRG explant and profiles of inflammatory markers during virus infection.

PubMed

Sharthiya, Harsh; Seng, Chanmoly; Van Kuppevelt, T H; Tiwari, Vaibhav; Fornaro, Michele

2017-06-01

The molecular mechanism of herpes simplex virus (HSV) entry and the associated inflammatory response in the nervous system remain poorly understood. Using mouse-derived ex vivo dorsal root ganglia (DRG) explant model and single cell neurons (SCNs), in this study, we provided a visual evidence for the expression of heparan sulfate (HS) and 3-O-sulfated heparan sulfate (3-OS HS) followed by their interactions with HSV-1 glycoprotein B (gB) and glycoprotein D (gD) during cell entry. Upon heparanase treatment of DRG-derived SCN, a significant inhibition of HSV-1 entry was observed suggesting the involvement of HS role during viral entry. Finally, a cytokine array profile generated during HSV-1 infection in DRG explant indicated an enhanced expression of chemokines (LIX, TIMP-2, and M-CSF)-known regulators of HS. Taken together, these results highlight the significance of HS during HSV-1 entry in DRG explant. Further investigation is needed to understand which isoforms of 3-O-sulfotransferase (3-OST)-generated HS contributed during HSV-1 infection and associated cell damage.

Development of pacemaker properties and rhythmogenic mechanisms in the mouse embryonic respiratory network

PubMed Central

Chevalier, Marc; Toporikova, Natalia; Simmers, John; Thoby-Brisson, Muriel

2016-01-01

Breathing is a vital rhythmic behavior generated by hindbrain neuronal circuitry, including the preBötzinger complex network (preBötC) that controls inspiration. The emergence of preBötC network activity during prenatal development has been described, but little is known regarding inspiratory neurons expressing pacemaker properties at embryonic stages. Here, we combined calcium imaging and electrophysiological recordings in mouse embryo brainstem slices together with computational modeling to reveal the existence of heterogeneous pacemaker oscillatory properties relying on distinct combinations of burst-generating INaP and ICAN conductances. The respective proportion of the different inspiratory pacemaker subtypes changes during prenatal development. Concomitantly, network rhythmogenesis switches from a purely INaP/ICAN-dependent mechanism at E16.5 to a combined pacemaker/network-driven process at E18.5. Our results provide the first description of pacemaker bursting properties in embryonic preBötC neurons and indicate that network rhythmogenesis undergoes important changes during prenatal development through alterations in both circuit properties and the biophysical characteristics of pacemaker neurons. DOI: http://dx.doi.org/10.7554/eLife.16125.001 PMID:27434668

Effective delivery of large genes to the retina by dual AAV vectors

PubMed Central

Trapani, Ivana; Colella, Pasqualina; Sommella, Andrea; Iodice, Carolina; Cesi, Giulia; de Simone, Sonia; Marrocco, Elena; Rossi, Settimio; Giunti, Massimo; Palfi, Arpad; Farrar, Gwyneth J; Polishchuk, Roman; Auricchio, Alberto

2014-01-01

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes. PMID:24150896

Constitutive Cyclin O deficiency results in penetrant hydrocephalus, impaired growth and infertility

PubMed Central

Núnez-Ollé, Marc; Jung, Carole; Terré, Berta; Balsiger, Norman A.; Plata, Cristina; Roset, Ramon; Pardo-Pastor, Carlos; Garrido, Marta; Rojas, Santiago; Alameda, Francesc; Lloreta, Josep; Martín-Caballero, Juan; Flores, Juana M.; Stracker, Travis H.; Valverde, Miguel A.; Muñoz, Francisco J.; Gil-Gómez, Gabriel

2017-01-01

Cyclin O (encoded by CCNO) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo, we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno-/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno+/- mice also developed hydrocephalus and affected Ccno-/- and Ccno+/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno-/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients. PMID:29245899

Constitutive Cyclin O deficiency results in penetrant hydrocephalus, impaired growth and infertility.

PubMed

Núnez-Ollé, Marc; Jung, Carole; Terré, Berta; Balsiger, Norman A; Plata, Cristina; Roset, Ramon; Pardo-Pastor, Carlos; Garrido, Marta; Rojas, Santiago; Alameda, Francesc; Lloreta, Josep; Martín-Caballero, Juan; Flores, Juana M; Stracker, Travis H; Valverde, Miguel A; Muñoz, Francisco J; Gil-Gómez, Gabriel

2017-11-21

Cyclin O (encoded by CCNO ) is a member of the cyclin family with regulatory functions in ciliogenesis and apoptosis. Homozygous CCNO mutations have been identified in human patients with Reduced Generation of Multiple Motile Cilia (RGMC) and conditional inactivation of Ccno in the mouse recapitulates some of the pathologies associated with the human disease. These include defects in the development of motile cilia and hydrocephalus. To further investigate the functions of Ccno in vivo , we have generated a new mouse model characterized by the constitutive loss of Ccno in all tissues and followed a cohort during ageing. Ccno -/- mice were growth impaired and developed hydrocephalus with high penetrance. In addition, some Ccno +/- mice also developed hydrocephalus and affected Ccno -/- and Ccno +/- mice exhibited additional CNS defects including cortical thinning and hippocampal abnormalities. In addition to the CNS defects, both male and female Ccno -/- mice were infertile and female mice exhibited few motile cilia in the oviduct. Our results further establish CCNO as an important gene for normal development and suggest that heterozygous CCNO mutations could underlie hydrocephalus or diminished fertility in some human patients.

Gangliosides and Ceramides Change in a Mouse Model of Blast Induced Traumatic Brain Injury

PubMed Central

2013-01-01

Explosive detonations generate atmospheric pressure changes that produce nonpenetrating blast induced “mild” traumatic brain injury (bTBI). The structural basis for mild bTBI has been extremely controversial. The present study applies matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging to track the distribution of gangliosides in mouse brain tissue that were exposed to very low level of explosive detonations (2.5–5.5 psi peak overpressure). We observed major increases of the ganglioside GM2 in the hippocampus, thalamus, and hypothalamus after a single blast exposure. Moreover, these changes were accompanied by depletion of ceramides. No neurological or brain structural signs of injury could be inferred using standard light microscopic techniques. The first source of variability is generated by the Latency between blast and tissue sampling (peak intensity of the blast wave). These findings suggest that subtle molecular changes in intracellular membranes and plasmalemma compartments may be biomarkers for biological responses to mild bTBI. This is also the first report of a GM2 increase in the brains of mature mice from a nongenetic etiology. PMID:23590251

Genetically engineered mouse models for studying inflammatory bowel disease.

PubMed

Mizoguchi, Atsushi; Takeuchi, Takahito; Himuro, Hidetomo; Okada, Toshiyuki; Mizoguchi, Emiko

2016-01-01

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory condition that is mediated by very complex mechanisms controlled by genetic, immune, and environmental factors. More than 74 kinds of genetically engineered mouse strains have been established since 1993 for studying IBD. Although mouse models cannot fully reflect human IBD, they have provided significant contributions for not only understanding the mechanism, but also developing new therapeutic means for IBD. Indeed, 20 kinds of genetically engineered mouse models carry the susceptibility genes identified in human IBD, and the functions of some other IBD susceptibility genes have also been dissected out using mouse models. Cutting-edge technologies such as cell-specific and inducible knockout systems, which were recently employed to mouse IBD models, have further enhanced the ability of investigators to provide important and unexpected rationales for developing new therapeutic strategies for IBD. In this review article, we briefly introduce 74 kinds of genetically engineered mouse models that spontaneously develop intestinal inflammation. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mouse Tumor Biology (MTB): a database of mouse models for human cancer.

PubMed

Bult, Carol J; Krupke, Debra M; Begley, Dale A; Richardson, Joel E; Neuhauser, Steven B; Sundberg, John P; Eppig, Janan T

2015-01-01

The Mouse Tumor Biology (MTB; http://tumor.informatics.jax.org) database is a unique online compendium of mouse models for human cancer. MTB provides online access to expertly curated information on diverse mouse models for human cancer and interfaces for searching and visualizing data associated with these models. The information in MTB is designed to facilitate the selection of strains for cancer research and is a platform for mining data on tumor development and patterns of metastases. MTB curators acquire data through manual curation of peer-reviewed scientific literature and from direct submissions by researchers. Data in MTB are also obtained from other bioinformatics resources including PathBase, the Gene Expression Omnibus and ArrayExpress. Recent enhancements to MTB improve the association between mouse models and human genes commonly mutated in a variety of cancers as identified in large-scale cancer genomics studies, provide new interfaces for exploring regions of the mouse genome associated with cancer phenotypes and incorporate data and information related to Patient-Derived Xenograft models of human cancers. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Yong, E-mail: yongzhao@uic.edu; Guo, Chengshan; Hwang, David

2010-09-03

Research highlights: {yields} Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2r{gamma}{sup null} mice. {yields} Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. {yields} The islet {beta} cells were selectively destroyed by infiltrated human T cells. {yields} The model can facilitate translational research to find a cure for type 1 diabetes. -- Abstract: Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing {beta} cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model inmore » NOD-scid IL2r{gamma}{sup null} mice. The selective destruction of pancreatic islet {beta} cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total {beta}-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the {beta} cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet {beta} cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4{sup +} T cell infiltration and clonal expansion, and the mouse islet {beta}-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet {beta} cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.« less

Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

PubMed

Ikeda, Koki; Koga, Tomoaki; Sasaki, Fumiyuki; Ueno, Ayumi; Saeki, Kazuko; Okuno, Toshiaki; Yokomizo, Takehiko

2017-05-13

DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

The latest animal models of ovarian cancer for novel drug discovery.

PubMed

Magnotti, Elizabeth; Marasco, Wayne A

2018-03-01

Epithelial ovarian cancer is a heterogeneous disease classified into five subtypes, each with a different molecular profile. Most cases of ovarian cancer are diagnosed after metastasis of the primary tumor and are resistant to traditional platinum-based chemotherapeutics. Mouse models of ovarian cancer have been utilized to discern ovarian cancer tumorigenesis and the tumor's response to therapeutics. Areas covered: The authors provide a review of mouse models currently employed to understand ovarian cancer. This article focuses on advances in the development of orthotopic and patient-derived tumor xenograft (PDX) mouse models of ovarian cancer and discusses current humanized mouse models of ovarian cancer. Expert opinion: The authors suggest that humanized mouse models of ovarian cancer will provide new insight into the role of the human immune system in combating and augmenting ovarian cancer and aid in the development of novel therapeutics. Development of humanized mouse models will take advantage of the NSG and NSG-SGM3 strains of mice as well as new strains that are actively being derived.

Integrating model behavior, optimization, and sensitivity/uncertainty analysis: overview and application of the MOUSE software toolbox

USDA-ARS?s Scientific Manuscript database

This paper provides an overview of the Model Optimization, Uncertainty, and SEnsitivity Analysis (MOUSE) software application, an open-source, Java-based toolbox of visual and numerical analysis components for the evaluation of environmental models. MOUSE is based on the OPTAS model calibration syst...

