Persistent hyperplastic tunica vasculosa lentis and persistent hyperplastic primary vitreous in transgenic line TgN3261Rpw

Persistent hyperplastic tunica vasculosa lentis and persistent hyperplastic primary vitreous are congenital ocular anomalies that can lead to cataract formation. A line of insertional mutant mice, TgN3261Rpw, generated at the Oak Ridge National Laboratory in a large-scale insertional mutagenesis program was found to have a low incidence (8/243; 3.29%) of multiple developmental ocular abnormalities. The ocular abnormalities include persistent hyperplastic primary vitreous, persistent hyperplastic tunica vasculosa lentis, failure of cleavage of the anterior segment, retrolental fibrovascular membrane, posterior polar cataract, and detached retina. This transgenic mouse line provides an ontogenetic model because of the high degree of similarity of this entity in humans, dogs, and mice.     

Knockout mouse production assisted by Blm knockdown

Production of knockout mice using targeted embryonic stem cells (ESCs) is a powerful approach for investigating the function of specific genes in vivo. Although the protocol for gene targeting via homologous recombination (HR) in ESCs is already well established, the targeting efficiency varies at different target loci and is sometimes too low. It is known that knockdown of the Bloom syndrome gene, BLM, enhances HR-mediated gene targeting efficiencies in various cell lines. However, it has not yet been investigated whether this approach in ESCs is applicable for successful knockout mouse production. Therefore, we attempted to answer this question. Consistent with previous reports, Blm knockdown enhanced gene targeting efficiencies for three gene loci that we examined by 2.3–4.1-fold. Furthermore, the targeted ESC clones generated good chimeras and were successful in germline transmission. These data suggest that Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production.     

Reduced formation of granulomata in gamma(delta) T cell knockout BALB/c mice inoculated with Mycobacterium avium subsp. paratuberculosis

The role of gamma(delta) T cells in the bovine immune response to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) infection is poorly understood. Accordingly, using BALB/c mice that are innately susceptible to M. paratuberculosis, we compared wild-type and gamma(delta) T cell knockout BALB/c mice to study the protective roles of gamma(delta) T cells in M. paratuberculosis infection. Ten-week-old mice were inoculated intraperitoneally with either a low dose (4 x 10(6) colony-forming units [CFU]/mouse) or a high dose (4 x 10(9) CFU/mouse) of M. paratuberculosis strain ATCC 19698. Histopathologic and morphometric examinations showed reductions in the number and area of granulomatous lesions in the liver of the knockout mice at 18 weeks after inoculation with either the low or the high dose of the mycobacteria. Furthermore, at 18 weeks after inoculation, the bacterial load in the spleens of the knockout mice inoculated with the high dose was significantly lower than that of wild-type mice. No differences were found in bacterial load between the knockout and the wild-type mice in the low-dose groups. In contrast, in the livers of wild-type mice inoculated with either the low or high mycobacterial dose, increased areas of epithelioid granulomata were observed and the granulomata became disseminated widely during the experimental period. These findings in model mice suggest that gamma(delta) T cells, rather than restricting mycobacterial growth, may play a crucial role in development of epithelioid granulomata similar to those seen consistently in bovine paratuberculosis. The results of this study may have relevance to our understanding of the pathogenesis of paratuberculosis in ruminants, in which a prominent number of gamma(delta) T cells exist in the lymphoid system.     

Hydrocephalus and hypertrichosis in Golden Retriever dogs

Congenital hydrocephalus has been reported in most species of domestic animals and is one of the most common congenital malformations of the canine central nervous system⁽¹⁾. Small, toy and brachycephalic breeds are at higher risk of hydrocephalus than larger breeds. Newborn and immature hydrocephalic puppies typically show an enlarged, domeshaped cranium, open fontanelles, visual and auditory impairment, poor growth rate and sometimes an uncoordinated gait. The pathogenesis of hydrocephalus is unclear but inheritance as an autosomal recessive trait has been identified in some breeds. Suspected inherited polymicrogyria and hydrocephalus has been identified in young Standard Poodles and a similar condition seen in a Golden Retriever dog in the USA⁽¹⁾. During the past 2 years, congenital and suspected inherited (as an autosomal recessive trait) hydrocephalus has been diagnosed in Golden Retriever puppies in New Zealand.     

