A simple PCR-based method for the rapid genotyping of inherited fifth complement component (C5)-deficient mice

The fifth component of complement (C5) is considered to be the center of complement activation and function. However, there are no genetically engineered knockout mice for this gene, and the only commercially available inherited C5-deficient mice, in which a “TA” nucleotide deletion in the coding frame was previously identified, are in theC57BL/10Sn genetic background rather than the commonly used backgrounds C57BL/6 and BALB/c. Therefore, these mice must be backcrossed into the desired genetic background. Here, we developed an ARMS (amplification refractory mutation system) PCR method using a specific primer pair that was able to discriminate between the genotypes when the resulting product was analyzed by agarose gel electrophoresis. These results were supported by quantitative RT-PCR and semi-quantitative PCR and were consistent with the results from sequencing each backcrossed generation. Using ARMS-PCR method, we generated C5-deficient mice in the C57BL/6 background over 9 backcrossed generations and further verified the phenotype using complement-mediated hemolytic assays. In this study, we describe a simple, rapid and reliable PCR-based method for genotyping inherited C5-deficient mice that may be used to backcross C57BL/10Sn mice into other genetic backgrounds.     

Targeted disruption of the gene for the PAK5 kinase in mice

PAK5 is a member of the group B family of PAK serine/threonine kinases and is an effector for the Rho GTPase Cdc42. PAK5 is highly expressed in the brain and is expressed at lower levels in several other tissues. In cell lines, PAK5 has been shown to play a role in filopodia formation and neurite outgrowth. To examine the biological function of PAK5, we deleted the PAK5 gene in mice. The phenotypes of the PAK5-null mice are completely different from those of mice null for PAK4, another member of the group B PAK family. Unlike PAK4-null mice, which are embryonic lethal, PAK5-null mice develop normally and are fertile. The nervous system appears normal in the absence of PAK5, as do other tissues in which PAK5 is normally expressed. Our results suggest functional redundancy between PAK5 and other Rho GTPase targets.     

A novel acoustic evaluation system of scratching in mouse dermatitis: rapid and specific detection of invisibly rapid scratch in an atopic dermatitis model mouse

Scratching is an essential and a skin specific behavior induced by itching, which is a common symptom of atopic dermatitis (AD) and other types of dermatitis. Itching sensation and scratching are closely associated and thus scratching times are currently used for evaluating itching in animal models. However, objective measuring systems of scratching to assess the grade of dermatitis and the effectiveness of anti-pruritic drugs in animal dermatitis models are lacking. To investigate a quantitative evaluation system for itching, we have developed a novel acoustic scratching counting system and compared its accuracy with time-consuming slow-motion video recording system. We have also objectively evaluated the efficacy of an antihistamine using this novel system. Scratching behavior of an AD model mouse (K14 driven IL-18 transgenic mouse) was recorded visually and acoustically. Specific scratching sound produced by mice was recorded and counted using a software we have developed, and the results obtained using our acoustic system were not statistically different from data obtained using slow motion video system. Surprisingly, mice scratched more than 10 times/second, which was invisibly rapid motion and revealed inaccuracy of conventional hand counting system. Results were identical to that of measured by 10 times time costing slow-motion video analysis. The antihistamine is clearly effective for suppression of scratching as demonstrated using this objective and accurate method. This novel motion analysis system will open a window for physiological and pathological analysis for animal models and development of anti-pruritic drugs.     

Isolation of a murine homologue of the Drosophila neuralized gene, a gene required for axonemal integrity in spermatozoa and terminal maturation of the mammary gland

The Drosophila neuralized gene shows genetic interactions with Notch, Enhancer of split, and other neurogenic genes and is thought to be involved in cell fate specification in the central nervous system and the mesoderm. In addition, a human homologue of the Drosophila neuralized gene has been described as a potential tumor suppressor gene in malignant astrocytomas. We have isolated a murine homologue of the Drosophila and human Neuralized genes and, in an effort to understand its physiological function, derived mice with a targeted deletion of this gene. Surprisingly, mice homozygous for the introduced mutation do not show aberrant cell fate specifications in the central nervous system or in the developing mesoderm. This is in contrast to mice with targeted deletions in other vertebrate homologues of neurogenic genes such as Notch, Delta, and Cbf-1. Male Neuralized null mice, however, are sterile due to a defect in axoneme organization in the spermatozoa that leads to highly compromised tail movement and sperm immotility. In addition, female Neuralized null animals are defective in the final stages of mammary gland maturation during pregnancy.     

