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The multifunctionality of genome is suggested at some loci in different species but not well understood. Here we identified a DES-K16 region in an intron of the Kctd16 gene as the chromatin highly marked with epigenetic modifications of both enhancers (H3K4me1 and H3K27ac) and silencers (H3K27me3) in mouse spermatocytes. In vitro reporter gene assay demonstrated that DES-K16 exhibited significant enhancer activity in spermatocyte-derived GC-2spd(ts) and hepatic tumor-derived Hepa1-6 cells, and a deletion of this sequence in GC-2spd(ts) cells resulted in a decrease and increase of Yipf5 and Kctd16 expression, respectively. This was consistent with increased and decreased expression of Yipf5 and Kctd16, respectively, in primary spermatocytes during testis development. While known dual enhancer-silencers exert each activity in different tissues, our data suggest that DES-K16 functions as both enhancer and silencer in a single cell type, GC-2spd(ts) cells. This is the first report on a dual enhancer-silencer element which activates and suppresses gene expression in a single cell type., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Depletion of yeast/fly Ataxin-2 rescues TDP-43 overexpression toxicity. In mouse models of Amyotrophic Lateral Sclerosis via TDP-43 overexpression, depletion of its ortholog ATXN2 mitigated motor neuron degeneration and extended lifespan from 25 days to >300 days. There is another ortholog in mammals, named ATXN2L (Ataxin-2-like), which is almost uncharacterized but also functions in RNA surveillance at stress granules. We generated mice with Crispr/Cas9-mediated deletion of Atxn2l exons 5-8, studying homozygotes prenatally and heterozygotes during aging. Our novel findings indicate that ATXN2L absence triggers mid-gestational embryonic lethality, affecting female animals more strongly. Weight and development stages of homozygous mutants were reduced. Placenta phenotypes were not apparent, but brain histology showed lamination defects and apoptosis. Aged heterozygotes showed no locomotor deficits or weight loss over 12 months. Null mutants in vivo displayed compensatory efforts to maximize Atxn2l expression, which were prevented upon nutrient abundance in vitro. Mouse embryonal fibroblast cells revealed more multinucleated giant cells upon ATXN2L deficiency. In addition, in human neural cells, transcript levels of ATXN2L were induced upon starvation and glucose and amino acids exposure, but this induction was partially prevented by serum or low cholesterol administration. Neither ATXN2L depletion triggered dysregulation of ATXN2, nor a converse effect was observed. Overall, this essential role of ATXN2L for embryogenesis raises questions about its role in neurodegenerative diseases and neuroprotective therapies.

Background and Aims: Biliary atresia (BA) is a devastating neonatal cholangiopathy that progresses to fibrosis and end-stage liver disease by 2 years of age. Portoenterostomy may reestablish biliary drainage, but, despite drainage, virtually all afflicted patients develop fibrosis and progress to end-stage liver disease requiring liver transplantation for survival., Approach and Results: In the murine model of BA, rhesus rotavirus (RRV) infection of newborn pups results in a cholangiopathy paralleling human BA and has been used to study mechanistic aspects of the disease. Unfortunately, nearly all RRV-infected pups succumb by day of life 14. Thus, in this study we generated an RRV-TUCH rotavirus reassortant (designated as T R(VP2,VP4) ) that when injected into newborn mice causes an obstructive jaundice phenotype with lower mortality rates. Of the mice that survived, 63% developed Ishak stage 3-5 fibrosis with histopathological signs of inflammation/fibrosis and bile duct obstruction., Conclusions: This model of rotavirus-induced neonatal fibrosis will provide an opportunity to study disease pathogenesis and has potential to be used in preclinical studies with an objective to identify therapeutic targets that may alter the course of BA., (© 2019 The Authors. Hepatology published by Wiley Periodicals, Inc. on behalf of American Association for the Study of Liver Diseases.)

Patient-derived xenograft (PDX) models are created by engraftment of patient tumor tissues into immunocompetent mice. Since a PDX model retains the characteristics of the primary patient tumor including gene expression profiles and drug responses, it has become the most reliable in vivo human cancer model. The engraftment rate increases with the introduction of Non-obese diabetic Severe combined immunodeficiency (NOD/SCID)-based immunocompromised mice, especially the NK-deficient NOD strains NOD/SCID/interleukin-2 receptor gamma chain(IL2Rγ) null ( NOG/NSG) and NOD/SCID/Jak3(Janus kinase 3) null (NOJ). Success rates differ with tumor origin: gastrointestinal tumors acquire a higher engraftment rate, while the rate is lower for breast cancers. Subcutaneous transplantation is the most popular method to establish PDX, but some tumors require specific environments, e.g., orthotropic or renal capsule transplantation. Human hormone treatment is necessary to establish hormone-dependent cancers such as prostate and breast cancers. PDX mice with human hematopoietic and immune systems (humanized PDX) are powerful tools for the analysis of tumor-immune system interaction and evaluation of immunotherapy response. A PDX biobank equipped with patients' clinical data, gene-expression patterns, mutational statuses, tumor tissue architects, and drug responsiveness will be an authoritative resource for developing specific tumor biomarkers for chemotherapeutic predictions, creating individualized therapy, and establishing precise cancer medicine., Competing Interests: The authors declare no conflict of interest.

Producing hair cells of the inner ear is the major goal of ongoing research that combines advances in developmental and stem cell biology. The recent advent of an inner ear organoid protocol-resulting in three-dimensional stem cell-derived tissues resembling vestibular sensory epithelia-has sparked interest in applications such as regeneration, drug discovery, and disease modeling. In this study, we adapted this protocol for a novel mouse embryonic stem cell line with a fluorescent reporter for Pax2 expression. We used Pax2 EGFP/+ organoid formation to model otic induction, the pivotal developmental event when preplacodal tissue adopts otic fate. We found upregulation of Pax2 and activation of ERK downstream of fibroblast growth factor signaling in organoid formation as in embryonic inner ear development. Pax2 expression was evident from the EGFP reporter beginning at the vesicle formation stage and persisting through generation of the sensory epithelium. The native ventralizing signal sonic hedgehog was largely absent from the cell aggregates as otic vesicles began to form, confirming the dorsal vestibular organoid fate. Nonetheless, cochlear- or vestibular-like neurons appeared to delaminate from the derived otic vesicles and formed synaptic contacts with hair cells in the organoids. Cell lines with transcriptional reporters such as Pax2 EGFP/+ facilitate direct evaluation of morphological changes during organoid production, a major asset when establishing and validating the culture protocol.

The system-level identification and analysis of molecular networks in mammals can be accelerated by 'next-generation' genetics, defined as genetics that does not require crossing of multiple generations of animals in order to achieve the desired genetic makeup. We have established a highly efficient procedure for producing knock-in (KI) mice within a single generation, by optimizing the genome-editing protocol for KI embryonic stem (ES) cells and the protocol for the generation of fully ES-cell-derived mice (ES mice). Using this protocol, the production of chimeric mice is eliminated, and, therefore, there is no requirement for the crossing of chimeric mice to produce mice that carry the KI gene in all cells of the body. Our procedure thus shortens the time required to produce KI ES mice from about a year to ∼3 months. Various kinds of KI ES mice can be produced with a minimized amount of work, facilitating the elucidation of organism-level phenomena using a systems biology approach. In this report, we describe the basic technologies and protocols for this procedure, and discuss the current challenges for next-generation mammalian genetics in organism-level systems biology studies.