Embryo implantation is blocked by intraperitoneal injection with anti-LIF antibody in mice

Leukemia inhibitory factor (LIF) is essential for embryo implantation in mice and plays an important role in other mammals including humans. Intraperitoneal (i.p.) injections with anti-LIF antibody (7.5 μg/g body weight, 3 times) between D3 (D1 = day of vaginal plug detection) and D4 effectively blocked embryo implantation; complete inhibition was achieved in C57BL/6J mice, and implantation was dramatically reduced in ICR mice (reduced to 27%). Normal rabbit IgG used as the control did not disturb embryo implantation. Anti-LIF antibody was localized not only in the stroma, but also in the luminal epithelium and the glandular lumen after i.p. injections. Growth-arrested blastocysts were recovered from the uterus without any implantation sites in both strains. Blastocysts made contact with the LE on the antimesometrial side; however, uterine stromal cells did not undergo secondary decidual reaction, and the uterine lumen was open, even at D7. Several regions of decidualization in ICR mice treated with anti-LIF antibody were smaller than those of the control, and development of blastocysts was delayed. The expression of LIF-regulated genes, such as immune-responsive gene-1 and insulin-like growth factor binding protein-3, was significantly decreased in C57BL/6J mice treated with anti-LIF antibody compared with the control, but not in ICR mice. The present study demonstrated that simple ip injections of an antibody are sufficient to block one of the important factors involved in embryo implantation in mice, and this method should also be easily applicable to the investigation of other factors involved in implantation.     

Expression and significance of p53, rb,p21/waf-1, p16/ink-4a,and PTEN tumor suppressors in canine melanoma

The role of tumor suppressor genes in the pathogenesis of canine melanoma is incompletely understood. The genes encoding the tumor suppressors p53, Rb, p21 (waf-1), p16 (ink-4a), and PTEN have been postulated to contribute to the pathogenesis of melanoma in humans and experimental animal models. To assess whether inactivation of these genes similarly contributes to the origin and progression of canine melanoma, we examined their expression in seven distinct canine melanoma cell lines and in 31 retrospective samples (representing 29 dogs) of spontaneous canine melanoma. Various patterns suggestive of loss of tumor suppressor function emerged in these cell lines. The most frequently observed abnormality was loss or significant reduction of p16 expression in six of seven cell lines and in 21 of 26 tumor samples. Loss or significant reduction of PTEN expression was seen in four of seven cell lines and in 13 of 27 tumor samples. Although p53 was detectable in all the cell lines and in 24 of 30 tumors, exclusion of p53 from the nuclear compartment was observed in each of the cell lines and in 18 of 25 tumor samples. These results indicate that loss of function of these tumor suppressor proteins is a common occurrence that may contribute to the origin of canine melanoma. In our sample population, abnormalities in the expression or localization of one or more tumor suppressor proteins occurred with similar frequency in malignant and benign tumors; thus, additional work is necessary to determine how these proteins may impact disease progression and response to therapy.     

Expression of hepatitis C virus core protein associated with malignant lymphoma in transgenic mice

Hepatitis C virus (HCV) is a major causative agent for chronic liver diseases leading to hepatocellular carcinoma (HCC) and has also been suggested to be a possible etiologic factor for different lymphoproliferative diseases, including mixed cryoglobulinemia (MC) and B-cell non-Hodgkin's lymphoma (NHL). To understand the roles of HCV core protein in the pathogenesis of HCV related diseases, we produced two lines of the transgenic mice (HC82310 and HC9053) that express the HCV core transgene. One of the lines, HC9053, developed malignant lymphoma (ML, follicular center cell type) with a high frequency (80%) at the ages over 20 months. Hepatocellular adenoma was also observed in this line of transgenic mouse. We demonstrated expression of HCV core protein and mRNA in the liver of transgenic mice, and also detected the core mRNA in the enlarged lymph nodes of the transgenic mice which developed ML. These results suggest that the core protein may play an important role in the development of ML, and that the HC9053 transgenic mice provide suitable models for understanding the mechanism of HCV-related lymphoproliferative diseases.     