Nuclear receptor TLX stimulates hippocampal neurogenesis and enhances learning and memory in a transgenic mouse model

PubMed Central

Murai, Kiyohito; Qu, Qiuhao; Sun, GuoQiang; Ye, Peng; Li, Wendong; Asuelime, Grace; Sun, Emily; Tsai, Guochuan E.; Shi, Yanhong

2014-01-01

The role of the nuclear receptor TLX in hippocampal neurogenesis and cognition has just begun to be explored. In this study, we generated a transgenic mouse model that expresses TLX under the control of the promoter of nestin, a neural precursor marker. Transgenic TLX expression led to mice with enlarged brains with an elongated hippocampal dentate gyrus and increased numbers of newborn neurons. Specific expression of TLX in adult hippocampal dentate gyrus via lentiviral transduction increased the numbers of BrdU+ cells and BrdU+NeuN+ neurons. Furthermore, the neural precursor-specific expression of the TLX transgene substantially rescued the neurogenic defects of TLX-null mice. Consistent with increased neurogenesis in the hippocampus, the TLX transgenic mice exhibited enhanced cognition with increased learning and memory. These results suggest a strong association between hippocampal neurogenesis and cognition, as well as significant contributions of TLX to hippocampal neurogenesis, learning, and memory. PMID:24927526

Transcriptional Mechanisms of Proneural Factors and REST in Regulating Neuronal Reprogramming of Astrocytes

PubMed Central

Masserdotti, Giacomo; Gillotin, Sébastien; Sutor, Bernd; Drechsel, Daniela; Irmler, Martin; Jørgensen, Helle F.; Sass, Steffen; Theis, Fabian J.; Beckers, Johannes; Berninger, Benedikt; Guillemot, François; Götz, Magdalena

2015-01-01

Summary Direct lineage reprogramming induces dramatic shifts in cellular identity, employing poorly understood mechanisms. Recently, we demonstrated that expression of Neurog2 or Ascl1 in postnatal mouse astrocytes generates glutamatergic or GABAergic neurons. Here, we take advantage of this model to study dynamics of neuronal cell fate acquisition at the transcriptional level. We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes. Within this subset, only NeuroD4 could by itself induce neuronal reprogramming in both mouse and human astrocytes, while co-expression with Insm1 was required for glutamatergic maturation. Cultured astrocytes gradually became refractory to reprogramming, in part by the repressor REST preventing Neurog2 from binding to the NeuroD4 promoter. Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4. Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming. PMID:26119235

Centella asiatica Extract Improves Behavioral Deficits in a Mouse Model of Alzheimer's Disease: Investigation of a Possible Mechanism of Action

PubMed Central

Soumyanath, Amala; Zhong, Yong-Ping; Henson, Edward; Wadsworth, Teri; Bishop, James; Gold, Bruce G.; Quinn, Joseph F.

2012-01-01

Centella asiatica (CA), commonly named gotu kola, is an Ayurvedic herb used to enhance memory and nerve function. To investigate the potential use of CA in Alzheimer's disease (AD), we examined the effects of a water extract of CA (GKW) in the Tg2576 mouse, a murine model of AD with high β-amyloid burden. Orally administered GKW attenuated β-amyloid-associated behavioral abnormalities in these mice. In vitro, GKW protected SH-SY5Y cells and MC65 human neuroblastoma cells from toxicity induced by exogenously added and endogenously generated β-amyloid, respectively. GKW prevented intracellular β-amyloid aggregate formation in MC65 cells. GKW did not show anticholinesterase activity or protect neurons from oxidative damage and glutamate toxicity, mechanisms of current AD therapies. GKW is rich in phenolic compounds and does not contain asiatic acid, a known CA neuroprotective triterpene. CA thus offers a unique therapeutic mechanism and novel active compounds of potential relevance to the treatment of AD. PMID:22506133

Centella asiatica Extract Improves Behavioral Deficits in a Mouse Model of Alzheimer's Disease: Investigation of a Possible Mechanism of Action.

PubMed

Soumyanath, Amala; Zhong, Yong-Ping; Henson, Edward; Wadsworth, Teri; Bishop, James; Gold, Bruce G; Quinn, Joseph F

2012-01-01

Centella asiatica (CA), commonly named gotu kola, is an Ayurvedic herb used to enhance memory and nerve function. To investigate the potential use of CA in Alzheimer's disease (AD), we examined the effects of a water extract of CA (GKW) in the Tg2576 mouse, a murine model of AD with high β-amyloid burden. Orally administered GKW attenuated β-amyloid-associated behavioral abnormalities in these mice. In vitro, GKW protected SH-SY5Y cells and MC65 human neuroblastoma cells from toxicity induced by exogenously added and endogenously generated β-amyloid, respectively. GKW prevented intracellular β-amyloid aggregate formation in MC65 cells. GKW did not show anticholinesterase activity or protect neurons from oxidative damage and glutamate toxicity, mechanisms of current AD therapies. GKW is rich in phenolic compounds and does not contain asiatic acid, a known CA neuroprotective triterpene. CA thus offers a unique therapeutic mechanism and novel active compounds of potential relevance to the treatment of AD.

Monitoring the dynamics of clonal tumour evolution in vivo using secreted luciferases.

PubMed

Charles, Joël P; Fuchs, Jeannette; Hefter, Mirjam; Vischedyk, Jonas B; Kleint, Maximilian; Vogiatzi, Fotini; Schäfer, Jonas A; Nist, Andrea; Timofeev, Oleg; Wanzel, Michael; Stiewe, Thorsten

2014-06-03

Tumours are heterogeneous cell populations that undergo clonal evolution during tumour progression, metastasis and response to therapy. Short hairpin RNAs (shRNAs) generate stable loss-of-function phenotypes and are versatile experimental tools to explore the contribution of individual genetic alterations to clonal evolution. In these experiments tumour cells carrying shRNAs are commonly tracked with fluorescent reporters. While this works well for cell culture studies and leukaemia mouse models, fluorescent reporters are poorly suited for animals with solid tumours--the most common tumour types in cancer patients. Here we develop a toolkit that uses secreted luciferases to track the fate of two different shRNA-expressing tumour cell clones competitively, both in vitro and in vivo. We demonstrate that secreted luciferase activities can be measured robustly in the blood stream of tumour-bearing mice to accurately quantify, in a minimally invasive manner, the dynamic evolution of two genetically distinct tumour subclones in preclinical mouse models of tumour development, metastasis and therapy.

In Vivo and In Vitro Characterization of a Plasmodium Liver Stage-Specific Promoter

PubMed Central

Horstmann, Sebastian; Annoura, Takeshi; del Portillo, Hernando A.; Khan, Shahid M.; Heussler, Volker T.

2015-01-01

Little is known about stage-specific gene regulation in Plasmodium parasites, in particular the liver stage of development. We have previously described in the Plasmodium berghei rodent model, a liver stage-specific (lisp2) gene promoter region, in vitro. Using a dual luminescence system, we now confirm the stage specificity of this promoter region also in vivo. Furthermore, by substitution and deletion analyses we have extended our in vitro characterization of important elements within the promoter region. Importantly, the dual luminescence system allows analyzing promoter constructs avoiding mouse-consuming cloning procedures of transgenic parasites. This makes extensive mutation and deletion studies a reasonable approach also in the malaria mouse model. Stage-specific expression constructs and parasite lines are extremely valuable tools for research on Plasmodium liver stage biology. Such reporter lines offer a promising opportunity for assessment of liver stage drugs, characterization of genetically attenuated parasites and liver stage-specific vaccines both in vivo and in vitro, and may be key for the generation of inducible systems. PMID:25874388

A live attenuated strain of Yersinia pestis ΔyscB provides protection against bubonic and pneumonic plagues in mouse model.

PubMed

Zhang, Xuecan; Qi, Zhizhen; Du, Zongmin; Bi, Yujing; Zhang, Qingwen; Tan, Yafang; Yang, Huiying; Xin, Youquan; Yang, Ruifu; Wang, Xiaoyi

2013-05-24

To develop a safe and effective live plague vaccine, the ΔyscB mutant was constructed based on Yersinia pestis biovar Microtus strain 201 that is avirulent to humans, but virulent to mice. The virulence, immunogenicity and protective efficacy of the ΔyscB mutant were evaluated in this study. The results showed that the ΔyscB mutant was severely attenuated, elicited a higher F1-specific antibody titer and provided protective efficacy against bubonic and pneumonic plague in mouse model. The ΔyscB mutant could induce the secretion of both Th1-associated cytokines (IFN-γ, IL-2 and TNF-α) and Th2-associated cytokines (IL-4 and IL-10). Taken together, the ΔyscB mutant represented a potential vaccine candidate based on its ability to generate strong humoral and cell-mediated immune responses and to provide good protection against both subcutaneous and intranasal Y. pestis challenge. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development of a functional thyroid model based on an organoid culture system.

PubMed

Saito, Yoshiyuki; Onishi, Nobuyuki; Takami, Hiroshi; Seishima, Ryo; Inoue, Hiroyoshi; Hirata, Yuki; Kameyama, Kaori; Tsuchihashi, Kenji; Sugihara, Eiji; Uchino, Shinya; Ito, Koichi; Kawakubo, Hirofumi; Takeuchi, Hiroya; Kitagawa, Yuko; Saya, Hideyuki; Nagano, Osamu

2018-03-04

The low turnover rate of thyroid follicular cells and the lack of a long-term thyroid cell culture system have hampered studies of thyroid carcinogenesis. We have now established a thyroid organoid culture system that supports thyroid cell proliferation in vitro. The established mouse thyroid organoids performed thyroid functions including thyroglobulin synthesis, iodide uptake, and the production and release of thyroid hormone. Furthermore, transplantation of the organoids into recipient mice resulted in the formation of normal thyroid-like tissue capable of iodide uptake and thyroglobulin production in vivo. Finally, forced expression of oncogenic NRAS (NRAS Q61R ) in thyroid organoids established from p53 knockout mice and transplantation of the manipulated organoids into mouse recipients generated a model of poorly differentiated thyroid cancer. Our findings suggest that this newly developed thyroid organoid culture system is a potential research tool for the study of thyroid physiology and pathology including thyroid cancer. Copyright © 2018 Elsevier Inc. All rights reserved.

Atm reactivation reverses ataxia telangiectasia phenotypes in vivo.

PubMed

Di Siena, Sara; Campolo, Federica; Gimmelli, Roberto; Di Pietro, Chiara; Marazziti, Daniela; Dolci, Susanna; Lenzi, Andrea; Nussenzweig, Andre; Pellegrini, Manuela

2018-02-22

Hereditary deficiencies in DNA damage signaling are invariably associated with cancer predisposition, immunodeficiency, radiation sensitivity, gonadal abnormalities, premature aging, and tissue degeneration. ATM kinase has been established as a central player in DNA double-strand break repair and its deficiency causes ataxia telangiectasia, a rare, multi-system disease with no cure. So ATM represents a highly attractive target for the development of novel types of gene therapy or transplantation strategies. Atm tamoxifen-inducible mouse models were generated to explore whether Atm reconstitution is able to restore Atm function in an Atm-deficient background. Body weight, immunodeficiency, spermatogenesis, and radioresistance were recovered in transgenic mice within 1 month from Atm induction. Notably, life span was doubled after Atm restoration, mice were protected from thymoma and no cerebellar defects were observed. Atm signaling was functional after DNA damage in vivo and in vitro. In summary, we propose a new Atm mouse model to investigate novel therapeutic strategies for ATM activation in ataxia telangiectasia disease.

