Evaluation of a plasmid-based transgenic mouse model for detecting in vivo mutations

To study in vivo somatic mutations a C57BL/6 transgenic mousemodel was constructed harboring multiple chromosomally integratedcopies of the plasmid pUR288, which carried the lacZ reportergene as the mutational target. We previously demonstrated thatlacZ-containing plasmids could be rescued from their integratedstate efficient enough to detect mutations in lacZ by positiveselection. The smaller size of the plasmid vector, as comparedwith our earlier transgenic mouse model based on bacteriophagelambda vectors, should offer considerable advantages in termsof rescue efficiency and sensitivity to large size alterationsin the lacZ gene. To evaluate the plasmid-based mouse modelfor its suitability to detect in vivo mutations, we determinedmutant frequencies in different organs of untreated and ethylnitrosourea (ENU)-treated animals using a new, improved protocol.The rescue efficiencies obtained were as high as 200 000/µggenomic DNA; millions of transformants could be obtained inone single experiment. The average spontaneous mutant frequencyin four different organs of 4- to 8-week-old mice ranged from4.41 to 6.82x10^–5;, compared with a mutant frequency ofthe same plasmid grown in Escherichia coli of     

β_1整合素在生后不同年龄阶段小鼠睾丸生精细胞分布的研究

目的　观察 β1 整合素在出生后不同年龄阶段小鼠睾丸生精细胞中的免疫组化定位。方法　用免疫组织化学观察 β1 整合素在生后 1d、4d、7d、1 1d、2周、3周、4周、5周、6周和成年昆明小鼠睾丸生精细胞中的分布。结果　出生后 1～ 1 0d的小鼠睾丸生精小管上皮细胞未见 β1 整合素的表达 ;出生后 1 1d到成年小鼠的不同年龄阶段均可检测到 β1 整合素在睾丸生精小管上皮细胞中的表达。在生后 1 1d到 4周小鼠 ,β1整合素主要表达于增殖能力旺盛的精原细胞的胞膜 ,5周后 β1整合素主要表达于精原细胞和拉长期精子细胞头部细胞膜。结论　β1 整合素是生后 1 1d～ 4周小鼠精原细胞的表面标志     

Clusterin expression during fetal and postnatal CNS development in mouse

Clusterin (or apolipoprotein J) is a widely distributed multifunctional glycoprotein involved in CNS plasticity and post-traumatic remodeling. Using biochemical and morphological approaches, we investigated the clusterin ontogeny in the CNS of wild-type (WT) mice and explored developmental consequences of clusterin gene knock-out in clusterin null (Clu-/-) mice. A punctiform expression of clusterin mRNA was detected through the hypothalamic region, neocortex and hippocampus at embryonic stages E14/E15. From embryonic stage E16 to the first week of the postnatal life, the vast majority of CNS neurons expressed low levels of clusterin mRNA. In contrast, a very strong hybridizing signal mainly localized in pontobulbar and spinal cord motor nuclei was observed from the end of the first postnatal week to adulthood. Astrocytes expressing clusterin mRNA were often detected through the hippocampus and neocortex in neonatal mice. Real-time polymerase chain amplification and clusterin-immunoreactivity dot-blot analyses indicated that clusterin levels paralleled mRNA expression. Comparative analyses between WT and Clu-/- mice during postnatal development showed no significant differences in brain weight, neuronal, synaptic and astrocyte markers as well myelin basic protein expression. However, quantitative estimation of large motor neuron populations in the facial nucleus revealed a significant deficit in motor cells (-16%) in Clu-/- compared with WT mice. Our data suggest that clusterin expression is already present in fetal life mainly in subcortical structures. Although the lack of this protein does not significantly alter basic aspects of the CNS development, it may have a negative impact on neuronal development in certain motor nuclei.     