Dihydrofolate reductase (DHFR) is a critical enzyme in the folate metabolism pathway and also plays a role in regulating nitric oxide (NO) signaling in endothelial cells. Although both coding and noncoding mutations with phenotypic effects have been identified in the human DHFR gene, no mouse model is currently available to study the consequences of perturbing DHFR in vivo In order to identify genes involved in definitive hematopoiesis, we performed a forward genetic screen and produced a mouse line, here referred to as Orana, with a point mutation in the Dhfr locus leading to a Thr136Ala substitution in the DHFR protein. Homozygote Orana mice initiate definitive hematopoiesis, but expansion of progenitors in the fetal liver is compromised, and the animals die between embryonic day 13.5 (E13.5) and E14.5. Heterozygote Orana mice survive to adulthood but have tissue-specific alterations in folate abundance and distribution, perturbed stress erythropoiesis, and impaired endothelium-dependent relaxation of the aorta consistent with the role of DHFR in regulating NO signaling. Orana mice provide insight into the dual roles of DHFR and are a useful model for investigating the role of environmental and dietary factors in the context of vascular defects caused by altered NO signaling., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Mice cannot be used as a model to evaluate HIV-1 therapeutics because they do not become infected by HIV-1 due to structural differences between several human and mouse proteins required for HIV-1 replication. This has limited their use for in vivo assessment of anti-HIV-1 therapeutics and the mechanism by which cofactors, such as illicit drug use accelerate HIV-1 replication and disease course in substance abusers. Here, we describe the development and application of two in vivo humanized mouse models that are highly sensitive and useful models for the in vivo evaluation of candidate anti-HIV therapeutics. The first model, hu-spl-PBMC-NSG mice, uses NOD-SCID IL2rγ(-/-) (NSG) mice intrasplenically injected with human peripheral blood mononuclear cells (PBMC) which develop productive splenic HIV-1 infection after intrasplenic inoculation with a replication-competent HIV-1 expressing Renilla reniformis luciferase (HIV-LucR) and enables investigators to use bioluminescence to visualize and quantitate the temporal effects of therapeutics on HIV-1 infection. The second model, hCD4/R5/cT1 mice, consists of transgenic mice carrying human CD4, CCR5 and cyclin T1 genes, which enables murine CD4-expressing cells to support HIV-1 entry, Tat-mediated LTR transcription and consequently develop productive infection. The hCD4/R5/cT1 mice develop disseminated infection of tissues including the spleen, small intestine, lymph nodes and lungs after intravenous injection with HIV-1-LucR. Because these mice can be infected with HIV-LucR expressing transmitted/founder and clade A/E and C Envs, these mouse models can also be used to evaluate the in vivo efficacy of broadly neutralizing antibodies and antibodies induced by candidate HIV-1 vaccines. Furthermore, because hCD4/R5/cT1 mice can be infected by vaginal inoculation with replication-competent HIV-1 expressing NanoLuc (HIV-nLucR)-, this mouse model can be used to evaluate the mechanisms by which substance abuse and other factors enhance mucosal transmission of HIV-1.

Autophagy is a major intracellular degradation system by which cytoplasmic components are enclosed by autophagosomes and delivered to lysosomes. Formation of the autophagosome requires a set of autophagy-related (Atg) proteins. Among these proteins, the ULK1 complex, which is composed of ULK1 (or ULK2), FIP200, Atg13, and Atg101, acts at an initial step. Previous studies showed that ULK1 and FIP200 also function in pathways other than autophagy. However, whether Atg13 and Atg101 act similarly to ULK1 and FIP200 remains unknown. In the present study, we generated Atg13 knockout mice. Like FIP200-deficient mice, Atg13-deficient mice die in utero, which is distinct from most other types of Atg-deficient mice. Atg13-deficient embryos show growth retardation and myocardial growth defects. In cultured fibroblasts, Atg13 deficiency blocks autophagosome formation at an upstream step. In addition, sensitivity to tumor necrosis factor alpha (TNF-α)-induced apoptosis is enhanced by deletion of Atg13 or FIP200, but not by other Atg proteins, as well as by simultaneous deletion of ULK1 and ULK2. These results suggest that Atg13 has both autophagic and nonautophagic functions and that the latter are essential for cardiac development and likely shared with FIP200 but not with ULK1/2., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

microRNAs (miRNAs or miRS) have been demonstrated to be essential for neural development. miR-125b-2, presented on human chromosome 21, is overexpressed in neurons of individuals with Down syndrome (DS) with cognitive impairments. It has been reported that miR-125b-2 promotes specific types of neuronal differentiation; however, the function of miR-125b-2 in the early development of the embryo has remained to be fully elucidated. In the present study, a mouse embryonic stem cell (mESC) line was stably transfected with a miR-125b-2 lentiviral expression vector and found that miR-125b-2 overexpression did not affect the self-renewal or proliferation of mESCs. However, miR-125b-2 overexpression inhibited the differentiation of mESCs into endoderm and ectoderm. Finally, miR-125b-2 overexpression was found to impair all-trans-retinoic acid-induced neuron development in embryoid bodies. The findings of the present study implied that miR-125b-2 overexpression suppressed the differentiation of mESCs into neurons, which highlights that miR‑125b-2 is important in the regulation of ESC differentiation. The present study provided a basis for the further identification of novel targets of miR-125b-2, which may contribute to an enhanced understanding of the molecular mechanisms of ESC differentiation.

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mongolian gerbils (Meriones unguiculatus) are widely used as animal models for a variety of infectious diseases. However, immunological reagents such as cytokines have not been characterized. Two heterohybridomas, D9(E6)C2B3 and D9(E4), obtained by fusion of gerbil splenocytes with mouse myeloma cells (P3-X63-Ag8.653), expressed gerbil CD3G mRNA. These cells were suggested to be T cell heterohybridomas. They also expressed gerbil IL6 [D9(E6)C2B3] and TGFB [D9(E4) and D9(E6)C2B3] mRNAs. The addition of conditioned medium (CM) obtained from the culture of D9(E6)C2B3 significantly enhanced antibody secretion and expression of gerbil Cγ1 and Cε IGHC mRNAs in the B11D2(C2) heterohybridoma, which secretes gerbil IgG1. However, the addition of CM from both heterohybridomas did not improve in proliferation of B11D2(C2) cells. These results indicate that CM from D9(E6)C2B3 improved the culture of gerbil-mouse heterohybridomas, possibly by secreting gerbil IL6.