Monitoring the dynamics of clonal tumour evolution in vivo using secreted luciferases

PubMed Central

Charles, Joël P.; Fuchs, Jeannette; Hefter, Mirjam; Vischedyk, Jonas B.; Kleint, Maximilian; Vogiatzi, Fotini; Schäfer, Jonas A.; Nist, Andrea; Timofeev, Oleg; Wanzel, Michael; Stiewe, Thorsten

2014-01-01

Tumours are heterogeneous cell populations that undergo clonal evolution during tumour progression, metastasis and response to therapy. Short hairpin RNAs (shRNAs) generate stable loss-of-function phenotypes and are versatile experimental tools to explore the contribution of individual genetic alterations to clonal evolution. In these experiments tumour cells carrying shRNAs are commonly tracked with fluorescent reporters. While this works well for cell culture studies and leukaemia mouse models, fluorescent reporters are poorly suited for animals with solid tumours—the most common tumour types in cancer patients. Here we develop a toolkit that uses secreted luciferases to track the fate of two different shRNA-expressing tumour cell clones competitively, both in vitro and in vivo. We demonstrate that secreted luciferase activities can be measured robustly in the blood stream of tumour-bearing mice to accurately quantify, in a minimally invasive manner, the dynamic evolution of two genetically distinct tumour subclones in preclinical mouse models of tumour development, metastasis and therapy. PMID:24889111

coq7/clk-1 regulates mitochondrial respiration and the generation of reactive oxygen species via coenzyme Q.

PubMed

Nakai, Daisuke; Shimizu, Takahiko; Nojiri, Hidetoshi; Uchiyama, Satoshi; Koike, Hideo; Takahashi, Mayumi; Hirokawa, Katsuiku; Shirasawa, Takuji

2004-10-01

coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, and shows sluggish behaviours and an extended lifespan. In C. elegans and Saccharomyces cerevisiae, coq7/clk-1 is required for the biosynthesis of coenzyme Q (CoQ), an essential co-factor in mitochondrial respiration. The clk-1 mutant contains dietary CoQ(8) from Escherichia coli and demethoxyubiquinone 9 (DMQ9) instead of CoQ(9). In a previous study, we generated COQ7-deficient mice by targeted disruption of the coq7 gene and reported that mouse coq7/clk-1 is also essential for CoQ synthesis, maintenance of mitochondrial integrity and neurogenesis. In the present study, we rescued COQ7-deficient mice from embryonic lethality and established a mouse model with decreased CoQ level by transgene expression of COQ7/CLK-1. A biochemical analysis showed a concomitant decrease in CoQ(9), mitochondrial respiratory enzyme activity and the generation of reactive oxygen species (ROS) in the mitochondria of CoQ-insufficient mice. This implied that the depressed activity of respiratory enzymes and the depressed production of ROS may play a physiological role in the control of lifespan in mammalian species and of C. elegans.

Cell Fusion Reprogramming Leads to a Specific Hepatic Expression Pattern during Mouse Bone Marrow Derived Hepatocyte Formation In Vivo

PubMed Central

Arza, Elvira; Alvarez-Barrientos, Alberto; Fabregat, Isabel; Garcia-Bravo, Maria; Meza, Nestor W.; Segovia, Jose C.

2012-01-01

The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation. PMID:22457803

Eggshell-inspired biomineralization generates vaccines that do not require refrigeration.

PubMed

Wang, Guangchuan; Li, Xiaofeng; Mo, Lijuan; Song, Zhiyong; Chen, Wei; Deng, Yongqiang; Zhao, Hui; Qin, Ede; Qin, Chengfeng; Tang, Ruikang

2012-10-15

We're not gonna bake it: In situ biomineralization creates an egg-like shell on vaccine particles to improve their thermostability. Different from the bare vaccine (squares), the biomineralized vaccine (red circles) can be stored at ambient temperature without refrigeration for up to a week and retain biological activity both in vitro (see graph), as well as in a mouse model. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genome-Wide Expression Profiling of Five Mouse Models Identifies Similarities and Differences with Human Psoriasis

PubMed Central

Swindell, William R.; Johnston, Andrew; Carbajal, Steve; Han, Gangwen; Wohn, Christian; Lu, Jun; Xing, Xianying; Nair, Rajan P.; Voorhees, John J.; Elder, James T.; Wang, Xiao-Jing; Sano, Shigetoshi; Prens, Errol P.; DiGiovanni, John; Pittelkow, Mark R.; Ward, Nicole L.; Gudjonsson, Johann E.

2011-01-01

Development of a suitable mouse model would facilitate the investigation of pathomechanisms underlying human psoriasis and would also assist in development of therapeutic treatments. However, while many psoriasis mouse models have been proposed, no single model recapitulates all features of the human disease, and standardized validation criteria for psoriasis mouse models have not been widely applied. In this study, whole-genome transcriptional profiling is used to compare gene expression patterns manifested by human psoriatic skin lesions with those that occur in five psoriasis mouse models (K5-Tie2, imiquimod, K14-AREG, K5-Stat3C and K5-TGFbeta1). While the cutaneous gene expression profiles associated with each mouse phenotype exhibited statistically significant similarity to the expression profile of psoriasis in humans, each model displayed distinctive sets of similarities and differences in comparison to human psoriasis. For all five models, correspondence to the human disease was strong with respect to genes involved in epidermal development and keratinization. Immune and inflammation-associated gene expression, in contrast, was more variable between models as compared to the human disease. These findings support the value of all five models as research tools, each with identifiable areas of convergence to and divergence from the human disease. Additionally, the approach used in this paper provides an objective and quantitative method for evaluation of proposed mouse models of psoriasis, which can be strategically applied in future studies to score strengths of mouse phenotypes relative to specific aspects of human psoriasis. PMID:21483750

β-Cell-specific pyruvate dehydrogenase deficiency impairs glucose-stimulated insulin secretion

PubMed Central

Srinivasan, Malathi; Choi, Cheol S.; Ghoshal, Pushpankur; Pliss, Lioudmila; Pandya, Jignesh D.; Hill, David; Cline, Gary

2010-01-01

Glucose-stimulated insulin secretion (GSIS) by β-cells requires the generation of ATP from oxidation of pyruvate as well as generation of coupling factors involving three different pyruvate cycling shuttles. The roles of several key enzymes involved in pyruvate cycling in β-cells have been documented using isolated islets and β-cell clonal lines. To investigate the role of the pyruvate dehydrogenase (PDH) complex (PDC) in GSIS, a murine model of β-cell-specific PDH deficiency (β-PDHKO) was created. Pancreatic insulin content was decreased in 1-day-old β-PDHKO male pups and adult male mice. The plasma insulin levels were decreased and blood glucose levels increased in β-PDHKO male mice from neonatal life onward. GSIS was reduced in isolated islets from β-PDHKO male mice with about 50% reduction in PDC activity. Impairment in a glucose tolerance test and in vivo insulin secretion during hyperglycemic clamp was evident in β-PDHKO adults. No change in the number or size of islets was found in pancreata from 4-wk-old β-PDHKO male mice. However, an increase in the mean size of individual β-cells in islets of these mice was observed. These findings show a key role of PDC in GSIS by pyruvate oxidation. This β-PDHKO mouse model represents the first mouse model in which a mitochondrial oxidative enzyme deletion by gene knockout has been employed to demonstrate an altered GSIS by β-cells. PMID:20841503

Graft function assessment in mouse models of single- and dual- kidney transplantation.

PubMed

Wang, Lei; Wang, Ximing; Jiang, Shan; Wei, Jin; Buggs, Jacentha; Fu, Liying; Zhang, Jie; Liu, Ruisheng

2018-05-23

Animal models of kidney transplantation (KTX) are widely used in studying immune response of hosts to implanted grafts. Additionally, KTX can be used in generating kidney-specific knockout animal models by transplantation of kidneys from donors with global knockout of a gene to wild type recipients or vise verse. Dual kidney transplantation (DKT) provides a more physiological environment for recipients than single kidney transplantation (SKT). However, DKT in mice is rare due to technical challenges. In this study, we successfully performed DKT in mice and compared the hemodynamic response and graft function with SKT. The surgical time, complications and survival rate of DKT were not significantly different from SKT, where survival rates were above 85%. Mice with DKT showed less injury and quicker recovery with lower plasma creatinine (Pcr) and higher GFR than SKT mice (Pcr = 0.34 and 0.17 mg/dl in DKT vs. 0.50 and 0.36 mg/dl in SKT at 1 and 3 days, respectively; GFR = 215 and 131 µl/min for DKT and SKT, respectively). In addition, the DKT exhibited better renal functional reserve and long-term outcome of renal graft function than SKT based on the response to acute volume expansion. In conclusion, we have successfully generated a mouse DKT model. The hemodynamic responses of DKT better mimic physiological situations with less kidney injury and better recovery than SKT because of reduced confounding factors such as single nephron hyperfiltration. We anticipate DKT in mice will provide an additional tool for evaluation of renal significance in physiology and disease.

Second-harmonic generation and fluorescence lifetime imaging microscopy through a rodent mammary imaging window

NASA Astrophysics Data System (ADS)

Young, Pamela A.; Nazir, Muhammad; Szulczewski, Michael J.; Keely, Patricia J.; Eliceiri, Kevin W.

2012-03-01

Tumor-Associated Collagen Signatures (TACS) have been identified that manifest in specific ways during breast tumor progression and that correspond to patient outcome. There are also compelling metabolic changes associated with carcinoma invasion and progression. We have characterized the difference in the autofluorescent properties of metabolic co-factors, NADH and FAD, between normal and carcinoma breast cell lines. Also, we have shown in vitro that increased collagen density alters metabolic genes which are associated with glycolysis and leads to a more invasive phenotype. Establishing the relationship between collagen density, cellular metabolism, and metastasis in physiologically relevant cancer models is crucial for developing cancer therapies. To study cellular metabolism with respect to collagen density in vivo, we use multiphoton fluorescence excitation microscopy (MPM) in conjunction with a rodent mammary imaging window implanted in defined mouse cancer models. These models are ideal for the study of collagen changes in vivo, allowing determination of corresponding metabolic changes in breast cancer invasion and progression. To measure cellular metabolism, we collect fluorescence lifetime (FLIM) signatures of NADH and FAD, which are known to change based on the microenvironment of the cells. Additionally, MPM systems are capable of collecting second harmonic generation (SHG) signals which are a nonlinear optical property of collagen. Therefore, MPM, SHG, and FLIM are powerful tools with great potential for characterizing key features of breast carcinoma in vivo. Below we present the current efforts of our collaborative group to develop intravital approaches based on these imaging techniques to look at defined mouse mammary models.

A medicinal extract of Scutellaria baicalensis and Acacia catechu acts as a dual inhibitor of cyclooxygenase and 5-lipoxygenase to reduce inflammation.