Generation of transgenic mice on an NOD/SCID background using the conventional microinjection technique

Humanized mice, which refers to immunodeficient mice repopulated with the human immune system, are powerful tools for study in the field of immunology. It has been difficult, however, to generate these transgenic (Tg) mice directly from such strains as the NOD/SCID mouse. In this study, we describe a method developed by us for the generation of Tg mice on an NOD/SCID background. First, we obtained fertilized eggs efficiently by means of in vitro fertilization (IVF); then, we attempted to generate CAG-EGFP Tg mice on an NOD/SCID background, finding that delayed timing of the microinjection after the IVF improved the time to development of the two-cell-stage embryos and the obtainment of newborns. We successfully generated Tg mice and confirmed the germ-line transmission in the offspring. In conclusion, we established a novel system for directly generating transgenic mice on an NOD/SCID background. This novel system is expected to allow improved efficiency of the generation of humanized mice.     

Creation of an experimental rearing environment for microbiome animal research using an individually ventilated cage system and bioBUBBLE enclosure

To avoid microbial contamination risk, vinyl film isolators are generally used in animal microbiome experiments involving germ-free (GF) mice and/or gnotobiotic (GB) mice. However, it can take several months to gain expertise in operating the isolator competently. Furthermore, sterilization and sterility testing, which are essential for isolator preparation, can take more than 20 days. Hence, we built an experimental rearing environment that combines an individual ventilation cage system and a bioBUBBLE clean room enclosure to easily set up an experimental animal microbiome environment for animal facilities. In this work, a three-step evaluation was conducted. First, we examined whether GF mice can be maintained in this rearing environment without bacterial contamination. Next, we examined whether GF and GB mice can be maintained without cross-contamination in one individual ventilation cage rack. Finally, we tested whether GF mice can be maintained in a biological safety cabinet controlled by negative pressure. In our series of experiments, no microbial contamination occurred over more than 3 months. These results indicated that our rearing system that combines the individual ventilation cage and bioBUBBLE systems can be used not only for experiments with GF mice but also for Biosafety Level 2 experiments that handle bacteria. Our system can mitigate various disadvantages of using vinyl film isolators. In conclusion, we established an experimental method with improved working time and efficiency compared with those of the previous vinyl isolator method.     

Nonfibrinolytic functions of plasminogen

Although the roles of plasminogen and plasmin in mediating blood clot dissolution are well known, the availability of mice deficient for components of the fibrinolytic system has allowed direct approaches to be made toward elucidating the role of these proteins in other diverse physiological and pathophysiological processes. A number of these studies have identified plasminogen as playing an important role in inflammation and other cell migratory processes. With the identification of receptors for plasminogen on a number of pathogens, and the ability to activate plasminogen through either endogenous production of plasminogen activators or utilization of host activators, mice deficient for components of the fibrinolytic system offer a unique approach toward further elucidating the importance of this system in pathogen infection and dissemination.     

Construction and characterization of a live, attenuated apxIICA inactivation mutant of Actinobacillus pleuropneumoniae lacking a drug resistance marker

The apxIIC gene of Actinobacillus pleuropneumoniae serotype 7 was inactivated by homologous recombination using a sucrose counter-selectable marker system, resulting in a mutant strain that had no antibiotic resistance marker and expressed an inactivated ApxII toxin. The safety and immunogenicity of the mutant were evaluated in mice. The mutant strain caused no adverse effects in mice at doses up to 2 x 10(9) CFU via the intraperitoneal route while the parental strain induced total mortality at a dose of 2 x 10(7) CFU. Mice vaccinated intraperitoneally with the mutant strain had 100% and 70% protection against homologous (serotype 7) or heterologous (serotype 1, 3) challenge with A. pleuropneumoniae, respectively. The A. pleuropneumoniae mutant strain HB04C- and the counterselection method used in the study show promise in developing effective live vaccines for porcine pleuropneumonia and for other infections diseases of the respiratory system.     