Background: The protein p49/STRAP (SRFBP1) is a transcription cofactor of serum response factor (SRF) which regulates cytoskeletal and muscle-specific genes., Results: Two conserved domains were found in the p49/STRAP protein. The SRF-binding domain was at its N-terminus and was highly conserved among mammalian species, xenopus and zebrafish. A BUD22 domain was found at its C-terminus in three sequence databases. The BUD22 domain was conserved among mammalian p49/STRAP proteins, and yeast cellular morphogenesis proteins, which is involved in ribosome biogenesis that affects growth rate and cell size. The endogenous p49/SRAP protein was localized mainly in the nucleus but also widely distributed in the cytoplasm, and was in close proximity to the actin. Transfected GFP-p49/STRAP protein co-localized with nucleolin within the nucleolus. Overexpression of p49/STRAP reduced actin content in cultured cells and resulted in smaller cell size versus control cells. Increased expression of p49/STRAP in transgenic mice resulted in newborns with malformations, which included asymmetric abdominal and thoracic cavities, and substantial changes in cardiac morphology. p49/STRAP altered the expression of certain muscle-specific genes, including that of the SRF gene, which is a key regulator of cardiac genes at the developmental, structural and maintenance level and has two SRE binding sites., Conclusions: Since p49/STRAP is a co-factor of SRF, our data suggest that p49/STRAP likely regulates cell size and morphology through SRF target genes. The function of its BUD22 domain warrants further investigation. The observed increase in p49/STRAP expression during cellular aging may contribute to observed morphological changes in senescence.

The observation of viral plaques is the standard method for determining the viral titer and understanding the behaviors of viruses. Here, we report the application of a wide field-of-view (FOV), time-lapse, on-chip imaging platform, termed the ePetri, for plaque analysis of murine norovirus 1 (MNV-1). The ePetri offers the ability to dynamically track plaques at the individual cell death event level over a wide FOV of 6 mm × 4 mm. As demonstration, we captured high-resolution time-lapse images of MNV-1-infected cells at 30 min intervals. We implemented a customized image-processing program containing a density-based clustering algorithm to analyze the spatial-temporal distribution of cell death events to identify plaques at their earliest stages. By using the results in a viral titer count format, we showed that our approach gives results that are comparable to conventional plaque assays. We further showed that the extra information collected by the ePetri can be used to monitor the dynamics of plaque formation and growth. Finally, we performed a demonstration experiment to show the relevance of such an experimental format for viral inhibitor study. We believe the ePetri is a simple and compact solution for the automation of viral plaque assays, plaque behavior analysis, and antiviral drug discovery and study.

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3'SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3'SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3'SIA-Le(X) identified in a previous glycan microarray screen.

A typical feature of scrapie in sheep and goats is the accumulation of disease-associated prion protein. Scrapie consists of many strains with different biological properties. Nine natural sheep scrapie cases were transmitted to wild-type mice and mouse-passaged isolates were classified into 2 types based on incubation time: short and long. These 2 types displayed a distinct difference in their pathology. We attempted to transmit these mouse-passaged isolates to 2 murine cell lines (GT1-7 and L929) to compare their properties. All of the isolates were transmitted to L929 cells. However, only mouse-passaged field isolates with a long incubation time were transmitted to GT1-7 cells. This specific susceptibility of GT1-7 cells was also confirmed with a primary-passaged isolate that was not completely adapted to the new host species. Characterization of the mechanisms of the specific susceptibility of GT1-7 cells to isolates with a long incubation time may lead to a greater understanding of the differences among prion strains.

On 29 March 2013, the Chinese Center for Disease Control and Prevention confirmed the first reported case of human infection with an avian influenza A(H7N9) virus. The recent human infections with H7N9 virus, totalling over 130 cases with 39 fatalities to date, have been characterized by severe pulmonary disease and acute respiratory distress syndrome (ARDS). This is concerning because H7 viruses have typically been associated with ocular disease in humans, rather than severe respiratory disease. This recent outbreak underscores the need to better understand the pathogenesis and transmission of these viruses in mammals. Here we assess the ability of A/Anhui/1/2013 and A/Shanghai/1/2013 (H7N9) viruses, isolated from fatal human cases, to cause disease in mice and ferrets and to transmit to naive animals. Both H7N9 viruses replicated to higher titre in human airway epithelial cells and in the respiratory tract of ferrets compared to a seasonal H3N2 virus. Moreover, the H7N9 viruses showed greater infectivity and lethality in mice compared to genetically related H7N9 and H9N2 viruses. The H7N9 viruses were readily transmitted to naive ferrets through direct contact but, unlike the seasonal H3N2 virus, did not transmit readily by respiratory droplets. The lack of efficient respiratory droplet transmission was corroborated by low receptor-binding specificity for human-like α2,6-linked sialosides. Our results indicate that H7N9 viruses have the capacity for efficient replication in mammals and human airway cells and highlight the need for continued public health surveillance of this emerging virus.

In multicellular organisms, developmental changes to replication timing occur in 400-800 kb domains across half the genome. While examples of epigenetic control of replication timing have been described, a role for DNA sequence in mammalian replication-timing regulation has not been substantiated. To assess the role of DNA sequences in directing developmental changes to replication timing, we profiled replication timing in mice carrying a genetically rearranged Human Chromosome 21 (Hsa21). In two distinct mouse cell types, Hsa21 sequences maintained human-specific replication timing, except at points of Hsa21 rearrangement. Changes in replication timing at rearrangements extended up to 900 kb and consistently reconciled with the wild-type replication pattern at developmental boundaries of replication-timing domains. Our results are consistent with DNA sequence-driven regulation of Hsa21 replication timing during development and provide evidence that mammalian chromosomes consist of multiple independent units of replication-timing regulation.

The SIV-infected rhesus macaque (Macaca mulatta) is the most established model of AIDS disease systems, providing insight into pathogenesis and a model system for testing novel vaccines. The understanding of cellular immune responses based on the identification and study of Major Histocompatibility Complex (MHC) molecules, including their MHC:peptide-binding motif, provides valuable information to decipher outcomes of infection and vaccine efficacy. Detailed characterization of Mamu-B*039:01, a common allele expressed in Chinese rhesus macaques, revealed a unique MHC:peptide-binding preference consisting of glycine at the second position. Peptides containing a glycine at the second position were shown to be antigenic from animals positive for Mamu-B*039:01. A similar motif was previously described for the D(d) mouse MHC allele, but for none of the human HLA molecules for which a motif is known. Further investigation showed that one additional macaque allele, present in Indian rhesus macaques, Mamu-B*052:01, shares this same motif. These "G2" alleles were associated with the presence of specific residues in their B pocket. This pocket structure was found in 6% of macaque sequences but none of 950 human HLA class I alleles. Evolutionary studies using the "G2" alleles points to common ancestry for the macaque sequences, while convergent evolution is suggested when murine and macaque sequences are considered. This is the first detailed characterization of the pocket residues yielding this specific motif in nonhuman primates and mice, revealing a new supertype motif not present in humans.

We isolated a novel murine norovirus (MNV), MT30-2 strain, from feces of conventional mice in Japan to evaluate the virucidal activity of four antiseptics. The MNV MT30-2 strain was inactivated by as little as 0.2% (w/v) povidone-iodine (PVP-I) and 0.1% (w/v) sodium hypochlorite (NaOCl) treatment as determined by a novel plaque assay. Importantly, PVP-I reduced the MNV titer by 4 log(10) within 15 s of exposure. The other two antiseptics, benzethonium chloride (BEC) and chlorhexidine gluconate (CHG), did not reduce the MNV titer even when treatment lasted for 60 s. When the virus titer was reduced by PVP-I or NaOCl treatment, the amount of MNV RNA was not reduced, indicating that the presence of viral RNA was not related to the virucidal activity of the antiseptics. PVP-I and NaOCl will be useful in controlling the spread of MNV, which is a common problem in mice colonies. In this study, we isolated a novel MNV and newly revealed that two antiseptics (PVP-I and NaOCl) were able to inactivate MNV at low concentrations and in a short contact time.