PubMed

Burnett, B P; Jia, Q; Zhao, Y; Levy, R M

2007-09-01

A mixed extract containing two naturally occurring flavonoids, baicalin from Scutellaria baicalensis and catechin from Acacia catechu, was tested for cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) inhibition via enzyme, cellular, and in vivo models. The 50% inhibitory concentration for inhibition of both ovine COX-1 and COX-2 peroxidase enzyme activities was 15 microg/mL, while the mixed extract showed a value for potato 5-LOX enzyme activity of 25 microg/mL. Prostaglandin E2 generation was inhibited by the mixed extract in human osteosarcoma cells expressing COX-2, while leukotriene production was inhibited in both human cell lines, immortalized THP-1 monocyte and HT-29 colorectal adenocarcinoma. In an arachidonic acid-induced mouse ear swelling model, the extract decreased edema in a dose-dependent manner. When arachidonic acid was injected directly into the intra-articular space of mouse ankle joints, the mixed extract abated the swelling and restored function in a rotary drum walking model. These results suggest that this natural, flavonoid mixture acts via "dual inhibition" of COX and LOX enzymes to reduce production of pro-inflammatory eicosanoids and attenuate edema in an in vivo model of inflammation.

Cockayne syndrome pathogenesis: lessons from mouse models.

PubMed

Jaarsma, Dick; van der Pluijm, Ingrid; van der Horst, Gijsbertus T J; Hoeijmakers, Jan H J

2013-01-01

Cockayne syndrome (CS) is a rare multisystem disorder characterized by cachectic dwarfism, nervous system abnormalities and features of premature aging. CS symptoms are associated with mutations in 5 genes, CSA, CSB, XPB, XPD and XPG encoding for proteins involved in the transcription-coupled subpathway of nucleotide excision DNA repair (NER). Mutant mice have been generated for all CS-associated genes and provide tools to examine how the cellular defects translate into CS symptoms. Mice deficient for Csa or Csb genetically mimic CS in man, and develop mild CS symptoms including reduced fat tissue, photoreceptor cell loss, and mild, but characteristic, nervous system pathology. These mild CS models are converted into severe CS models with short life span, progressive nervous system degeneration and cachectic dwarfism after simultaneous complete inactivation of global genome NER. A spectrum of mild-to-severe CS-like symptoms occurs in Xpb, Xpd, and Xpg mice that genetically mimic patients with a disorder that combines CS symptoms with another NER syndrome, xeroderma pigmentosum. In conclusion, CS mouse models mice develop a range of CS phenotypes and open promising perspectives for testing interventional approaches. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Learning and behavioral deficits associated with the absence of the fragile X mental retardation protein: what a fly and mouse model can teach us

PubMed Central

Santos, Ana Rita; Kanellopoulos, Alexandros K.

2014-01-01

The Fragile X syndrome (FXS) is the most frequent form of inherited mental disability and is considered a monogenic cause of autism spectrum disorder. FXS is caused by a triplet expansion that inhibits the expression of the FMR1 gene. The gene product, the Fragile X Mental Retardation Protein (FMRP), regulates mRNA metabolism in brain and nonneuronal cells. During brain development, FMRP controls the expression of key molecules involved in receptor signaling, cytoskeleton remodeling, protein synthesis and, ultimately, spine morphology. Symptoms associated with FXS include neurodevelopmental delay, cognitive impairment, anxiety, hyperactivity, and autistic-like behavior. Twenty years ago the first Fmr1 KO mouse to study FXS was generated, and several years later other key models including the mutant Drosophila melanogaster, dFmr1, have further helped the understanding of the cellular and molecular causes behind this complex syndrome. Here, we review to which extent these biological models are affected by the absence of FMRP, pointing out the similarities with the observed human dysfunction. Additionally, we discuss several potential treatments under study in animal models that are able to partially revert some of the FXS abnormalities. PMID:25227249

Interaction between emotion and memory: importance of mammillary bodies damage in a mouse model of the alcoholic Korsakoff syndrome.

PubMed

Béracochéa, Daniel

2005-01-01

Chronic alcohol consumption (CAC) can lead to the Korsakoff syndrome (KS), a memory deficiency attributed to diencephalic damage and/or to medial temporal or cortical related dysfunction. The etiology of KS remains unclear. Most animal models of KS involve thiamine-deficient diets associated with pyrithiamine treatment. Here we present a mouse model of CAC-induced KS. We demonstrate that CAC-generated retrieval memory deficits in working/ episodic memory tasks, together with a reduction of fear reactivity, result from damage to the mammillary bodies (MB). Experimental lesions of MB in non-alcoholic mice produced the same memory and emotional impairments. Drugs having anxiogenic-like properties counteract such impairments produced by CAC or by MB lesions. We suggest (a) that MB are the essential components of a brain network underlying emotional processes, which would be critically important in the retrieval processes involved in working/ episodic memory tasks, and (b) that failure to maintain emotional arousal due to MB damage can be a main factor of CAC-induced memory deficits. Overall, our animal model fits well with general neuropsychological and anatomic impairments observed in KS.

Interaction Between Emotion and Memory: Importance of Mammillary Bodies Damage in a Mouse Model of the Alcoholic Korsakoff Syndrome

PubMed Central

Béracochéa, Daniel

2005-01-01

Chronic alcohol consumption (CAC) can lead to the Korsakoff syndrome (KS), a memory deficiency attributed to diencephalie damage and/or to medial temporal or cortical related dysfunction. The etiology of KS remains unclear. Most animal models of KS involve thiaminedeficient diets associated with pyrithiamine treatment. Here we present a mouse model of CAC-induced KS. We demonstrate that CAC-generated retrieval memory deficits in working/ episodic memory tasks, together with a reduction of fear reactivity, result from damage to the mammillary bodies (MB). Experimental lesions of MB in non-alcoholic mice produced the same memory and emotional impairments. Drugs having anxiogenic-like properties counteract such impairments produced by CAC or by MB lesions. We suggest (a) that MB are the essential components of a brain network underlying emotional processes, which would be critically important in the retrieval processes involved in working/ episodic memory tasks, and (b) that failure to maintain emotional arousal due to MB damage can be a main factor of CAC-induced memory deficits. Overall, our animal model fits well with general neuropsychological and anatomic impairments observed in KS. PMID:16444899

[Effect of topical application of a recombinant adenovirus carrying promyelocytic leukemia gene in a psoriasis-like mouse model].

PubMed

Wang, Qiongyu; Zhang, Aijun; Ma, Huiqun; Wang, Shijie; Ma, Yunyun; Zou, Xingwei; Li, Ruilian

2013-03-01

To investigate the effects of topical treatment with adenovirus-mediated promyelocytic leukemia gene (PML) gene in a psoriasis-like mouse model. The effect of adenovirus-mediated PML gene on the granular layer of mouse tail scale epidermis and epithelial mitosis were observed on longitudinal histological sections prepared from the tail skin and vaginal epithelium of the mice. Adenovirus-mediated PML gene significantly inhibited mitosis of mouse vaginal epithelial cells and promoted the formation of granular layer in mouse tail scale epidermis. The therapeutic effect of PML gene in the psoriasis-like mouse model may be associated with increased granular cells and suppressed epidemic cell proliferation.

Evaluation of the Genetic and Nutritional Control of Obesity and Type 2 Diabetes in a Novel Mouse Model on Chromosome 7: An Insight into Insulin Signaling and Glucose Homeostasis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nelson, S.; Dhar, M.

Obesity is the main cause of type 2 diabetes, accounting for 90-95% of all diabetes cases in the US. Human obesity is a complex trait and can be studied using appropriate mouse models. A novel polygenic mouse model for studying the genetic and environmental contributions to and the physiological ramifications of obesity and related phenotypes is found in specific lines of mice bred and maintained at Oak Ridge National Laboratory. Heterozygous mice with a maternally inherited copy of two radiation-induced deletions in the p region of mouse chromosome 7, p23DFioD and p30PUb, have significantly greater body fat and show hyperinsulinemiamore » compared to the wild-type. A single gene, Atp10c, maps to this critical region and codes for a putative aminophospholipid translocase. Biochemical and molecular studies were initiated to gain insight into obesity and glucose homeostasis in these animals and to study the biological role of Atp10c in creating these phenotypes. Glucose and insulin tolerance tests were standardized for the heterozygous p23DFioD and control mice on a custom-made diet containing 20% protein, 70% carbohydrate, and 10% fat (kcal). Atp10c expression profiles were also generated using Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR). Heterozygous p23DFioD animals showed insulin resistance after receiving a dose of either 0.375 or 0.75 U/kg Illetin R insulin. RT-PCR data also shows differences in Atp10c expression in the mutants versus control mice. Using these standardized biochemical assays, future studies will further the understanding of genetic and nutritional controls of glucose homeostasis and obesity in animal models and subsequently in human populations.« less

Accelerated renal disease is associated with the development of metabolic syndrome in a glucolipotoxic mouse model

PubMed Central

Martínez-García, Cristina; Izquierdo, Adriana; Velagapudi, Vidya; Vivas, Yurena; Velasco, Ismael; Campbell, Mark; Burling, Keith; Cava, Fernando; Ros, Manuel; Orešič, Matej; Vidal-Puig, Antonio; Medina-Gomez, Gema

2012-01-01

SUMMARY Individuals with metabolic syndrome are at high risk of developing chronic kidney disease (CKD) through unclear pathogenic mechanisms. Obesity and diabetes are known to induce glucolipotoxic effects in metabolically relevant organs. However, the pathogenic role of glucolipotoxicity in the aetiology of diabetic nephropathy is debated. We generated a murine model, the POKO mouse, obtained by crossing the peroxisome proliferator-activated receptor gamma 2 (PPARγ2) knockout (KO) mouse into a genetically obese ob/ob background. We have previously shown that the POKO mice showed: hyperphagia, insulin resistance, hyperglycaemia and dyslipidaemia as early as 4 weeks of age, and developed a complete loss of normal β-cell function by 16 weeks of age. Metabolic phenotyping of the POKO model has led to investigation of the structural and functional changes in the kidney and changes in blood pressure in these mice. Here we demonstrate that the POKO mouse is a model of renal disease that is accelerated by high levels of glucose and lipid accumulation. Similar to ob/ob mice, at 4 weeks of age these animals exhibited an increased urinary albumin:creatinine ratio and significantly increased blood pressure, but in contrast showed a significant increase in the renal hypertrophy index and an associated increase in p27Kip1 expression compared with their obese littermates. Moreover, at 4 weeks of age POKO mice showed insulin resistance, an alteration of lipid metabolism and glomeruli damage associated with increased transforming growth factor beta (TGFβ) and parathyroid hormone-related protein (PTHrP) expression. At this age, levels of proinflammatory molecules, such as monocyte chemoattractant protein-1 (MCP-1), and fibrotic factors were also increased at the glomerular level compared with levels in ob/ob mice. At 12 weeks of age, renal damage was fully established. These data suggest an accelerated lesion through glucolipotoxic effects in the renal pathogenesis in POKO mice. PMID:22773754

Accelerated renal disease is associated with the development of metabolic syndrome in a glucolipotoxic mouse model.