小鼠出生后肾脏发育过程中的细胞增殖与凋亡

目的：观察小鼠出生后肾脏发育过程中增殖细胞核抗原(PCNA)的表达及细胞凋亡的特征,探讨出生后小鼠肾脏发育过程中细胞增殖与凋亡的规律及其关系。方法：应用免疫组织化学技术和原位末端标记法(TUNEL法)分别检测小鼠出生后1～70d肾脏中PCNA阳性的细胞和凋亡细胞。结果：小鼠出生后1～70d,皮质中的肾小体、肾小管、髓放线以及髓质中的肾小管和集合管的细胞,早期增殖活跃,随着肾脏发育成熟而表达逐渐减弱。同时,也存在着细胞凋亡现象,且凋亡高峰一般出现在增殖高峰之后。结论：细胞增殖与凋亡在小鼠生后肾脏发育的整个过程中普遍存在,生后1～7d细胞增殖旺盛,增殖高峰之后出现凋亡高峰,生后28～70d两者活动均减弱。     

Localization of Thy-1 expression during postnatal development of the mouse cerebellar cortex

Summary Thy-1 is a cell membrane differentiation antigen with a restricted distribution in murine tissues. In both mice and rats the antigen is widely expressed in the CNS, while in the neonatal cerebellum it is expressed at very low levels. We have devised a protocol of immersion fixation by freeze-substitution that preserves both antigenicity and tissue morphology. We have stained freeze-substituted tissue sections of developing mouse cerebella with monoclonal anti-Thy-1. Thy-1 is faintly detectable at birth in Purkinje cells and in the molecular layer. The intensity in these two sites increases to a maximum at day 9; this subsequently decreases in the Purkinje cell cytoplasm until most are negative by day 21, but persists in the molecular layer into adulthood. Thy-1 is not detectable in the external granular layer and is only detectable in the glomeruli of the internal granular layer. Ascending fibre tracts are positive from day 5 onwards. The chronologic and anatomic expressions of Thy-1 are compatible with a role of Thy-1 in the generation and maintenance of synapses.     

TGF-beta1 is increased in a transgenic mouse model of familial Alzheimer's disease and causes neuronal apoptosis

Alzheimer's disease (AD) is a neurodegenerative disorder, due to excess amyloid-beta peptide (Abeta). TGF-beta1 and beta-catenin signaling pathways have been separately implicated in modulating Abeta-neurotoxicity. However, the underlying mechanisms remain unclear. Here, we report that TGF-beta1 and nuclear Smad7 and beta-catenin levels were markedly upregulated in cortical brain regions of the TgCRND8 mice, a mouse model of familial Alzheimer's disease. Coimmunoprecipitation of cortical brain tissue lysates revealed an interaction between Smad7 and beta-catenin. This interaction which was significantly enhanced in the TgCRND8 mice was also associated with an increase in TCF/LEF DNA-shift binding activity. TCF/LEF reporter gene activity was significantly increased in mouse primary cortical neuronal cultures (MCN) from the TgCRND8 mice, compared to controls. Interestingly, exposure of MCN to Abeta(1-42) led to an increase in TGF-beta1 and nuclear levels of both beta-catenin and Smad7. Furthermore, addition of TGF-beta1 to the MCN caused an increase in apoptosis and Smad7 levels. When Smad7 or beta-catenin levels were reduced by siRNA, TGF-beta1-induced apoptosis was suppressed, indicating that both Smad7 and beta-catenin are required for TGF-beta1-induced neurotoxicity. Since Abeta(1-42)-induced TGF-beta1, we suggest that TGF-beta1 may amplify Abeta(1-42)-mediated neurodegeneration in AD via Smad7 and beta-catenin interaction and nuclear localization.     

嗜酸性粒细胞增多症转基因小鼠模型的病理学观察

目的 研究过表达IL-5的嗜酸性粒细胞增多症转基因小鼠模型的生物学特性和病理学变化.方法 记录转基因小鼠的繁育情况,通过计数小鼠外周血中嗜酸性粒细胞的变化规律;观察嗜酸性粒细胞增多对器官损害的病理变化特征.结果 小鼠已成功繁育,外周血中嗜酸性粒细胞占白细胞数的比例持续增长到48周龄时的49.9±3.5%,脏器组织内出现嗜酸性粒细胞浸润和组织细胞变性、坏死等病理改变,出现体表被毛缺失和直肠脱垂现象.结论 小鼠体内高水平的IL-5能显著影响外周血内嗜酸性粒细胞的数目和比值,造成了肺脏、肝脏和脾脏等脏器的严重损坏.表明该小鼠是研究嗜酸性粒细胞增多症良好的转基因小鼠模型.     

Validation studies with Muta Mouse: a transgenic mouse model for detecting mutations in vivo.