肌肉生长抑制素在小鼠不同发育阶段心肌组织中的表达

本试验旨在探究肌肉生长抑制素（myostatin，MSTN）基因在小鼠不同发育阶段心肌组织中的表达情况，分别选取幼年（7日龄）、性成熟（21~28日龄）、体成熟（42~56日龄）和老年（84日龄以上）4个发育阶段的健康小鼠（各3只）为研究对象，采用实时荧光定量PCR、HE染色、免疫组织化学等方法研究小鼠不同发育时期MSTN基因在心肌组织中的表达及不同时期心肌组织的发育。实时荧光定量PCR结果显示，MSTN基因在小鼠不同发育阶段心肌组织中均有表达，且其在小鼠老年时期的表达量最高，体成熟时期的表达量最低；幼年小鼠和性成熟小鼠心肌组织中MSTN基因表达水平显著低于老年小鼠（P<0.05），而体成熟小鼠则极显著性低于老年小鼠（P<0.01）。HE染色结果显示，小鼠心肌细胞核及肌纤维的生长在不同发育时期明显不同，其中幼年时期的细胞核最多，肌纤维排列相对较紧密，而在性成熟、体成熟、老年时期心肌的发育指标依次降低。免疫组织化学结果显示，MSTN基因主要在心肌细胞核中表达，其各个时期表达量与实时荧光定量PCR的结果相符合。     

Limited susceptibility of three different mouse (Mus musculus) lines to Porcine circovirus-2 infection and associated lesions

Porcine circovirus associated disease (PCVAD), a major global problem for pork producers, is characterized microscopically by depletion and histiocytic replacement of follicles in the lymphoid tissues. The objectives of this study were to determine 1) if Porcine circovirus-2 (PCV-2) inoculated mice (Mus musculus) can develop PCV-2 associated lymphoid lesions and serve as a model for PCVAD, and 2) if differences in PCV-2 host susceptibility exist among mice lines. Three groups (n = 48/group) of 4-wk-old male mice were used: BALB/c, C57BL/6, and C3H/HeJ. A 2 × 2 factorial analysis was designed for each group using PCV-2 inoculation and keyhole limpet hemocyanin in incomplete Freund’s adjuvant injections on day 0 and 7 as factors. Necropsies were performed on days 12, 17, 22, 27, 32, and 37. Serum samples collected at each necropsy tested negative for anti-IgG PCV-2 antibodies in all mice at all time points by 2 different PCV-2 enzyme-linked immunosorbent assays (ELISA). The PCV-2 DNA was detected by polymerase chain reaction (PCR) in 93% (100/108) of tissues and 42.6% (46/108) of serum samples from PCV-2-inoculated mice from days 12 to 37. Microscopic lesions consistent with PCV-2 infection were not observed in any mice and PCV-2 DNA and PCV-2 antigen were not detected in tissues by in-situ-hybridization or immunohistochemistry assays, respectively. Based on incidence of PCV-2 DNA in serum samples, the C57BL/6 mouse line was more resistant to PCV-2 infection than the other lines. The results indicate the mouse model likely has limited utility to advance understanding of the pathogenesis of PCV-2 associated lesions, but mice could potentially be important in the epidemiology of PCV-2.     

蛋白酪氨酸磷酸酶Shp2基因mRNA在小鼠睾丸发育过程中的表达

为探讨蛋白酪氨酸磷酸酶Shp2 mRNA在小鼠出生后睾丸发育过程中的表达规律,本试验以16组出生后不同发育阶段的昆明种健康小鼠的睾丸组织为材料,经Real-time PCR检测小鼠出生后睾丸中Shp2基因mRNA的表达变化。结果显示,Shp2基因mRNA在小鼠出生后睾丸发育过程中连续表达,且第20~31天出现明显波动,20 d时Shp2表达量极显著增加(P<0.01),然后下降(P<0.01),在31 d再次极显著增加(P<0.01),31 d后,Shp2的表达量又逐渐恢复平稳(P>0.05)。上述结果表明,Shp2基因的连续性表达及在特定发育阶段表达量的明显波动,预示着Shp2参与小鼠出生后睾丸发育的全过程,但具体作用如何还有待进一步研究。     

Pathology methods for the evaluation of embryonic and perinatal developmental defects and lethality in genetically engineered mice

The normal embryonic development of organs and other tissues in mice and all species is preprogrammed by genes. Inactivation of a gene involved in any stage of normal embryonic development can have severe consequences leading to embryonic or postnatal developmental defects and lethality. Pathology methods are reviewed for evaluating normal and abnormal placenta and embryo, especially after E12.5. These methods include pathology protocols for necropsy and histopathology in addition to references that will provide additional knowledge for embryo assessment including histology atlases and advanced embryo imaging techniques.     