PubMed

Martínez-García, Cristina; Izquierdo, Adriana; Velagapudi, Vidya; Vivas, Yurena; Velasco, Ismael; Campbell, Mark; Burling, Keith; Cava, Fernando; Ros, Manuel; Oresic, Matej; Vidal-Puig, Antonio; Medina-Gomez, Gema

2012-09-01

Individuals with metabolic syndrome are at high risk of developing chronic kidney disease (CKD) through unclear pathogenic mechanisms. Obesity and diabetes are known to induce glucolipotoxic effects in metabolically relevant organs. However, the pathogenic role of glucolipotoxicity in the aetiology of diabetic nephropathy is debated. We generated a murine model, the POKO mouse, obtained by crossing the peroxisome proliferator-activated receptor gamma 2 (PPARγ2) knockout (KO) mouse into a genetically obese ob/ob background. We have previously shown that the POKO mice showed: hyperphagia, insulin resistance, hyperglycaemia and dyslipidaemia as early as 4 weeks of age, and developed a complete loss of normal β-cell function by 16 weeks of age. Metabolic phenotyping of the POKO model has led to investigation of the structural and functional changes in the kidney and changes in blood pressure in these mice. Here we demonstrate that the POKO mouse is a model of renal disease that is accelerated by high levels of glucose and lipid accumulation. Similar to ob/ob mice, at 4 weeks of age these animals exhibited an increased urinary albumin:creatinine ratio and significantly increased blood pressure, but in contrast showed a significant increase in the renal hypertrophy index and an associated increase in p27(Kip1) expression compared with their obese littermates. Moreover, at 4 weeks of age POKO mice showed insulin resistance, an alteration of lipid metabolism and glomeruli damage associated with increased transforming growth factor beta (TGFβ) and parathyroid hormone-related protein (PTHrP) expression. At this age, levels of proinflammatory molecules, such as monocyte chemoattractant protein-1 (MCP-1), and fibrotic factors were also increased at the glomerular level compared with levels in ob/ob mice. At 12 weeks of age, renal damage was fully established. These data suggest an accelerated lesion through glucolipotoxic effects in the renal pathogenesis in POKO mice.

Tauopathy induced by low level expression of a human brain-derived tau fragment in mice is rescued by phenylbutyrate.

PubMed

Bondulich, Marie K; Guo, Tong; Meehan, Christopher; Manion, John; Rodriguez Martin, Teresa; Mitchell, Jacqueline C; Hortobagyi, Tibor; Yankova, Natalia; Stygelbout, Virginie; Brion, Jean-Pierre; Noble, Wendy; Hanger, Diane P

2016-08-01

Human neurodegenerative tauopathies exhibit pathological tau aggregates in the brain along with diverse clinical features including cognitive and motor dysfunction. Post-translational modifications including phosphorylation, ubiquitination and truncation, are characteristic features of tau present in the brain in human tauopathy. We have previously reported an N-terminally truncated form of tau in human brain that is associated with the development of tauopathy and is highly phosphorylated. We have generated a new mouse model of tauopathy in which this human brain-derived, 35 kDa tau fragment (Tau35) is expressed in the absence of any mutation and under the control of the human tau promoter. Most existing mouse models of tauopathy overexpress mutant tau at levels that do not occur in human neurodegenerative disease, whereas Tau35 transgene expression is equivalent to less than 10% of that of endogenous mouse tau. Tau35 mice recapitulate key features of human tauopathies, including aggregated and abnormally phosphorylated tau, progressive cognitive and motor deficits, autophagic/lysosomal dysfunction, loss of synaptic protein, and reduced life-span. Importantly, we found that sodium 4-phenylbutyrate (Buphenyl®), a drug used to treat urea cycle disorders and currently in clinical trials for a range of neurodegenerative diseases, reverses the observed abnormalities in tau and autophagy, behavioural deficits, and loss of synapsin 1 in Tau35 mice. Our results show for the first time that, unlike other tau transgenic mouse models, minimal expression of a human disease-associated tau fragment in Tau35 mice causes a profound and progressive tauopathy and cognitive changes, which are rescued by pharmacological intervention using a clinically approved drug. These novel Tau35 mice therefore represent a highly disease-relevant animal model in which to investigate molecular mechanisms and to develop novel treatments for human tauopathies. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain.

Tauopathy induced by low level expression of a human brain-derived tau fragment in mice is rescued by phenylbutyrate

PubMed Central

Bondulich, Marie K.; Guo, Tong; Meehan, Christopher; Manion, John; Rodriguez Martin, Teresa; Mitchell, Jacqueline C.; Hortobagyi, Tibor; Yankova, Natalia; Stygelbout, Virginie; Brion, Jean-Pierre; Noble, Wendy

2016-01-01

Abstract Human neurodegenerative tauopathies exhibit pathological tau aggregates in the brain along with diverse clinical features including cognitive and motor dysfunction. Post-translational modifications including phosphorylation, ubiquitination and truncation, are characteristic features of tau present in the brain in human tauopathy. We have previously reported an N-terminally truncated form of tau in human brain that is associated with the development of tauopathy and is highly phosphorylated. We have generated a new mouse model of tauopathy in which this human brain-derived, 35 kDa tau fragment (Tau35) is expressed in the absence of any mutation and under the control of the human tau promoter. Most existing mouse models of tauopathy overexpress mutant tau at levels that do not occur in human neurodegenerative disease, whereas Tau35 transgene expression is equivalent to less than 10% of that of endogenous mouse tau. Tau35 mice recapitulate key features of human tauopathies, including aggregated and abnormally phosphorylated tau, progressive cognitive and motor deficits, autophagic/lysosomal dysfunction, loss of synaptic protein, and reduced life-span. Importantly, we found that sodium 4-phenylbutyrate (Buphenyl®), a drug used to treat urea cycle disorders and currently in clinical trials for a range of neurodegenerative diseases, reverses the observed abnormalities in tau and autophagy, behavioural deficits, and loss of synapsin 1 in Tau35 mice. Our results show for the first time that, unlike other tau transgenic mouse models, minimal expression of a human disease-associated tau fragment in Tau35 mice causes a profound and progressive tauopathy and cognitive changes, which are rescued by pharmacological intervention using a clinically approved drug. These novel Tau35 mice therefore represent a highly disease-relevant animal model in which to investigate molecular mechanisms and to develop novel treatments for human tauopathies. PMID:27297240

seq-ImmuCC: Cell-Centric View of Tissue Transcriptome Measuring Cellular Compositions of Immune Microenvironment From Mouse RNA-Seq Data.

PubMed

Chen, Ziyi; Quan, Lijun; Huang, Anfei; Zhao, Qiang; Yuan, Yao; Yuan, Xuye; Shen, Qin; Shang, Jingzhe; Ben, Yinyin; Qin, F Xiao-Feng; Wu, Aiping

2018-01-01

The RNA sequencing approach has been broadly used to provide gene-, pathway-, and network-centric analyses for various cell and tissue samples. However, thus far, rich cellular information carried in tissue samples has not been thoroughly characterized from RNA-Seq data. Therefore, it would expand our horizons to better understand the biological processes of the body by incorporating a cell-centric view of tissue transcriptome. Here, a computational model named seq-ImmuCC was developed to infer the relative proportions of 10 major immune cells in mouse tissues from RNA-Seq data. The performance of seq-ImmuCC was evaluated among multiple computational algorithms, transcriptional platforms, and simulated and experimental datasets. The test results showed its stable performance and superb consistency with experimental observations under different conditions. With seq-ImmuCC, we generated the comprehensive landscape of immune cell compositions in 27 normal mouse tissues and extracted the distinct signatures of immune cell proportion among various tissue types. Furthermore, we quantitatively characterized and compared 18 different types of mouse tumor tissues of distinct cell origins with their immune cell compositions, which provided a comprehensive and informative measurement for the immune microenvironment inside tumor tissues. The online server of seq-ImmuCC are freely available at http://wap-lab.org:3200/immune/.

Generation of tooth-periodontium complex structures using high-odontogenic potential dental epithelium derived from mouse embryonic stem cells.

PubMed

Zhang, Yancong; Li, Yongliang; Shi, Ruirui; Zhang, Siqi; Liu, Hao; Zheng, Yunfei; Li, Yan; Cai, Jinglei; Pei, Duanqing; Wei, Shicheng

2017-06-08

A number of studies have shown that tooth-like structures can be regenerated using induced pluripotent stem cells and mouse embryonic stem (mES) cells. However, few studies have reported the regeneration of tooth-periodontium complex structures, which are more suitable for clinical tooth transplantation. We established an optimized approach to induce high-odontogenic potential dental epithelium derived from mES cells by temporally controlling bone morphogenic protein 4 (BMP4) function and regenerated tooth-periodontium complex structures in vivo. First, immunofluorescence and quantitative reverse transcription-polymerase chain reaction were used to identify the watershed of skin and the oral ectoderm. LDN193189 was then used to inhibit the BMP4 receptor around the watershed, followed by the addition of exogenous BMP4 to promote BMP4 function. The generated dental epithelium was confirmed by western blot analysis and immunofluorescence. The generated epithelium was ultimately combined with embryonic day 14.5 mouse mesenchyme and transplanted into the renal capsules of nude mice. After 4 weeks, the tooth-periodontium complex structure was examined by micro-computed tomography (CT) and hematoxylin and eosin (H&E) staining. Our study found that the turning point of oral ectoderm differentiation occurred around day 3 after the embryoid body was transferred to a common culture plate. Ameloblastin-positive dental epithelial cells were detected following the temporal regulation of BMP4. Tooth-periodontium complex structures, which included teeth, a periodontal membrane, and alveolar bone, were formed when this epithelium was combined with mouse dental mesenchyme and transplanted into the renal capsules of nude mice. Micro-CT and H&E staining revealed that the generated tooth-periodontium complex structures shared a similar histological structure with normal mouse teeth. An optimized induction method was established to promote the differentiation of mES cells into dental epithelium by temporally controlling the function of BMP4. A novel tooth-periodontium complex structure was generated using the epithelium.

Generation of an allelic series of knock-in mice using recombinase-mediated cassette exchange (RMCE).

PubMed

Roebroek, Anton J M; Van Gool, Bart

2014-01-01

Molecular genetic strategies applying embryonic stem cell (ES cell) technologies to study the function of a gene in mice or to generate a mouse model for a human disease are continuously under development. Next to (conditional) inactivation of genes the application and importance of approaches to generate knock-in mutations are increasing. In this chapter the principle and application of recombinase-mediated cassette exchange (RMCE) are discussed as being a new emerging knock-in strategy, which enables easy generation of a series of different knock-in mutations within one gene. An RMCE protocol, which was used to generate a series of different knock-in mutations in the Lrp1 gene of ES cells, is described in detail as an example of how RMCE can be used to generate highly efficiently an allelic series of differently modified ES cell clones from a parental modified ES cell clone. Subsequently the differently modified ES cell clones can be used to generate an allelic series of mutant knock-in mice.