SARS coronavirus (SARS-CoV) causes severe acute respiratory tract disease characterized by diffuse alveolar damage and hyaline membrane formation. This pathology often progresses to acute respiratory distress (such as acute respiratory distress syndrome [ARDS]) and atypical pneumonia in humans, with characteristic age-related mortality rates approaching 50% or more in immunosenescent populations. The molecular basis for the extreme virulence of SARS-CoV remains elusive. Since young and aged (1-year-old) mice do not develop severe clinical disease following infection with wild-type SARS-CoV, a mouse-adapted strain of SARS-CoV (called MA15) was developed and was shown to cause lethal infection in these animals. To understand the genetic contributions to the increased pathogenesis of MA15 in rodents, we used reverse genetics and evaluated the virulence of panels of derivative viruses encoding various combinations of mouse-adapted mutations. We found that mutations in the viral spike (S) glycoprotein and, to a much less rigorous extent, in the nsp9 nonstructural protein, were primarily associated with the acquisition of virulence in young animals. The mutations in S likely increase recognition of the mouse angiotensin-converting enzyme 2 (ACE2) receptor not only in MA15 but also in two additional, independently isolated mouse-adapted SARS-CoVs. In contrast to the findings for young animals, mutations to revert to the wild-type sequence in nsp9 and the S glycoprotein were not sufficient to significantly attenuate the virus compared to other combinations of mouse-adapted mutations in 12-month-old mice. This panel of SARS-CoVs provides novel reagents that we have used to further our understanding of differential, age-related pathogenic mechanisms in mouse models of human disease.

Background: Focal Dermal Hypoplasia (FDH) is a genetic disorder characterized by developmental defects in skin, skeleton and ectodermal appendages. FDH is caused by dominant loss-of-function mutations in X-linked PORCN. PORCN orthologues in Drosophila and mice encode endoplasmic reticulum proteins required for secretion and function of Wnt proteins. Wnt proteins play important roles in embryo development, tissue homeostasis and stem cell maintenance. Since features of FDH overlap with those seen in mouse Wnt pathway mutants, FDH likely results from defective Wnt signaling but molecular mechanisms by which inactivation of PORCN affects Wnt signaling and manifestations of FDH remain to be elucidated., Results: We introduced intronic loxP sites and a neomycin gene in the mouse Porcn locus for conditional inactivation. Porcn-ex3-7flox mice have no apparent developmental defects, but chimeric mice retaining the neomycin gene (Porcn-ex3-7Neo-flox) have limb, skin, and urogenital abnormalities. Conditional Porcn inactivation by EIIa-driven or Hprt-driven Cre recombinase results in increased early embryonic lethality. Mesenchyme-specific Prx-Cre-driven inactivation of Porcn produces FDH-like limb defects, while ectodermal Krt14-Cre-driven inactivation produces thin skin, alopecia, and abnormal dentition. Furthermore, cell-based assays confirm that human PORCN mutations reduce WNT3A secretion., Conclusions: These data indicate that Porcn inactivation in the mouse produces a model for human FDH and that phenotypic features result from defective WNT signaling in ectodermal- and mesenchymal-derived structures.

Background: Jamestown Canyon virus (JCV), family Bunyaviridae, is a mosquito-borne pathogen endemic in the United States and Canada that can cause encephalitis in humans and is considered an emerging threat to public health. The virus is genetically similar to Inkoo virus circulating in Europe, suggesting that much of the northern hemisphere contains JCV or similar variants., Results: We have completed the sequence of three isolates of JCV collected in geographically diverse locations over a 57 year time span. The nucleotide identity for the three strains is 90, 83, and 85% for the S, M, and L segments respectively whereas the percent identify for the predicted amino acid sequences of the N, NSS, M poly, GN, NSM, GC, and L proteins was 97, 91, 94, 98, 91, 94, and 97%, respectively. In Swiss Webster mice, each JCV isolate exhibits low neuroinvasiveness but high infectivity. Two of the three JCV isolates were highly neurovirulent after IC inoculation whereas one isolate, JCV/03/CT, exhibited low neurovirulence. In rhesus monkeys, JCV infection is accompanied by a low-titered viremia, lack of clinical disease, but a robust neutralizing antibody response., Conclusions: The first complete sequence of JCV is reported for three separate isolates, and a relatively high level of amino acid sequence conservation was observed even for viruses isolated 57 years apart indicating that the virus is in relative evolutionary stasis. JCV is highly infectious for mice and monkeys, and these animals, especially mice, represent useful experimental hosts for further study.

Background: Rotaviruses are the single most important cause of severe diarrhea in young children worldwide. The current study was conducted to assess whether colostrum containing rotavirus-specific antibodies (Gastrogard-R®) could protect against rotavirus infection. In addition, this illness model was used to study modulatory effects of intervention on several immune parameters after re-infection., Methods: BALB/c mice were treated by gavage once daily with Gastrogard-R® from the age of 4 to 10 days, and were inoculated with rhesus rotavirus (RRV) at 7 days of age. A secondary inoculation with epizootic-diarrhea infant-mouse (EDIM) virus was administered at 17 days of age. Disease symptoms were scored daily and viral shedding was measured in fecal samples during the post-inoculation periods. Rotavirus-specific IgM, IgG and IgG subclasses in serum, T cell proliferation and rotavirus-specific delayed-type hypersensitivity (DTH) responses were also measured., Results: Primary inoculation with RRV induced a mild but consistent level of diarrhea during 3-4 days post-inoculation. All mice receiving Gastrogard-R® were 100% protected against rotavirus-induced diarrhea. Mice receiving both RRV and EDIM inoculation had a lower faecal-viral load following EDIM inoculation then mice receiving EDIM alone or Gastrogard-R®. Mice receiving Gastrogard-R® however displayed an enhanced rotavirus-specific T-cell proliferation whereas rotavirus-specific antibody subtypes were not affected., Conclusions: Preventing RRV-induced diarrhea by Gastrogard-R® early in life showed a diminished protection against EDIM re-infection, but a rotavirus-specific immune response was developed including both B cell and T cell responses. In general, this intervention model can be used for studying clinical symptoms as well as the immune responses required for protection against viral re-infection.

Small and large intestinal epithelial cell (IEC) lines were established from adult murine intestinal crypts. Both established small and large IECs line (named aMoS7 and aMoC1 respectively) expressed epithelial markers. Similarly to IECs isolated from adult mouse intestines, the expression of major histocompatibility complex (MHC) class II molecules was induced by interferon-γ-treatment in both established cell lines. This expression of MHC class II molecules was higher in small intestinal aMoS7 cells than in large intestinal aMoC1 cells. Treatment with lipopolysaccharide and with ligands of Toll-like receptors 1, 2, 3, and 7 induced secretion of interleukin-6 from both adult IEC lines. These results suggest that the aMoS7 and aMoC1 cell lines can serve as useful tools in analyzing the immunological functions of IECs, especially in studying the IEC response to microbial components and its antigen presenting ability.