Mechanisms of resistance and susceptibility to experimental visceral leishmaniosis: BALB/c mouse versus syrian hamster model

ABSTRACT: Several animal models have been established to study visceral leishmaniosis (VL), a worldwide vector-borne disease affecting humans and domestic animals that constitutes a serious public health problem. BALB/c mice and Syrian hamsters are the most widely used experimental models. In this paper, we summarize the advantages and disadvantages of these two experimental models and discuss the results obtained using these models in different studies of VL. Studies using the BALB/c mouse model have underscored differences between the liver and spleen in the course of VL, indicating that pathological evaluation of the visceral organs is essential for understanding the immune mechanisms induced by Leishmania infantum infection. The main goal of this review is to collate the relevant literature on Leishmania pathogenesis into a sequence of events, providing a schematic view of the main components of adaptive and innate immunity in the liver and spleen after experimental infection with L. infantum or L. donovani. This review also presents several viewpoints and reflections about some controversial aspects of Leishmania research, including the choice of experimental model, route of administration, inoculum size and the relevance of pathology (intimately linked to parasite persistence): a thorough understanding of which is essential for future VL research and the successful development of efficient control strategies for Leishmania spp.     

Gene expression responses to a Salmonella infection in the chicken intestine differ between lines

Poultry products are an important source of Salmonella enterica. An effective way to reduce food poisoning due to Salmonella would be to breed chickens more resistant to Salmonella. Unfortunately host responses to Salmonella are complex with many factors involved. To learn more about responses to Salmonella in young chickens, a cDNA microarray analysis was performed to compare gene expression profiles between two chicken lines under control and Salmonella infected conditions. Newly hatched chickens were orally infected with S. enterica serovar Enteritidis. Since the intestine is the first barrier the bacteria encounter after oral inoculation, intestinal gene expression was investigated at different timepoints. Differences in gene expression between the two chicken lines were found in control as well as Salmonella infected conditions. In response to the Salmonella infection a fast growing chicken broiler line induced genes that affect T-cell activation, whereas in a slow growing broiler line genes involved in macrophage activation seemed to be more affected at day 1 post-infection. At days 7 and 9 most gene expression differences between the two chicken lines were identified under control conditions, indicating a difference in the intestinal development between the two chicken lines which might be linked to the difference in Salmonella susceptibility. The findings in this study have lead to the identification of novel genes and possible cellular pathways, which are host dependent.     

Conditional knockout of brca1/2 and p53 in mouse ovarian surface epithelium: Do they play a role in ovarian carcinogenesis?

Alterations of genes are known to be critical for the induction of tumorigenesis, but the mechanism of ovarian carcinogenesis is little understood and remains to be elucidated. In this study, we investigated the roles of brca1, brca2 and p53 genes in the development of ovarian cancer using conditional knockout mice generated by a Cre-loxP recombinant system. Following the application of recombinant adenovirus expressing Cre in vitro, the proliferation of ovarian surface epithelium (OSE) was increased. For instance, a significant increase in cell growth was observed in OSE cells in vitro by conditional knockout isolated from the mice bearing concurrent floxed copies of brca1 and brca2/p53. However, the proliferative effect of the ovarian cells was not observed in concurrent brca1/brca2 or p53 knockout mice in vivo, indicating that we could not observe the direct evidence of the involvement of brca1, brca2, and p53 in ovarian carcinogenesis. Since morphological changes including tumor formation were not observed in mice bearing floxed copies of concurrent brca1/brca2 or p53, the inactivation of brca1/2 or p53 is not sufficient for the induction of tumor formation. Taken together, these results suggest that the deficiency of these genes may not be involved directly in the mechanism of ovarian carcinogenesis.     

Immunopathogenic pathways in canine inflammatory myopathies resemble human myositis

Progress in the treatment of inflammatory myopathies is impeded by the lack of suitable animal models. Inflammatory myopathies occur spontaneously in the dog, are a heterogeneous group of disorders, and are more common than in humans. Clinical signs of weakness and muscle atrophy are reliably present, and there are histological and immunohistological similarities to forms of human myositis. In this study, microarray technology followed by quantitative real-time PCR and immunohistochemistry on muscle biopsy sections was used to investigate gene expression in cases of canine inflammatory myopathies. Several genes involved with innate and adaptive immunity were highly upregulated including those that participate in macrophage and dendritic cell activation and migration, and antigen processing and presentation. Other genes including those that participate in B cell growth, development, migration and activation, immunoglobulin genes, genes in pro-inflammatory and anti-inflammatory pathways, and genes involved with tissue remodeling were upregulated. In previous reports utilizing microarray technology in human myositis, there was activation of similar pathways involved in the immune response. This study strengthens the argument that forms of canine myositis may be important animal models of human myositis and suggests useful biomarkers for therapeutic response using the dog in pre-clinical trials.     