Fibrin activates GPVI in human and mouse platelets

PubMed Central

Alshehri, Osama M.; Montague, Samantha; Watson, Stephanie K.; Frampton, Jon; Bender, Markus; Watson, Steve P.

2015-01-01

The glycoprotein VI (GPVI)-Fc receptor γ (FcRγ) chain is the major platelet signaling receptor for collagen. Paradoxically, in a FeCl3 injury model, occlusion, but not initiation of thrombus formation, is delayed in GPVI-deficient and GPVI-depleted mice. In this study, we demonstrate that GPVI is a receptor for fibrin and speculate that this contributes to development of an occlusive thrombus. We observed a marked increase in tyrosine phosphorylation, including the FcRγ chain and Syk, in human and mouse platelets induced by thrombin in the presence of fibrinogen and the αIIbβ3 blocker eptifibatide. This was not seen in platelets stimulated by a protease activated receptor (PAR)-4 peptide, which is unable to generate fibrin from fibrinogen. The pattern of tyrosine phosphorylation was similar to that induced by activation of GPVI. Consistent with this, thrombin did not induce tyrosine phosphorylation of Syk and the FcRγ chain in GPVI-deficient mouse platelets. Mouse platelets underwent full spreading on fibrin but not fibrinogen, which was blocked in the presence of a Src kinase inhibitor or in the absence of GPVI. Spreading on fibrin was associated with phosphatidylserine exposure (procoagulant activity), and this too was blocked in GPVI-deficient platelets. The ectodomain of GPVI was shown to bind to immobilized monomeric and polymerized fibrin. A marked increase in embolization was seen following FeCl3 injury in GPVI-deficient mice, likely contributing to the delay in occlusion in this model. These results demonstrate that GPVI is a receptor for fibrin and provide evidence that this interaction contributes to thrombus growth and stability. PMID:26282541

Thymidine kinase 2 deficiency-induced mtDNA depletion in mouse liver leads to defect β-oxidation.

PubMed

Zhou, Xiaoshan; Kannisto, Kristina; Curbo, Sophie; von Döbeln, Ulrika; Hultenby, Kjell; Isetun, Sindra; Gåfvels, Mats; Karlsson, Anna

2013-01-01

Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2(-/-)) that progressively loses its mtDNA. The TK2(-/-) mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2(-/-) mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2(-/-) mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2(-/-) mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial β-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2(-/-) mice causes impaired mitochondrial function that leads to defect β-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies.

Thymidine Kinase 2 Deficiency-Induced mtDNA Depletion in Mouse Liver Leads to Defect β-Oxidation

PubMed Central

von Döbeln, Ulrika; Hultenby, Kjell; Isetun, Sindra; Gåfvels, Mats; Karlsson, Anna

2013-01-01

Thymidine kinase 2 (TK2) deficiency in humans causes mitochondrial DNA (mtDNA) depletion syndrome. To study the molecular mechanisms underlying the disease and search for treatment options, we previously generated and described a TK2 deficient mouse strain (TK2−/−) that progressively loses its mtDNA. The TK2−/− mouse model displays symptoms similar to humans harboring TK2 deficient infantile fatal encephalomyopathy. Here, we have studied the TK2−/− mouse model to clarify the pathological role of progressive mtDNA depletion in liver for the severe outcome of TK2 deficiency. We observed that a gradual depletion of mtDNA in the liver of the TK2−/− mice was accompanied by increasingly hypertrophic mitochondria and accumulation of fat vesicles in the liver cells. The levels of cholesterol and nonesterified fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2−/− mice. In mice with hepatic mtDNA levels below 20%, the blood sugar and the ketone levels dropped. These mice also exhibited reduced mitochondrial β-oxidation due to decreased transport of long chain acylcarnitines into the mitochondria. The gradual loss of mtDNA in the liver of the TK2−/− mice causes impaired mitochondrial function that leads to defect β-oxidation and, as a result, insufficient production of ketone bodies and glucose. This study provides insight into the mechanism of encephalomyopathy caused by TK2 deficiency-induced mtDNA depletion that may be used to explore novel therapeutic strategies. PMID:23505564

Gene expression profile of mouse prostate tumors reveals dysregulations in major biological processes and identifies potential murine targets for preclinical development of human prostate cancer therapy.

PubMed

Haram, Kerstyn M; Peltier, Heidi J; Lu, Bin; Bhasin, Manoj; Otu, Hasan H; Choy, Bob; Regan, Meredith; Libermann, Towia A; Latham, Gary J; Sanda, Martin G; Arredouani, Mohamed S

2008-10-01

Translation of preclinical studies into effective human cancer therapy is hampered by the lack of defined molecular expression patterns in mouse models that correspond to the human counterpart. We sought to generate an open source TRAMP mouse microarray dataset and to use this array to identify differentially expressed genes from human prostate cancer (PCa) that have concordant expression in TRAMP tumors, and thereby represent lead targets for preclinical therapy development. We performed microarrays on total RNA extracted and amplified from eight TRAMP tumors and nine normal prostates. A subset of differentially expressed genes was validated by QRT-PCR. Differentially expressed TRAMP genes were analyzed for concordant expression in publicly available human prostate array datasets and a subset of resulting genes was analyzed by QRT-PCR. Cross-referencing differentially expressed TRAMP genes to public human prostate array datasets revealed 66 genes with concordant expression in mouse and human PCa; 56 between metastases and normal and 10 between primary tumor and normal tissues. Of these 10 genes, two, Sox4 and Tubb2a, were validated by QRT-PCR. Our analysis also revealed various dysregulations in major biologic pathways in the TRAMP prostates. We report a TRAMP microarray dataset of which a gene subset was validated by QRT-PCR with expression patterns consistent with previous gene-specific TRAMP studies. Concordance analysis between TRAMP and human PCa associated genes supports the utility of the model and suggests several novel molecular targets for preclinical therapy.

EWS-FLI1 regulates a transcriptional program in cooperation with Foxq1 in mouse Ewing sarcoma.

PubMed

Shimizu, Rikuka; Tanaka, Miwa; Tsutsumi, Shuichi; Aburatani, Hiroyuki; Yamazaki, Yukari; Homme, Mizuki; Kitagawa, Yoshimasa; Nakamura, Takuro

2018-06-26

EWS-FLI1 constitutes an oncogenic transcription factor that plays key roles in Ewing sarcoma development and maintenance. We have recently succeeded in generating an ex vivo mouse model for Ewing sarcoma by introducing EWS-FLI1 into embryonic osteochondrogenic progenitors. The model well recapitulates the biological characteristics, small round cell morphology, and gene expression profiles of human Ewing sarcoma. Here we clarified the global DNA binding properties of EWS-FLI1 in mouse Ewing sarcoma. GGAA microsatellites were found to serve as binding sites of EWS-FLI1 albeit with less frequency than that in human Ewing sarcoma; moreover, genomic distribution was not conserved between human and mouse. Nevertheless, EWS-FLI1 binding sites within GGAA microsatellites were frequently associated with the histone H3K27Ac enhancer mark, suggesting that EWS-FLI1 could affect global gene expression by binding its target sites. In particular, the Fox transcription factor binding motif was frequently observed within EWS-FLI1 peaks and Foxq1 was identified as the cooperative partner that interacts with the EWS portion of EWS-FLI1. Trib1 and Nrg1 were demonstrated as target genes that are co-regulated by EWS-FLI1 and Foxq1, and are important for cell proliferation and survival of Ewing sarcoma. Collectively, our findings present novel aspects of EWS-FLI1 function as well as the importance of GGAA microsatellites. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

In vivo bioluminescence tomography based on multi-view projection and 3D surface reconstruction

NASA Astrophysics Data System (ADS)

Zhang, Shuang; Wang, Kun; Leng, Chengcai; Deng, Kexin; Hu, Yifang; Tian, Jie

2015-03-01

Bioluminescence tomography (BLT) is a powerful optical molecular imaging modality, which enables non-invasive realtime in vivo imaging as well as 3D quantitative analysis in preclinical studies. In order to solve the inverse problem and reconstruct inner light sources accurately, the prior structural information is commonly necessary and obtained from computed tomography or magnetic resonance imaging. This strategy requires expensive hybrid imaging system, complicated operation protocol and possible involvement of ionizing radiation. The overall robustness highly depends on the fusion accuracy between the optical and structural information. In this study we present a pure optical bioluminescence tomographic system (POBTS) and a novel BLT method based on multi-view projection acquisition and 3D surface reconstruction. The POBTS acquired a sparse set of white light surface images and bioluminescent images of a mouse. Then the white light images were applied to an approximate surface model to generate a high quality textured 3D surface reconstruction of the mouse. After that we integrated multi-view luminescent images based on the previous reconstruction, and applied an algorithm to calibrate and quantify the surface luminescent flux in 3D.Finally, the internal bioluminescence source reconstruction was achieved with this prior information. A BALB/C mouse with breast tumor of 4T1-fLuc cells mouse model were used to evaluate the performance of the new system and technique. Compared with the conventional hybrid optical-CT approach using the same inverse reconstruction method, the reconstruction accuracy of this technique was improved. The distance error between the actual and reconstructed internal source was decreased by 0.184 mm.

Induced pluripotent stem cells from goat fibroblasts.

PubMed

Song, Hui; Li, Hui; Huang, Mingrui; Xu, Dan; Gu, Chenghao; Wang, Ziyu; Dong, Fulu; Wang, Feng

2013-12-01

Embryonic stem cells (ESCs) are a powerful model for genetic engineering, studying developmental biology, and modeling disease. To date, ESCs have been established from the mouse (Evans and Kaufman, 1981, Nature 292:154-156), non-human primates (Thomson et al., , Proc Nat Acad Sci USA 92:7844-7848), humans (Thomson et al., 1998, Science 282:1145-1147), and rats (Buehr et al., , Cell 135:1287-1298); however, the derivation of ESCs from domesticated ungulates such as goats, sheep, cattle, and pigs have not been successful. Alternatively, induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with several combinations of genes encoding transcription factors (OCT3/4, SOX2, KLF4, cMYC, LIN28, and NANOG). To date, iPSCs have been isolated from various species, but only limited information is available regarding goat iPSCs (Ren et al., 2011, Cell Res 21:849-853). The objectives of this study were to generate goat iPSCs from fetal goat primary ear fibroblasts using lentiviral transduction of four human transcription factors: OCT4, SOX2, KLF4, and cMYC. The goat iPSCs were successfully generated by co-culture with mitomycin C-treated mouse embryonic fibroblasts using medium supplemented with knockout serum replacement and human basic fibroblast growth factor. The goat iPSCs colonies are flat, compact, and closely resemble human iPSCs. They have a normal karyotype; stain positive for alkaline phosphatase, OCT4, and NANOG; express endogenous pluripotency genes (OCT4, SOX2, cMYC, and NANOG); and can spontaneously differentiate into three germ layers in vitro and in vivo. © 2013 Wiley Periodicals, Inc.

STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity.

PubMed

Demaria, Olivier; De Gassart, Aude; Coso, Sanja; Gestermann, Nicolas; Di Domizio, Jeremy; Flatz, Lukas; Gaide, Olivier; Michielin, Olivier; Hwu, Patrick; Petrova, Tatiana V; Martinon, Fabio; Modlin, Robert L; Speiser, Daniel E; Gilliet, Michel

2015-12-15

Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma.