Interactions between gut-associated lymphoid tissue and colonization levels of indigenous, segmented, filamentous bacteria in the small intestine of mice.

Unlike most other indigenous bacteria, segmented filamentous bacteria (SFB) are potent activators of the mucosal immune system. SFB are strongly anchored to the epithelial cells of the small intestine where they have a preference for mucosal lymphoid epithelium. Since SFB are only present in high numbers shortly after weaning, it was investigated whether an SFB-induced immune reaction results in the removal of these bacteria from the small intestine. A correlation was found between age and colonization levels in the small intestines of SFB monoassociated Swiss mice. Five-week-old athymic BALB/c (nu/nu) mice showed lower colonization levels than their heterozygous littermates, but the opposite was found at the age of 12 weeks. However, SFB inoculation of germfree Swiss mice resulted in higher colonization levels in 5-week-old mice when compared with 4-month-old mice. We conclude that SFB colonization levels in the small intestine are likely influenced by the activity of the mucosal immune system. However, an additional age-dependent factor that modulates SFB colonization levels cannot be excluded.     

Human MxA Protein Protects Mice Lacking a Functional Alpha/Beta Interferon System against La Crosse Virus and Other Lethal Viral Infections

The human MxA protein is part of the antiviral state induced by alpha/beta interferon (IFN-alpha/beta). MxA inhibits the multiplication of several RNA viruses in cell culture. However, its antiviral potential in vivo has not yet been fully explored. We have generated MxA-transgenic mice that lack a functional IFN system by crossing MxA-transgenic mice constitutively expressing MxA with genetically targeted (knockout) mice lacking the beta subunit of the IFN-alpha/beta receptor (IFNAR-1(-/-) mice). These mice are an ideal animal model to investigate the unique antiviral activity of human MxA in vivo, because they are unable to express other IFN-induced proteins. Here, we show that MxA confers resistance to Thogoto virus, La Crosse virus, and Semliki Forest virus. No Thogoto virus progeny was detectable in MxA-transgenic mice, indicating an efficient block of virus replication at the primary site of infection. In the case of La Crosse virus, MxA restricted invasion of the central nervous system. In contrast, Semliki Forest virus multiplication in the brain was detectable in both MxA-expressing and nonexpressing IFNAR-1(-/-) mice. However, viral titers were clearly reduced in MxA-transgenic mice. Our results demonstrate that MxA does not need the help of other IFN-induced proteins for activity but is a powerful antiviral agent on its own. Moreover, the results suggest that MxA may protect humans from potential fatal infections by La Crosse virus and other viral pathogens.     

Determination of free D-proline and D-leucine in the brains of mutant mice lacking D-amino acid oxidase activity.

A new procedure to accurately measure a trace amount of d-proline in biological samples has been developed. This D-amino acid was derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and was determined by a column-switching HPLC system, a combination of a micro-ODS column and a chiral column. The detection limit for D-proline spiked in a mouse cerebrum sample is 1 fmol (injection amount, S/N = 3). Within-day precision and day-to-day precision obtained for spiked d-proline (10 fmol) are 2.14 and 5.35% (RSD), respectively. Using the new method, the amount of free D-proline in eight brain regions and sera of mutant ddY/DAO- mice, lacking D-amino acid oxidase activity, and control ddY/DAO+ mice was determined. The amount of free D-leucine was also investigated. The amount and distribution of D-proline in the brains of ddY/DAO+ mice and ddY/DAO- mice are almost the same, and relatively high amounts of D-proline have been observed in the pituitary gland and in the pineal gland. On the other hand, the amount of D-leucine is different between the two strains. In the brains of ddY/DAO+ mice, a relatively high amount of D-leucine has been observed in the pineal gland compared with other regions. In the brains of ddY/DAO- mice, D-leucine amounts are approximately 10 times higher than those obtained in ddY/DAO+ mice and regional difference has not been observed, while the amounts of L-proline and L-leucine are not significantly different between the two strains. In the serum, the amounts of both free D-proline and d-leucine are significantly higher in the ddY/DAO- mice than those obtained in ddY/DAO+ mice.     