Tescalcin, an EF-hand calcium binding protein that regulates the Na(+)/H(+) exchanger 1 (NHE1), is highly expressed in various mouse tissues such as heart and brain. Despite its potentially important role in cell physiology, the mechanisms that regulate tescalcin gene (Tesc) expression are unknown. In this study, we report two new Tesc mRNA variants (V2 and V3) and characterize the mouse Tesc promoter. The V2 and V3 transcripts result from alternative splicing of intron 5. Our results show that Tesc mRNA variants are expressed in various mouse tissues. Primer extension analysis located the transcription start site at 94 nucleotides upstream of the translation start codon. The DNA nucleotide sequence of the 5'-flanking region contained a CpG island spanning the promoter region from nucleotides -372 to +814, a canonical TATA box (-38/-32), and putative transcription factor binding sites for Sp1, EGR1, ZBP-89, KLF3, MZF1, AP2, ZF5, and CDF-1. Transient transfection of the Y1 and msc-1 cell lines with a series of 5'-deleted promoter constructs indicated that the minimal promoter region was between nucleotides -130 and -40. Electrophoresis mobility shift assays, supershift assays, and mutation studies demonstrated that Sp1 and Sp3 bind to the GC-rich motifs, a CACCC box and three GC boxes, located within the Tesc proximal promoter. Nonetheless, mutations that abolished interaction of Sp1 and Sp3 with the GC-rich motifs located within the minimal promoter region did not abrogate promoter activity in Y1 cells. Mithramycin A, an inhibitor of Sp1-DNA interaction, reduced Tesc promoter activity in msc-1 cells in a dose-dependent manner. Sp3 was a weaker transactivator compared to Sp1 in Drosophila D.mel-2 cells. However, when Sp1 and Sp3 were coexpressed, they transactivated the Tesc promoter in a synergistic manner. In Y1 cells, mutation analysis of a putative ZF5 motif located within the Tesc minimal promoter indicated that this motif was critical for activity of Tesc promoter. Taken together, the data demonstrated that Sp1 and Sp3 transcription factors cooperate positively in the regulation of Tesc promoter, and that the putative ZF5 motif is critical for its activation., (Copyright 2010 Elsevier B.V. All rights reserved.)

Accurate chromosome segregation during meiosis requires that homologous chromosomes pair and become physically connected so that they can orient properly on the meiosis I spindle. These connections are formed by homologous recombination closely integrated with the development of meiosis-specific, higher-order chromosome structures. The yeast Pch2 protein has emerged as an important factor with roles in both recombination and chromosome structure formation, but recent analysis suggested that TRIP13, the mouse Pch2 ortholog, is not required for the same processes. Using distinct Trip13 alleles with moderate and severe impairment of TRIP13 function, we report here that TRIP13 is required for proper synaptonemal complex formation, such that autosomal bivalents in Trip13-deficient meiocytes frequently displayed pericentric synaptic forks and other defects. In males, TRIP13 is required for efficient synapsis of the sex chromosomes and for sex body formation. Furthermore, the numbers of crossovers and chiasmata are reduced in the absence of TRIP13, and their distribution along the chromosomes is altered, suggesting a role for TRIP13 in aspects of crossover formation and/or control. Recombination defects are evident very early in meiotic prophase, soon after DSB formation. These findings provide evidence for evolutionarily conserved functions for TRIP13/Pch2 in both recombination and formation of higher order chromosome structures, and they support the hypothesis that TRIP13/Pch2 participates in coordinating these key aspects of meiotic chromosome behavior., Competing Interests: The authors have declared that no competing interests exist.

Bacterial CpG motifs are known to induce both innate and adaptive immunity in infected hosts via toll-like receptor 9 (TLR9). Because small oligonucleotides (ODNs) mimicking bacterial CpG motifs are easily synthesized, they have found use as immunomodulatory agents in a number of disease models. We have developed a novel bioinformatics approach to identify effective CpG ODN sequences and evaluate their function as TLR9 ligands in a murine system. Among the CpG ODNs we identified, M5-30 and M6-395 showed significant ability to stimulate TNF-alpha and IFN-gamma production in a mouse macrophage cell line and mouse splenocytes, respectively. We also found that these CpG ODNs activated cells through the canonical NF-kappa B signaling pathway. Moreover, both CpG ODNs were able to induce Th1-mediated immunity in Mycobacterium tuberculosis (Mtb)-infected mice. Our results demonstrate that M5-30 and M6-395 function as TLR9-specific ligands, making them useful in the study of TLR9 functionality and signaling in mice.

Evolutionary expansion of a gene family may occur at both the DNA and RNA levels. The rat testis-specific Rtdpoz-T2 and -T1 (rT2 and rT1) retrogenes are members of the TD/POZ gene family which also includes the well-characterized SPOP gene. In this study, rT2/rT1 transcriptional activation in cancer cells is demonstrated; the cancer rT2/rT1 transcripts are structurally similar to the embryonic transcripts reported previously in frequent exonization of transposed elements. On database interrogation, we have identified an uncharacterized rT2/rT1-like SPOP paralog, designated as SPOP-like (SPOPL), in the human and rodent genomes. Ka/Ks analysis indicates that the SPOPL genes are under functional constraints implicating biological functions. Phylogenetic analyses further suggest that segmental duplication and retrotransposition events had occurred giving rise to new gene members or retrogenes in the human-rodent ancestors during the evolution of the TD/POZ gene family. Based on this and previous works, a model is proposed to map the routes of evolutionary expansion of the TD/POZ gene family. More importantly, different gene expression patterns of members of the family are depicted: intron-harboring members are ubiquitously expressed whereas retrogenes are expressed in tissue-specific and developmentally regulated manner, and are fortuitously re-activated in cancer cells involving exonization of transposed elements., (Copyright 2010 Elsevier B.V. All rights reserved.)

Based on the online bioinformatics analysis and alignment results, three RNAi sequences target to the Mus musculus Bicc1 gene were obtained. The three interference fragments were synthesized and cloned into pRS-Hush shRNA Vector. The Bicc1 eukaryotic expression vector pEGFP-C3-Bicc1 was constructed, tagging the GFP to the N-terminal of the Bicc1 protein. The pEGFP-C3-Bicc1 and three pRS-Hush-RNAi were co-transfected into the cultured HEK-293 cells line, respectively. The two RNAi (pRS-Hush-RNAi-Bicc1-N/-C) that could knock-down the Bicc1 expression levels in HEK-293 cells significantly were confirmed by cell immunofluorescent staining, semi-quantitative PCR and Western blotting. The results demonstrate that we have successfully obtained two efficent Bicc1 RNAi sequences, which lays a foundation for further studying on the construction of Bicc1 knock-down stable cell lines and biological function of mouse Bicc1 product.