Restoration of visual function by expression of a light-gated mammalian ion channel in retinal ganglion cells or ON-bipolar cells

PubMed Central

Gaub, Benjamin M.; Berry, Michael H.; Holt, Amy E.; Reiner, Andreas; Kienzler, Michael A.; Dolgova, Natalia; Nikonov, Sergei; Aguirre, Gustavo D.; Beltran, William A.; Flannery, John G.; Isacoff, Ehud Y.

2014-01-01

Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0460). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0460 was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0460 was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0460 in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0460 in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0460 was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation. PMID:25489083

What Is the Predictive Value of Animal Models for Vaccine Efficacy in Humans? Reevaluating the Potential of Mouse Models for the Human Immune System.

PubMed

Jameson, Stephen C; Masopust, David

2018-04-02

Much of what we understand about immunology, including the response to vaccines, come from studies in mice because they provide many practical advantages compared with research in higher mammals and humans. Nevertheless, modalities for preventing or treating disease do not always translate from mouse to humans, which has led to increasing scrutiny of the continued merits of mouse research. Here, we summarize the pros and cons of current laboratory mouse models for immunology research and discuss whether overreliance on nonphysiological, ultra-hygienic animal husbandry approaches has limited the ultimate translation potential of mouse-derived data to humans. Alternative approaches are discussed that may extend the use of the mouse model for preclinical studies. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD.

PubMed

Esanov, Rustam; Cabrera, Gabriela Toro; Andrade, Nadja S; Gendron, Tania F; Brown, Robert H; Benatar, Michael; Wahlestedt, Claes; Mueller, Christian; Zeier, Zane

2017-06-12

Amyotrophic Lateral Sclerosis (ALS) is a fatal and progressive neurodegenerative disorder with identified genetic causes representing a significant minority of all cases. A GGGGCC hexanucleotide repeat expansion (HRE) mutation within the C9ORF72 gene has recently been identified as the most frequent known cause of ALS. The expansion leads to partial heterochromatinization of the locus, yet mutant RNAs and dipeptide repeat proteins (DPRs) are still produced in sufficient quantities to confer neurotoxicity. The levels of these toxic HRE products positively correlate with cellular toxicity and phenotypic severity across multiple disease models. Moreover, the degree of epigenetic repression inversely correlates with some facets of clinical presentation in C9-ALS patients. Recently, bacterial artificial chromosomes (BAC) have been used to generate transgenic mice that harbor the HRE mutation, complementing other relevant model systems such as patient-derived induced pluripotent stem cells (iPSCs). While epigenetic features of the HRE have been investigated in various model systems and post-mortem tissues, epigenetic dysregulation at the expanded locus in C9-BAC mice remains unexplored. Here, we sought to determine whether clinically relevant epigenetic perturbations caused by the HRE are mirrored in a C9-BAC mouse model. We used complementary DNA methylation assessment and immunoprecipitation methods to demonstrate that epigenetic aberrations caused by the HRE, such as DNA and histone methylation, are recapitulated in the C9-BAC mice. Strikingly, we found that cytosine hypermethylation within the promoter region of the human transgene occurred in a subset of C9-BAC mice similar to what is observed in patient populations. Moreover, we show that partial heterochromatinization of the C9 HRE occurs during the first weeks of the mouse lifespan, indicating age-dependent epigenetic repression. Using iPSC neurons, we found that preventing R-loop formation did not impede heterochromatinization of the HRE. Taken together, these observations provide further insight into mechanism and developmental time-course of epigenetic perturbations conferred by the C9ORF72 HRE. Finally, we suggest that epigenetic repression of the C9ORF72 HRE and nearby gene promoter could impede or delay motor neuron degeneration in C9-BAC mouse models of ALS/FTD.

Comparative biology of cystic fibrosis animal models.

PubMed

Fisher, John T; Zhang, Yulong; Engelhardt, John F

2011-01-01

Animal models of human diseases are critical for dissecting mechanisms of pathophysiology and developing therapies. In the context of cystic fibrosis (CF), mouse models have been the dominant species by which to study CF disease processes in vivo for the past two decades. Although much has been learned through these CF mouse models, limitations in the ability of this species to recapitulate spontaneous lung disease and several other organ abnormalities seen in CF humans have created a need for additional species on which to study CF. To this end, pig and ferret CF models have been generated by somatic cell nuclear transfer and are currently being characterized. These new larger animal models have phenotypes that appear to closely resemble human CF disease seen in newborns, and efforts to characterize their adult phenotypes are ongoing. This chapter will review current knowledge about comparative lung cell biology and cystic fibrosis transmembrane conductance regulator (CFTR) biology among mice, pigs, and ferrets that has implications for CF disease modeling in these species. We will focus on methods used to compare the biology and function of CFTR between these species and their relevance to phenotypes seen in the animal models. These cross-species comparisons and the development of both the pig and the ferret CF models may help elucidate pathophysiologic mechanisms of CF lung disease and lead to new therapeutic approaches.

Fluorescent Arc/Arg3.1 indicator mice: a versatile tool to study brain activity changes in vitro and in vivo

PubMed Central

Grinevich, Valery; Kolleker, Alexander; Eliava, Marina; Takada, Naoki; Takuma, Hiroshi; Fukazawa, Yugo; Shigemoto, Ryuichi; Kuhl, Dietmar; Waters, Jack; Seeburg, Peter H.; Osten, Pavel

2014-01-01

The brain-specific immediate early gene Arc/Arg3.1 is induced in response to a variety of stimuli, including sensory and behavior-linked neural activity. Here we report the generation of transgenic mice, termed TgArc/Arg3.1-d4EGFP, expressing a 4-hour half-life form of enhanced green fluorescent protein (d4EGFP) under the control of the Arc/Arg3.1 promoter. We show that d4EGFP-mediated fluorescence faithfully reports Arc/Arg3.1 induction in response to physiological, pathological and pharmacological stimuli, and that this fluorescence permits electrical recording from activated neurons in the live mouse. Moreover, the fluorescent Arc/Arg3.1 indicator revealed activity changes in circumscribed brain areas in distinct modes of stress and in a mouse model of Alzheimer’s disease. These findings identify the TgArc/Arg3.1-d4EGFP mouse as a versatile tool to monitor Arc/Arg3.1 induction in neural circuits, both in vitro and in vivo. PMID:19628007

Imaging of cardiovascular dynamics in early mouse embryos with swept source optical coherence tomography

NASA Astrophysics Data System (ADS)

Larina, Irina V.; Liebling, Michael; Dickinson, Mary E.; Larin, Kirill V.

2009-02-01

Congenital cardiovascular defects are very common, occurring in 1% of live births, and cardiovascular failures are the leading cause of birth defect-related deaths in infants. To improve diagnostics, prevention and treatment of cardiovascular abnormalities, we need to understand not only how cells form the heart and vessels but also how physical factors such as heart contraction and blood flow influence heart development and changes in the circulatory network. Mouse models are an excellent resource for studying cardiovascular development and disease because of the resemblance to humans, rapid generation time, and availability of mutants with cardiovascular defects linked to human diseases. In this work, we present results on development and application of Doppler Swept Source Optical Coherence Tomography (DSS-OCT) for imaging of cardiovascular dynamics and blood flow in the mouse embryonic heart and vessels. Our studies demonstrated that the spatial and temporal resolution of the DSS-OCT makes it possible to perform sensitive measurements of heart and vessel wall movements and to investigate how contractile waves facilitate the movement of blood through the circulatory system.

Mouse bioassay for palytoxin. Specific symptoms and dose-response against dose-death time relationships.

PubMed

Riobó, P; Paz, B; Franco, J M; Vázquez, J A; Murado, M A; Cacho, E

2008-08-01

Nowadays, a variety of protocols are applied to quantitate palytoxin. However, there is not desirable agreement among them, the confidence intervals of the basic toxicological parameters are too wide and the formal descriptions lack the necessary generality to establish comparisons. Currently, the mouse bioassay is the most accepted one to categorize marine toxins and it must constitute the reference for other methods. In the present work, the mouse bioassay for palytoxin is deeply analyzed and carefully described showing the initial symptoms of injected mice which are presented here in the first time. These symptoms clearly differ from the more common marine toxins described up to now. Regarding to the toxicological aspects two considerations are taking into account: (i) the empiric models based in the dose-death time relationships cause serious ambiguities and (ii) the traditional moving average method contains in its regular use any inaccuracy elements. Herein is demonstrated that the logistic equation and the accumulative function of Weibull's distribution (with the modifications proposed) generate satisfactory toxicological descriptions in all the respects.

Development of ghrelin transgenic mice for elucidation of clinical implication of ghrelin.

PubMed

Aotani, Daisuke; Ariyasu, Hiroyuki; Shimazu-Kuwahara, Satoko; Shimizu, Yoshiyuki; Nomura, Hidenari; Murofushi, Yoshiteru; Kaneko, Kentaro; Izumi, Ryota; Matsubara, Masaki; Kanda, Hajime; Noguchi, Michio; Tanaka, Tomohiro; Kusakabe, Toru; Miyazawa, Takashi; Nakao, Kazuwa

2017-01-01

To elucidate the clinical implication of ghrelin, we have been trying to generate variable models of transgenic (Tg) mice overexpressing ghrelin. We generated Tg mice overexpressing des-acyl ghrelin in a wide variety of tissues under the control of β-actin promoter. While plasma des-acyl ghrelin level in the Tg mice was 44-fold greater than that of control mice, there was no differences in the plasma ghrelin level between des-acyl ghrelin Tg and the control mice. The des-acyl ghrelin Tg mice exhibited the lower body weight and the shorter body length due to modulation of GH-IGF-1 axis. We tried to generate Tg mice expressing a ghrelin analog, which possessed ghrelin-like activity (Trp 3 -ghrelin Tg mice). The plasma Trp 3 -ghrelin concentration in Trp 3 -ghrelin Tg mice was approximately 85-fold higher than plasma ghrelin (acylated ghrelin) concentration seen in the control mice. Because Trp 3 -ghrelin is approximately 24-fold less potent than ghrelin, the plasma Trp 3 -ghrelin concentration in Trp 3 -ghrelin Tg mice was calculated to have approximately 3.5-fold biological activity greater than that of ghrelin (acylated ghrelin) in the control mice. Trp 3 -ghrelin Tg mice did not show any phenotypes except for reduced insulin sensitivity in 1-year old. After the identification of ghrelin O-acyltransferase (GOAT), we generated doubly Tg mice overexpressing both mouse des-acyl ghrelin and mouse GOAT in the liver by cross-mating the two kinds of Tg mice. The plasma ghrelin concentration of doubly Tg mice was approximately 2-fold higher than that of the control mice. No apparent phenotypic changes in body weight and food intake were observed in doubly Tg mice. Further studies are ongoing in our laboratory to generate Tg mice with the increased plasma ghrelin level to a greater extent. The better understanding of physiological and pathophysiological significance of ghrelin from experiments using an excellent animal model may provide a new therapeutic approach for human diseases.