The comparison of sensitivity of motion sickness between retinal degeneration fast mice and normal mice

Recent studies report that a conflict between information from the visual system and vestibular system is one of the main reasons for induction of motion sickness (MS). We may be able to clarify the integration mechanism of visual and vestibular information using an animal model with a visual defect, the retinal degeneration fast (rdf) mouse, and the role of vestibular information in the pathogenesis of MS. The rdf mice and wild-type Kunming mice were subjected to rotary stimulation to induce MS. Conditioned taste anorexia to saccharin solution and behavior score were used to observe the differences in MS sensitivity between two types of mice. The decrease in intake of saccharin solution and the behavior score in rdf mice were greater than those in normal mice. After rotatory stimulation, the reduction of intake mass and the behavior score were greater in rdf mice compared to those of normal mice. The rdf mice were more sensitive to rotation than normal mice. We conclude that visual information plays a role in the pathogenesis of MS. Visual information and vestibular information impact each other and integrate through certain channels in the central nervous system in mice.     

The mouse mutation claw paw: forelimb deformity and delayed myelination throughout the peripheral nervous system.

We describe a murine autosomal recessive mutation claw paw (gene symbol clp), which in homozygous clp/clp mice produces striking abnormalities of limb posture within the first one or two postnatal days. Affected animals have delayed and abnormal myelination in the peripheral nervous system but not in the central nervous system, and also have persistently blocked myelination of small caliber axons that are myelinated in normal mice. Both abnormalities suggest that an important effect of the clp mutation is to impair the putative signaling mechanism by which an axon instructs a Schwann cell whether or not to myelinate it. The early onset of behavioral abnormalities in clp/clp mutant mice, as well as certain other features of the disorder, suggest that some effects of the clp gene are not accounted for by the pathological findings. The clp gene has been mapped to chromosome 7 near the Gpi-1 locus.     

Encephalomyelitis in mice experimentally infected with Akabane virus.

Lesions in the central nervous system of mice, induced by intracerebral injection of Akabane virus, were observed by the fluorescent antibody technique and histological method. Fluorescent antigens were recognized in the cytoplasm of nerve cells, but were not detected exactly in any other part. Fluoresced nerve cells were distributed almost all over the central nervous system, especially in medulla oblongata and spinal cord. The appearance of fluorescent antigens was followed by histological changes. So-called Nissl's acute severe degeneration was observed in nerve cells in the area where the fluorescent antigens were distributed. Spongy foci were seen in medulla oblongata and spinal cord. Virus was recovered from brain and spinal cord, but not from any other visceral organ or blood. Akabane virus showed an affinity to nerve cells and caused primary nonpurulent encephalomyelitis when inoculated intracerebrally to mice.     

神经特异性转录因子DAT1基因的表达

采用生物信息学分析、细胞培养和RT—PCR等方法,以初步解DAT1基因在小鼠主要组织及神经系统相关肿瘤细胞中的表达．生物信息学的基因表达谱分析发现与DAT1同源的EST有100多条,大多数EST分布于胎脑、成年脑、脑肿瘤、肺及肺部的良性肿瘤等组织．SAGE分析发现DAT1在脑及神经系统肿瘤组织中表达非常广泛;RT—PCR方法检测发现DAT1只在脑组织中特异表达。而其它组织如心脏、肺、肝脏、骨骼肌、肾脏、睾丸、卵巢等未见表达;DAT1在培养的正常星形胶质细胞C8中不表达,在胶质瘤、神经母细胞瘤等细胞株中有不同水平的表达．由于DAT1是一种LIM蛋白,而LIM蛋白在细胞的分化、发育调控和肿瘤形成中有重要作用,DAT1在脑及神经系统相关肿瘤中的较高表达提示,DAT1在神经系统中有重要功能。并可能与神经系统肿瘤的发生相关．     