Rodents, mice and rats in particular, are the species of choice for evaluating chemical carcinogenesis. However, different species and strains often respond very differently, undermining the logic of extrapolation of animal results to humans and complicating risk assessment. Intracisternal A particles (IAPs), endogenous retroviral sequences, are an important class of transposable elements that induce genomic mutations and cell transformation by disrupting gene expression. Several lines of evidence support a role of IAPs as mouse-specific genetic factors in responses to toxicity and expression of disease phenotypes. Since multiple subtypes and copies of IAPs are present in the mouse genome, their activity and locations relative to functional genes are of critical importance. This study identified the major "active" subtypes of IAPs (subtype 1/1a) that are responsible for newly transposed IAP insertions described in the literature, and confirmed that (1) polymorphisms for IAP insertions exist among different mouse strains and (2) promoter activity of the LTRs can be modulated by chemicals. This study further identified all the genes in the C57BL/6 mouse genome with IAP subtype 1 and 1a sequences inserted in their proximity, and the major biofunctional categories and cellular signaling networks of those genes. Since many "IAP-associated genes" play important roles in the regulation of cell proliferation, cell cycle, and cell death, the associated IAPs, upon activation, can affect cellular responses to xenobiotics and disease processes, especially carcinogenesis. This systemic analysis provides a solid foundation for further investigations of the role of IAPs as species- and strain-specific disease susceptibility factors., (2009 Wiley-Liss, Inc.)

The attachment of murine norovirus 1 (MNV) in biosolids, swine manure, and dairy manure to Romaine lettuce and internalization of this virus were evaluated. The MNV in animal manures had behavior similar to that of pure MNV; however, MNV in biosolids had significantly higher levels of attachment and internalization than pure MNV or MNV in manures. The incubation time did not affect the attachment of MNV in biosolids or manure. Confocal microscopy was used to observe MNV on lettuce after SYBR gold-labeled MNV was added directly to lettuce or after lettuce was submersed in labeled virus. MNV was observed on the lettuce surface, inside open cuts, and occasionally within stomata. In general, lettuce pieces with a long cut on the edge and short cuts on the stem was more likely to contain internalized MNV than intact lettuce pieces, as observed by confocal microscopy; however, while the difference was visible, it was not statistically significant. This study showed that the presence of MNV in biosolids may increase the risk of fresh produce contamination and that the MNV in open cuts and stomata is likely to be protected from sanitization.

Podosome-type adhesions are actin-based membrane protrusions involved in cell-matrix adhesion and extracellular matrix degradation. Despite growing knowledge of many proteins associated with podosome-type adhesions, much remains unknown concerning the function of podosomal proteins at the level of the whole animal. In this study, the spontaneous mouse mutant nee was used to identify a component of podosome-type adhesions that is essential for normal postnatal growth and development. Mice homozygous for the nee allele exhibited runted growth, craniofacial and skeletal abnormalities, ocular anterior segment dysgenesis, and hearing impairment. Adults also exhibited infertility and a form of lipodystrophy. Using genetic mapping and DNA sequencing, the cause of nee phenotypes was identified as a 1-bp deletion within the Sh3pxd2b gene on mouse Chromosome 11. Whereas the wild-type Sh3pxd2b gene is predicted to encode a protein with one PX domain and four SH3 domains, the nee mutation is predicted to cause a frameshift and a protein truncation altering a portion of the third SH3 domain and deleting all of the fourth SH3 domain. The SH3PXD2B protein is believed to be an important component of podosomes likely to mediate protein-protein interactions with membrane-spanning metalloproteinases. Testing this directly, SH3PXD2B localized to podosomes in constitutively active Src-transfected fibroblasts and through its last SH3 domain associated with a transmembrane member of a disintegrin and metalloproteinase family of proteins, ADAM15. These results identify SH3PXD2B as a podosomal-adaptor protein required for postnatal growth and development, particularly within physiologic contexts involving extracellular matrix regulation.

Background: Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF). Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency., Results: By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i) LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [Pluri genes], ii) LIF induced genes [Lifind genes] whose expression is differentially regulated depending upon cell context and iii) genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of Pluri and Lifind genes, and revealed their differential modulation by Oct4 or Nanog master genes. Individual knock down of a selection of Pluri and Lifind genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed., Conclusion: We have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment), which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG) is still expressed and which might be down-regulated together for driving cells towards differentiation.

Contemporary influenza B virus strains were generated encoding C-terminally truncated NS1 proteins. Viable viruses containing the N-terminal 14, 38, 57 or 80 aa of the NS1 protein were rescued in Vero cells. The influenza B virus NS1-truncated mutants were impaired in their ability to counteract interferon (IFN) production, induce antiviral pro-inflammatory cytokines early after infection and show attenuated or restricted growth in IFN-competent hosts. In Vero cells, all of the mutant viruses replicated to high titres comparable to the wild-type influenza B virus. Mice that received a single, intranasal immunization of the NS1-truncated mutants elicited an antibody response and protection against wild-type virus challenge. Therefore, these NS1-truncated mutants should prove useful as potential candidates for live-attenuated influenza virus vaccines.

The serotonin (5-HT) 5-HT(7) receptors are expressed in both the central nervous system and in peripheral tissues. Receptor distribution studies and pharmacological studies have established that 5-HT(7) receptors play an important role in the control of circadian rhythms and thermoregulation. Selective 5-HT(7) receptor ligands have potential therapeutic applications for the treatment of pain and migraine, schizophrenia, anxiety, cognitive disturbances and inflammation. We have cloned two novel C-terminal splice variants of the 5-HT(7) receptor from mouse brain. These two new splice variants have almost identical sequences as the rat 5-HT(7(b)) and 5-HT(7(c)) splice variants and so were given the same name. Ligand binding assays ([(3)H]5-CT), membrane localization and functional studies in transiently transfected cells indicated that all three splice variants are well expressed on the membrane and no major differences in their respective pharmacology and their ability to activate adenylyl cyclase were observed. This is in analogy with previous reports comparing either the rat or the human variants.

Parietal epithelial cells (PEC) are major constituents of crescents in crescentic glomerulonephritis. The purpose of these studies was to establish an immortalized PEC cell line with similar characteristics to PEC in vivo for use in future mechanistic studies. Glomeruli were isolated from H-2Kb tsA58 transgenic mice (ImmortoMouse) by standard differential sieving, and several candidate PEC cell lines were obtained by subcloning outgrowths of cells from capsulated glomeruli. One clone, designated mouse PEC (mPEC), was extensively characterized. mPEC exhibited a compact cell body with typical epithelial morphology when grown in permissive conditions, but the cell shape changed to polygonal after 14 d in growth-restrictive conditions. mPEC but not podocytes used as a negative control expressed claudin-1, claudin-2, and protein gene product 9.5, which are proteins specific to PEC in vivo, and did not express the podocyte-specific proteins synaptopodin and nephrin. The junctional proteins zonula occludens-1 and beta-catenin stained positively in both mPEC and podocytes, but the staining pattern at cell-cell contacts was intermittent in mPEC and linear in podocytes. Finally, mPEC had thin bundled cortical F-actin filaments and no F-actin projections compared with podocytes, which exhibited thick bundled cortical F-actin filaments and interdigitating F-actin projections at cell-cell contacts. We conclude that immortalized mPEC in culture exhibit specific features of PEC in vivo and that these cells are distinct from podocytes, despite having the same mesenchymal origin. This mPEC line will assist in future mechanistic studies of PEC and enhance our understanding of glomerular injury.