HCN4-Overexpressing Mouse Embryonic Stem Cell-Derived Cardiomyocytes Generate a New Rapid Rhythm in Rats with Bradycardia.

PubMed

Saito, Yukihiro; Nakamura, Kazufumi; Yoshida, Masashi; Sugiyama, Hiroki; Takano, Makoto; Nagase, Satoshi; Morita, Hiroshi; Kusano, Kengo F; Ito, Hiroshi

2018-05-30

A biological pacemaker is expected to solve the persisting problems of an artificial cardiac pacemaker including short battery life, lead breaks, infection, and electromagnetic interference. We previously reported HCN4 overexpression enhances pacemaking ability of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) in vitro. However, the effect of these cells on bradycardia in vivo has remained unclear. Therefore, we transplanted HCN4-overexpressing mESC-CMs into bradycardia model animals and investigated whether they could function as a biological pacemaker. The rabbit Hcn4 gene was transfected into mouse embryonic stem cells and induced HCN4-overexpressing mESC-CMs. Non-cardiomyocytes were removed under serum/glucose-free and lactate-supplemented conditions. Cardiac balls containing 5 × 10 3 mESC-CMs were made by using the hanging drop method. One hundred cardiac balls were injected into the left ventricular free wall of complete atrioventricular block (CAVB) model rats. Heart beats were evaluated using an implantable telemetry system 7 to 30 days after cell transplantation. The result showed that ectopic ventricular beats that were faster than the intrinsic escape rhythm were often observed in CAVB model rats transplanted with HCN4-overexpressing mESC-CMs. On the other hand, the rats transplanted with non-overexpressing mESC-CMs showed sporadic single premature ventricular contraction but not sustained ectopic ventricular rhythms. These results indicated that HCN4-overexpressing mESC-CMs produce rapid ectopic ventricular rhythms as a biological pacemaker.

A Computational Clonal Analysis of the Developing Mouse Limb Bud

PubMed Central

Marcon, Luciano; Arqués, Carlos G.; Torres, Miguel S.; Sharpe, James

2011-01-01

A comprehensive spatio-temporal description of the tissue movements underlying organogenesis would be an extremely useful resource to developmental biology. Clonal analysis and fate mappings are popular experiments to study tissue movement during morphogenesis. Such experiments allow cell populations to be labeled at an early stage of development and to follow their spatial evolution over time. However, disentangling the cumulative effects of the multiple events responsible for the expansion of the labeled cell population is not always straightforward. To overcome this problem, we develop a novel computational method that combines accurate quantification of 2D limb bud morphologies and growth modeling to analyze mouse clonal data of early limb development. Firstly, we explore various tissue movements that match experimental limb bud shape changes. Secondly, by comparing computational clones with newly generated mouse clonal data we are able to choose and characterize the tissue movement map that better matches experimental data. Our computational analysis produces for the first time a two dimensional model of limb growth based on experimental data that can be used to better characterize limb tissue movement in space and time. The model shows that the distribution and shapes of clones can be described as a combination of anisotropic growth with isotropic cell mixing, without the need for lineage compartmentalization along the AP and PD axis. Lastly, we show that this comprehensive description can be used to reassess spatio-temporal gene regulations taking tissue movement into account and to investigate PD patterning hypothesis. PMID:21347315

Severely altered guanidino compound levels, disturbed body weight homeostasis and impaired fertility in a mouse model of guanidinoacetate N-methyltransferase (GAMT) deficiency.

PubMed

Schmidt, Andreas; Marescau, Bart; Boehm, Ernest A; Renema, W Klaas Jan; Peco, Ruben; Das, Anib; Steinfeld, Robert; Chan, Sharon; Wallis, Julie; Davidoff, Michail; Ullrich, Kurt; Waldschütz, Ralph; Heerschap, Arend; De Deyn, Peter P; Neubauer, Stefan; Isbrandt, Dirk

2004-05-01

We generated a knockout mouse model for guanidinoacetate N-methyltransferase (GAMT) deficiency (MIM 601240), the first discovered human creatine deficiency syndrome, by gene targeting in embryonic stem cells. Disruption of the open reading frame of the murine GAMT gene in the first exon resulted in the elimination of 210 of the 237 amino acids present in mGAMT. The creation of an mGAMT null allele was verified at the genetic, RNA and protein levels. GAMT knockout mice have markedly increased guanidinoacetate (GAA) and reduced creatine and creatinine levels in brain, serum and urine, which are key findings in human GAMT patients. In vivo (31)P magnetic resonance spectroscopy showed high levels of PGAA and reduced levels of creatine phosphate in heart, skeletal muscle and brain. These biochemical alterations were comparable to those found in human GAMT patients and can be attributed to the very similar GAMT expression patterns found by us in human and mouse tissues. We provide evidence that GAMT deficiency in mice causes biochemical adaptations in brain and skeletal muscle. It is associated with increased neonatal mortality, muscular hypotonia, decreased male fertility and a non-leptin-mediated life-long reduction in body weight due to reduced body fat mass. Therefore, GAMT knockout mice are a valuable creatine deficiency model for studying the effects of high-energy phosphate depletion in brain, heart, skeletal muscle and other organs.

HSC extrinsic sex-related and intrinsic autoimmune disease-related human B-cell variation is recapitulated in humanized mice.

PubMed

Borsotti, Chiara; Danzl, Nichole M; Nauman, Grace; Hölzl, Markus A; French, Clare; Chavez, Estefania; Khosravi-Maharlooei, Mohsen; Glauzy, Salome; Delmotte, Fabien R; Meffre, Eric; Savage, David G; Campbell, Sean R; Goland, Robin; Greenberg, Ellen; Bi, Jing; Satwani, Prakash; Yang, Suxiao; Bathon, Joan; Winchester, Robert; Sykes, Megan

2017-10-24

B cells play a major role in antigen presentation and antibody production in the development of autoimmune diseases, and some of these diseases disproportionally occur in females. Moreover, immune responses tend to be stronger in female vs male humans and mice. Because it is challenging to distinguish intrinsic from extrinsic influences on human immune responses, we used a personalized immune (PI) humanized mouse model, in which immune systems were generated de novo from adult human hematopoietic stem cells (HSCs) in immunodeficient mice. We assessed the effect of recipient sex and of donor autoimmune diseases (type 1 diabetes [T1D] and rheumatoid arthritis [RA]) on human B-cell development in PI mice. We observed that human B-cell levels were increased in female recipients regardless of the source of human HSCs or the strain of immunodeficient recipient mice. Moreover, mice injected with T1D- or RA-derived HSCs displayed B-cell abnormalities compared with healthy control HSC-derived mice, including altered B-cell levels, increased proportions of mature B cells and reduced CD19 expression. Our study revealed an HSC-extrinsic effect of recipient sex on human B-cell reconstitution. Moreover, the PI humanized mouse model revealed HSC-intrinsic defects in central B-cell tolerance that recapitulated those in patients with autoimmune diseases. These results demonstrate the utility of humanized mouse models as a tool to better understand human immune cell development and regulation.

Variation in Airway Responsiveness of Male C57BL/6 Mice from 5 Vendors

PubMed Central

Chang, Herng-Yu Sucie; Mitzner, Wayne; Watson, Julie

2012-01-01

Mice are now the most commonly used animal model for the study of asthma. The mouse asthma model has many characteristics of the human pathology, including allergic sensitization and airway hyperresponsiveness. Inbred strains are commonly used to avoid variations due to genetic background, but variations due to rearing environment are not as well recognized. After a change in mouse vendors and a switch from C57BL/6J mice to C57BL/6N mice, we noted significant differences in airway responsiveness between the substrains. To further investigate the effect of vendor, we tested C57BL/6N mice from 3 other vendors and found significant differences between several of the substrains. To test whether this difference was due to genetic drift or rearing environment, we purchased new groups of mice from all 5 vendors, bred them in separate vendor-specific groups under uniform environmental conditions, and tested male first generation (F1) offspring at 8 to 10 wk of age. These F1 mice showed no significant differences in airway responsiveness, indicating that the rearing environment rather than genetic differences was responsible for the initial variation in pulmonary phenotype. The environmental factors that caused the phenotypic variation are unknown. However, differences between vendor in feed components, bedding type, or microbiome could have contributed. Whatever the basis, investigators using mouse models of asthma should be cautious in comparing data from mice obtained from different vendors. PMID:23043804

Tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury in different models possibly through suppression of NF-κB activation.

PubMed

Zhao, Zhanzhong; Tang, Xiangfang; Zhao, Xinghui; Zhang, Minhong; Zhang, Weijian; Hou, Shaohua; Yuan, Weifeng; Zhang, Hongfu; Shi, Lijun; Jia, Hong; Liang, Lin; Lai, Zhi; Gao, Junfeng; Zhang, Keyu; Fu, Ling; Chen, Wei

2014-07-01

Tylvalosin, a new broad-spectrum, third-generation macrolides, may exert a variety of pharmacological activities. Here, we report on its anti-oxidative and anti-inflammatory activity in RAW 264.7 macrophages and mouse treated with lipopolysaccharide (LPS) as well as piglet challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Tylvalosin treatment markedly decreased IL-8, IL-6, IL-1β, PGE2, TNF-α and NO levels in vitro and in vivo. LPS and PRRSV-induced reactive oxygen species (ROS) production, and the lipid peroxidation in mice lung tissues reduced after tylvalosin treatments. In mouse acute lung injury model induced by LPS, tylvalosin administration significantly attenuated tissues injury, and reduced the inflammatory cells recruitment and activation. The evaluated phospholipase A2 (PLA2) activity and the increased expressions of cPLA2-IVA, p-cPLA2-IVA and sPLA2-IVE were lowered by tylvalosin. Consistent with the mouse results, tylvalosin pretreatment attenuated piglet lung scores with improved growth performance and normal rectal temperature in piglet model induced by PRRSV. Furthermore, tylvalosin attenuated the IκBα phosphorylation and degradation, and blocked the NF-κB p65 translocation. These results indicate that in addition to its direct antimicrobial effect, tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury through suppression of NF-κB activation. Copyright © 2014 Elsevier Inc. All rights reserved.

Atmospheric Oxygen Inhibits Growth and Differentiation of Marrow-Derived Mouse Mesenchymal Stem Cells via a p53 Dependent Mechanism: Implications for Long-Term Culture Expansion

PubMed Central

Boregowda, Siddaraju; Krishnappa, Veena; Chambers, Jeremy; LoGrasso, Phillip V.; Lai, Wen-Tzu; Ortiz, Luis A.; Phinney, Donald G.

2013-01-01

Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long-term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A and BAX expression and mitochondrial ROS generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45−ve/CD44+ve cell fraction in marrow, produced increased MSC yields following immuno-depletion, and supported sustained MSC growth resulting in a 2300-fold increase in cumulative cell yield by 4th passage. MSCs cultured in 5% oxygen also exhibited enhanced tri-lineage differentiation. The oxygen-induced stress response was dependent upon p53 since siRNA mediated knockdown of p53 in wild type cells or exposure of p53−/− MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long-term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen-induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long-term expansion in vitro. PMID:22367737