宿主和病原体间遗传学关系小鼠动物模型研究进展

遗传因子决定着各种病原引起的感染性疾病的最终结果。人类基因的抗性和易感性的分子机制在许多方面还不清楚 ,宿主对某一种病原的反应的影响多基因控制就变得更为复杂。动物模型在宿主与病原之间复杂遗传系统解剖成单一的因素控制方面具有巨大的潜力。在克隆和鉴定宿主抗性基因方面 ,小鼠能成为一个无价的实验工具。通过三个途径能够建立人类感染性疾病的小鼠模型。 (1 )目标基因小鼠突变体的培育 ;(2 )在突变小鼠中 ,宿主抗性基因位置克隆 ;(3 )绘制和描述控制宿主与病原之间复杂性的数量特性位点图。文章通过上述三种方法的介绍进一步阐述人的感染性疾病 ,特别对细菌感染性疾病的抗性或易感性的机制研究。     

Isolation of a human erythrocyte-adapted substrain of Babesia rodhaini and analysis of the merozoite surface protein gene sequences

Babesia rodhaini is a rodent hemoparasite closely related to B. microti, the major causative agent of human babesiosis. We tested the infectivity of B. rodhaini for human erythrocytes by using the SCID mouse model in which the circulating erythrocytes were replaced with those of humans. Initially, parasites grew very poorly in the mouse model, but a variant capable of propagating in human erythrocytes emerged after an adaptation period of three weeks. In an attempt to identify parasite proteins involved in the alteration of host cell preference, an expression cDNA library of B. rodhaini was constructed and screened with immune mouse sera. Although we were able to obtain three merozoite surface protein (MSP) genes, sequences of these genes from both the parental strain and human erythrocyte-adapted substrain were identical. Our results suggest that B. rodhaini has potential ability to infect human erythrocytes, but development of this ability may not be brought about by an amino acid change in MSPs.     

Evaluation of the protection conferred by a naturally attenuated Neospora caninum isolate against congenital and cerebral neosporosis in mice

The parasite Neospora caninum is an important abortifacient agent in cattle worldwide. At present, the development of an effective and safe vaccine against bovine neosporosis is of great relevance. Recently, a new isolate of N. caninum (Nc-Spain 1 H) which was obtained from the brain of an asymptomatic congenitally infected calf, exhibited non-virulent behaviour in mouse and bovine infection models. The aim of this study was to determine the safety and efficacy of Nc-Spain 1 H when used as a vaccinal isolate in well-established BALB/c models of congenital and cerebral neosporosis. Mice were subcutaneously immunised twice at 3-week intervals and were challenged with 2 × 10⁶ tachyzoites of the virulent Nc-Liv isolate. After immunisation with live Nc-Spain 1 H tachyzoites, no parasitic DNA was detected in the dams’ brains before challenge and microsatellite analysis performed in PCR-positive mice showed that the profiles corresponded to the challenge isolate Nc-Liv, indicating the Nc-Spain 1 H isolate to be a safe vaccine candidate. The efficacy of the live vaccine was evaluated in the first experiment after the immunisation of mice with 5 × 10⁵ live Nc-Spain 1 H tachyzoites. This immunisation protocol significantly reduced the neonatal mortality to 2.4%, reduced the vertical transmission from 89.1% to 2.3% and completely limited the cerebral infection. These results were associated with a Th1-type immune response. In the second experiment, the effect of various immunising doses was established using ten-fold dilutions of the tachyzoites (from 5 × 10⁵ to 5 × 10). In all the cases, congenital protection rates above 60% were observed, and the mice that were immunised with the lowest dose (5 × 10) presented the highest protection rate (86%). Moreover, low immunising doses of Nc-Spain 1 H induced an IgG2a response, and high parasitic doses induced an IgG1 response. These results evidence the safety and the efficient protection that was conferred by Nc-Spain 1 H against congenital neosporosis, even when the mice were immunised with low parasitic doses.