A reduction in the DNA modification of cytosine methylation has been linked directly to chromosome rearrangements concomitant with retroelement amplification in several marsupial hybrid genomes. While phenotypes observed for interspecific eutherian hybrids are suggestive of methylation perturbations and retroelement instability, no link between retroelements, DNA methylation, and chromosome instability has yet been identified. Previous studies in eutherian hybrids, however, have been limited to a gross examination of methylation using methylation-sensitive restriction enzyme analysis or focused on single-copy genes and/or have avoided examination of repetitive DNA. Methylation changes and retroelements are proposed as mechanisms for double minute chromosome formation and oncogene amplification, both present in the genome of a Mus hybrid model, thus making it an ideal system to evaluate methylation status more closely. We have used the PCR-based methodologies methylation-sensitive amplicon subtraction (MS-AS) and methylation-sensitive representational difference analysis (MS-RDA) to detect differentially methylated sequences between three complex genomes and to isolate methylation perturbations in a Mus musculusxMus caroli hybrid. This novel application of MS-AS and MS-RDA resulted in the isolation of differentially methylated retroelements surrounding the locus on Chromosome 10 responsible for double minute chromosome formation within this interspecific eutherian hybrid.

In mammals, the hormonal regulation of water homeostasis is mediated by the aquaporin-2 water channel (Aqp2) of the collecting duct (CD). Vasopressin induces redistribution of Aqp2 from intracellular vesicles to the apical membrane of CD principal cells, accompanied by increased water permeability. Mutations of AQP2 gene in humans cause both recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. In this study, we generated a line of mice with the distal COOH-terminal tail of the Aqp2 deleted (Aqp2(Delta230)), including the protein kinase A phosphorylation site (S256), but still retaining the putative apical localization signal (221-229) at the COOH-terminal. Mice heterozygous for the truncation appear normal. Homozygotes are viable to adulthood, with reduced urine concentrating capacity, increased urine output, decreased urine osmolality, and increased daily water consumption. Desmopressin increased urine osmolality in wild-type mice but had no effect on Aqp2(Delta230/Delta230) mice. Kidneys from affected mice showed CD and pelvis dilatation and papillary atrophy. By immunohistochemical and immunoblot analyses using antibody against the NH(2)-terminal region of the protein Aqp2(Delta230/Delta230) mice had a markedly reduced protein abundance. Expression of the truncated protein in MDCK cells was consistent with a small amount of functional expression but no stimulation. Thus we have generated a mouse model of NDI that may be useful in studying the physiology and potential therapy of this disease.

MATE1 was the first mammalian example of the multidrug and toxin extrusion (MATE) protein family to be identified. Human MATE1 (hMATE1) is predominantly expressed and localized to the luminal membranes of the urinary tubules and bile canaliculi and mediates H(+)-coupled electroneutral excretion of toxic organic cations (OCs) into urine and bile (Otsuka M, Matsumoto T, Morimoto R, Arioka S, Omote H, and Moriyama Y. Proc Natl Acad Sci USA 102: 17923-17928, 2005). mMATE1, a mouse MATE ortholog, is also predominantly expressed in kidney and liver, although its transport properties are not yet characterized. In the present study, we investigated the transport properties and localization of mMATE1. Upon expression of this protein in HEK-293 cells, mMATE1 mediated electroneutral H(+)/tetraethylammonium exchange and showed a substrate specificity similar to that of hMATE1. Immunological techniques with specific antibodies against mMATE1 combined with RT-PCR revealed that mMATE1 is also expressed in various cells, including brain glia-like cells and capillaries, pancreatic duct cells, urinary bladder epithelium, adrenal gland cortex, alpha cells of the islets of Langerhans, Leydig cells, and vitamin A-storing Ito cells. These results indicate that mMATE1 is a polyspecific H(+)/OC exchanger. The unexpectedly wide distribution of mMATE1 suggests involvement of this transporter protein in diverse biological functions other than excretion of OCs from the body.

PCR-based testing for infectious agents in mouse cell lines and tissues has recently been developed as an alternative to the traditional MAP test. One drawback to currently available PCR-based assays is the lack of appropriate positive controls for PCR detection of the infectious agents. When negative samples are the norm and positive controls are absent, it is very difficult to feel confident detecting infectious agents. To alleviate this problem, the authors developed a panel of primers and positive-control DNA plasmids that enable rapid testing of biological samples, such as cell lines, tissues, or animal sera, for presence of the infectious agents most damaging to mouse colonies.

High-throughput gene trapping is a random approach for inducing insertional mutations across the mouse genome. This approach uses gene trap vectors that simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene. Gene trapping has been used by both public and private institutions to produce libraries of embryonic stem (ES) cells harboring mutations in single genes. Presently, approximately 66% of the protein coding genes in the mouse genome have been disrupted by gene trap insertions. Among these, however, genes encoding signal peptides or transmembrane domains (secretory genes) are underrepresented because they are not susceptible to conventional trapping methods. Here, we describe a high-throughput gene trapping strategy that effectively targets secretory genes. We used this strategy to assemble a library of ES cells harboring mutations in 716 unique secretory genes, of which 61% were not trapped by conventional trapping, indicating that the two strategies are complementary. The trapped ES cell lines, which can be ordered from the International Gene Trap Consortium (http://www.genetrap.org), are freely available to the scientific community.

We previously reported that CORS26 gene, isolated from C3H10T1/2 cells treated with transforming growth factor-beta1, was predominantly expressed in cartilage. Because the gene product is a kind of secretory protein produced by cartilage tissue, we named it "cartducin". Cartducin shares a similar modular organization to adipocyte-derived hormone, adiponectin. In this study, we investigated cartducin function during chondrogenesis and cartilage development. In situ hybridization analysis showed that cartducin transcripts were restricted to the proliferating chondrocytes in the growth plate cartilage. Whole-mount in situ hybridization revealed that the first significant induction of cartducin expression occurred in the sclerotome, which contains a chondrogenic cell lineage between days 9.5 and 10.5 postcoitus (p.c.) during mouse embryogenesis. Chondrogenic differentiation by combined treatment with bone morphogenetic protein-2 and insulin induced cartducin expression along with type II and IX collagen expression in chondrogenic progenitor N1511 cells. To elucidate the direct action of cartducin on the cells, recombinant cartducin protein was expressed in and purified from Escherichia coli. The recombinant cartducin potentially forms homo-oligomers and promoted the proliferation of chondrogenic progenitor N1511 cells, and chondrocytic HCS-2/8 cells in a dose-dependent manner. On the other hand, cartducin did not affect the production of sulfated glycosaminoglycan (sGAG) in these cells. These findings indicate that cartducin is a novel growth factor and plays important roles in regulating both chondrogenesis and cartilage development by its direct stimulatory action on the proliferation of chondrogenic precursors and chondrocytes., (Copyright (c) 2005 Wiley-Liss, Inc.)