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult cardiomyocytes are widely accepted as a good model for cardiac cellular physiology and pathophysiology, as well as for pharmaceutical intervention. Genetically modified mice preclude the need for complicated cardiomyocyte infection processes to generate the desired genotype, which are inefficient due to cardiomyocytes’ terminal differentiation. Isolation and culture of high quantity and quality functional cardiomyocytes will dramatically benefit cardiovascular research and provide an important tool for cell signaling transduction research and drug development. Here, we describe a well-established method for isolation of adult mouse cardiomyocytes that can be implemented with little training. The mouse heart is excised and cannulated to an isolated heart system, then perfused with a calcium-free and high potassium buffer followed by type II collagenase digestion in Langendorff retrograde perfusion mode. This protocol yields a consistent result for the collection of functional adult mouse cardiomyocytes from a variety of genetically modified mice.     

Culture and characterization of thymic epithelium from autoimmune NZB and NZB/W mice

Autoimmune NZB and NZB/W mice display early abnormalities in thymus histology, T cell development, and mature T cell function. Abnormalities in the subcapsular/medullary thymic epithelium (TE) can also be inferred from the early disappearance of thymulin from NZB. It has also been reported that NZB thymic epithelial cells do not grow in culture conditions that support the growth of these cells from other strains of mice. In order to study the contribution of TE to the abnormal T cell development and function in NZB and NZB/W mice, we have devised a culture system which supports the growth of TE cells from these mice. The method involves the use of culture vessels coated with extracellular matrix produced by a rat thymic epithelial cell line. TEA3A1, and selective low-calcium, low-serum medium. In addition TEA3A1 cells have been used as an antigen to generate monoclonal antibodies specific for subcapsular/medullary TE. These antibodies, as well as others already available, have been used to show that the culture conditions described here select for cells displaying subcapsular/medullary TE markers, whereas markers for cortical TE and macrophages are absent.     

Repair of dihydrouracil supported by base excision repair in mNTH1 knock-out cell extracts

In mammalian cells, thymine glycols and other oxidized pyrimidines such as 5,6-dihydrouracil are removed from DNA by the NTH1 protein, a bifunctional DNA-N-glycosylase. However, mNTH1 knock-out mice in common with other DNA glycosylase-deficient mice do not show any severe abnormalities associated with accumulation of DNA damage and mutations. In the present study we used an in vitro repair system to investigate the mechanism for the removal of 5,6-dihydrouracil from DNA by mNTH1-deficient cell-free extracts derived from testes of mNTH1 knock-out mice. We found that these extracts are able to support the removal of 5,6-dihydrouracil from DNA at about 20% of the efficiency of normal extracts. Furthermore, we also found that single-nucleotide patch base excision repair is the major pathway for removal of 5,6-dihydrouracil in mNTH1-deficient cell extracts, suggesting the involvement of other DNA glycosylase(s) in the removal of oxidized pyrimidines.     

An In Vitro Micromethod for the Quantitative Assessment of Central Demyelination

We report the development of a simple and reliable method for the study of demyelination in vitro based on the measurement of 2':3'-cyclic nucleotide 3'-phosphodiesterase in isolated myelin. Using only small quantities of myelin (equivalent to 100 micrograms of myelin protein) the system was tested under conditions that are believed to approximate those found at the site of an inflammatory demyelinating lesion. Treatment with a combination of trypsin, phospholipase A2, and lysophosphatidylcholine was used to evaluate the method. This microsystem has the potential not only for testing the myelinotoxicity of soluble factors but also for investigating the involvement of inflammatory cells in the demyelinating process. Myelin degradation by elicited peritoneal macrophages could be demonstrated at relatively high densities of these cells. Nylon wool purified lymph node T cells from myelin basic protein-primed SJL/J mice, after selective expansion with antigen and interleukin 2, failed to induce any significant myelin breakdown unless a limited number of syngeneic activated macrophages were also present. T cells from mice that had been inoculated with keyhole limpet haemocyanin failed to show any effect. The advantages of this technique over other in vitro systems are that it enables the study of demyelination using syngeneic sources of myelin and defined cell populations.