Gene trapping is a method of generating murine embryonic stem (ES) cell lines containing insertional mutations in known and novel genes. A number of international groups have used this approach to create sizeable public cell line repositories available to the scientific community for the generation of mutant mouse strains. The major gene trapping groups worldwide have recently joined together to centralize access to all publicly available gene trap lines by developing a user-oriented Website for the International Gene Trap Consortium (IGTC). This collaboration provides an impressive public informatics resource comprising approximately 45 000 well-characterized ES cell lines which currently represent approximately 40% of known mouse genes, all freely available for the creation of knockout mice on a non-collaborative basis. To standardize annotation and provide high confidence data for gene trap lines, a rigorous identification and annotation pipeline has been developed combining genomic localization and transcript alignment of gene trap sequence tags to identify trapped loci. This information is stored in a new bioinformatics database accessible through the IGTC Website interface. The IGTC Website (www.genetrap.org) allows users to browse and search the database for trapped genes, BLAST sequences against gene trap sequence tags, and view trapped genes within biological pathways. In addition, IGTC data have been integrated into major genome browsers and bioinformatics sites to provide users with outside portals for viewing this data. The development of the IGTC Website marks a major advance by providing the research community with the data and tools necessary to effectively use public gene trap resources for the large-scale characterization of mammalian gene function.

We describe a versatile genetic system for rapid analysis of mammalian gene function. In this, loss of reporter activity in a novel embryonic stem (ES) cell line enables rapid identification of targeting to the ubiquitously expressed Rosa26 locus. Subsequent regulation of gene activity is governed by a dual regulatory strategy utilizing two drugs, Tamoxifen and Doxycycline. To illustrate this approach, a dominant allele of Smoothened was introduced into this cell line, enabling regulated activation of Hedgehog signaling. By coupling Cre-loxP dependent activation with tetracycline dependent transcription in a single allele, we established a conditional method to control Smoothened activity and neural progenitor specification in differentiating ES cells in vitro and in chimeric embryos in vivo When crossed to an appropriate Cre driver strain, gene activity can also be temporally regulated within a specific cell lineage. This platform will facilitate rapid analysis of gene function in the mouse.

Pluripotent embryonic stem (ES) cells cultivated as cellular aggregates, so called embryoid bodies (EBs), differentiate spontaneously into different cell types of all three germ layers in vitro resembling processes of cellular differentiation during embryonic development. Regarding chondrogenic differentiation, murine ES cells differentiate into progenitor cells, which form pre-cartilaginous condensations in the EB-outgrowths and express marker molecules characteristic for mesenchymal cell types such as Sox5 and Sox6. Later, mature chondrocytes appear which express collagen type II, and the collagen fibers show a typical morphology as demonstrated by electron-microscopical analysis. These mature chondrogenic cells are organized in cartilage nodules and produce large amounts of extracellular proteoglycans as revealed by staining with cupromeronic blue. Finally, cells organized in nodules express collagen type X, indicating the hypertrophic stage. In conclusion, differentiation of murine ES cells into chondrocytes proceeds from the undifferentiated stem cell via progenitor cells up to mature chondrogenic cells, which then undergo hypertrophy. Furthermore, because the ES-cell-derived chondrocytes did not express elastin, a marker for elastic cartilage tissue, we suggest the cartilage nodules to resemble hyaline cartilage tissue.

Red-green color vision in primates is unique in the sense that it is mediated by two photoreceptor cells that are indistinguishable in all aspects except for their visual pigments. In order to generate an animal model for investigation of the interaction between red-green inputs at the molecular level, we applied knock-in technology and X-chromosome inactivation machinery to make a mouse model with cone cells possessing visual pigments with different spectral sensitivities. We introduced a S308A point mutation into the Green opsin gene allele on the X-chromosome. This manipulation generated a 24 nm red-shift of absorption maximum in the cone pigment with negligible functional differences in other molecular properties. Amplitudes of responses in ERG and ganglion cell recordings of homozygotes were similar to those of wild-types, although the spectral sensitivities differed. Heterozygotes showed variable spectral sensitivities of ganglion cell responses due to the different integration of the native and the S308A cone inputs on the dendritic fields. In situ hybridization experiments showed that cone cells with respective pigments formed patch-like clusters of specific L cone-types, approximately 30 mum in diameter, which were randomly distributed in the dorsal region of the retinas. Since the patch-like clustering was arranged by X-inactivation, such clustering could be present in the peripheral retinas of New World monkeys with polymorphic L pigments, indicating that our mice would be a suitable model to study evolution of the mammalian color vision system.

A type of retroviral gene trap vectors has been developed that can induce conditional mutations in most genes expressed in mouse embryonic stem (ES) cells. The vectors rely on directional site-specific recombination systems that can repair and re-induce gene trap mutations when activated in succession. After the gene traps are inserted into the mouse genome, genetic mutations can be produced at a particular time and place in somatic cells. In addition to their conditional features, the vectors create multipurpose alleles amenable to a wide range of post-insertional modifications. Here we have used these directional recombination vectors to assemble the largest library of ES cell lines with conditional mutations in single genes yet assembled, presently totaling 1,000 unique genes. The trapped ES cell lines, which can be ordered from the German Gene Trap Consortium, are freely available to the scientific community.

Tap, a member of the evolutionarily conserved nuclear RNA export factor (NXF) family of proteins, has been implicated in the nuclear export of bulk poly(A)+ RNAs. cDNAs encoding the mouse NXF proteins (Tap, NXF7, NXF2, and NXF3) were prepared and the gene products were characterized in terms of their genomic organization, expression patterns, and biochemical properties. Mouse Tap was found to be ubiquitously expressed, whereas tissue- and developmental stage specific expression of mouse Nxf2, Nxf3, and Nxf7 was observed. Although mouse Tap and NXF2 bound to the phenylalanine-glycine repeat sequences of nucleoporins, NXF7 and NXF3 did not. GFP-tagged mouse Tap and NXF2 were localized predominantly in the nucleus. In contrast, GFP-tagged NXF7 and NXF3 were localized exclusively in the cytoplasm. As shown for the human counterpart, disruption of the leucine-rich nuclear export signal or leptomycin B treatment abolishes the cytoplasmic localization of mouse NXF3. p15/NXT1, an essential cofactor for human Tap in the export of mRNAs, was able to bind to mouse Tap, NXF2, and NXF3, but NXF7 did not form a stable heterodimeric complex. Transient transfection experiments indicated that only mouse Tap and NXF2 enhance the nuclear export of an otherwise inefficiently exported mRNA substrate. The orthologous relationship between human and mouse Nxf genes is discussed on the basis of these data.

The growing use of mouse embryonic stem (ES) cells in research emphasizes their importance in studies of molecular mechanisms that maintain pluripotency and direct cellular differentiation. Although systems for regulatable transgene expression are essential for fine analysis of cellular processes at the molecular level, a strategy for the establishment of multiple ES cell lines carrying any of these systems has not yet been described. Here, we report our development of the ROSA-TET system, an effective system for the establishment of multiple ES cell lines carrying a tetracycline (Tc)-regulatable transgene at the Gt (ROSA)26asSor (ROSA26) locus. This system contains a knock-in step of a construct carrying both loxP and its mutant sequences into the ROSA26 locus, followed by a subsequent exchange step that introduces a cDNA to be Tc-regulated to the locus using the recombinase-mediated cassette exchange reaction. Both steps are demonstrated to give desired clones with high efficiency, suggesting that this system can be introduced readily into any ES cell lines, leading to the simultaneous establishment of multiple cell lines carrying different Tc-regulated cDNAs. We believe that use of this system will strongly accelerate molecular biological research using ES cells.