MutaMouse is a transgenic mouse engineered to detect mutations in vivo in any tissue of choice by using simple laboratory methods. The target is a bacterial lacZ gene incorporated via lambda phage into the genome of each mouse cell such that a concatamer of approximately 40 copies exists at a single site on both chromosomes of a homologous pair. In order to assess the potential usefulness of MutaMouse in detecting in vivo mutagenesis, several known mutagens/carcinogens were applied to male animals of 8-10 weeks in age. Intraperitoneal injections (single or 5 daily doses) of N-ethyl-N-nitrosourea (ENU), chlorambucil, procarbazine, cyclophosphamide, and acrylamide were investigated for mutagenic effects in bone marrow, liver, and testes. In addition, skin painting studies (single application) were performed with dimethylbenzanthracene (DMBA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and acetic acid. Increases in mutant frequency were clearly induced by all eight chemicals, the magnitudes of which were dependent on the chemical, dose, method of dosing, tissue analyzed, and the time lapse between treatment and isolation of DNA. Data on variability in mutant frequency was presented relative to the analyzed population of lacZ genes and number of animals per treatment group. Application of the MutaMouse model to the detection of heritable mutations was discussed.     

WISP-1 is an osteoblastic regulator expressed during skeletal development and fracture repair

Wnt-1-induced secreted protein 1 (WISP-1) is a member of the CCN (connective tissue growth factor, Cyr61, NOV) family of growth factors. Experimental evidence suggests that CCN family members are involved in skeletogenesis and bone healing. To investigate the role of WISP-1 in osteogenic processes, we characterized its tissue and cellular expression and evaluated its activity in osteoblastic and chondrocytic cell culture models. During embryonic development, WISP-1 expression was restricted to osteoblasts and to osteoblastic progenitor cells of the perichondral mesenchyme. In vitro, we showed that WISP-1 expression in differentiating osteoblasts promotes BMP-2-induced osteoblastic differentiation. Using in situ and cell binding analysis, we demonstrated WISP-1 interaction with perichondral mesenchyme and undifferentiated chondrocytes. We evaluated the effect of WISP-1 on chondrocytes by generating stably transfected mouse chondrocytic cell lines. In these cells, WISP-1 increased proliferation and saturation density but repressed chondrocytic differentiation. Because of the similarity between skeletogenesis and bone healing, we also analyzed WISP-1 spatiotemporal expression in a fracture repair model. We found that WISP-1 expression recapitulates the pattern observed during skeletal development. Our data demonstrate that WISP-1 is an osteogenic potentiating factor promoting mesenchymal cell proliferation and osteoblastic differentiation while repressing chondrocytic differentiation. Therefore, we propose that WISP-1 plays an important regulatory role during bone development and fracture repair.     

Dietary 4-HPR suppresses the development of bone metastasis in vivo in a mouse model of prostate cancer progression

The effects of the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) on prostate cancer metastasis in vivo were evaluated in the mouse prostate reconstitution (MPR) model. MPRs were produced by infection of either heterozygous (+/−) or nullizygous (−/−) p53-mutant fetal prostatic epithelial cells with the recombinant retrovirus Zipras/myc 9. Previous studies have documented that loss of p53 function potentiates metastasis in this model system. MPRs were grafted into homozygous (+/+) p53 male mice, fed a 4-HPR containing diet or a control diet and maintained until the status of tumor progression dictated sacrifice. Under these experimental conditions, treatment with 4-HPR did not have a significant effect on primary tumor wet weight for either p53 +/− or p53 −/− MPRs. For, p53 +/− MPRs the animals fed the 4-HPR diet had a slight improvement in survival and a significant reduction in the number of mesenteric metastases (P=0.0477, t-test). Notably, in p53 +/− MPRs the incidence of metastasis to lumbar spine and sternum was 92% in the control animals compared to 54% in the 4-HPR treated animals (P=0.035, χ²-test). In p53 −/− MPRs there was a trend toward a reduction in the number of soft tissue metastases to lung and liver in the 4-HPR group relative to the control diet group and a statistically significant reduction in the incidence of metastasis to bone was demonstrated in that 50% of control animals versus 30% of 4-HPR treated p53 −/− animals harbored bone metastases (P=0<0.05, χ²-test). Cell lines were established from portions of the primary tumor and from selected metastatic deposits in each experimental group. Clonal analysis, by retroviral integration pattern, indicated increased clonal diversity in both the primary tumors and metastasis-derived cell lines from 4-HPR treated animals relative to the control animals. In vitro treatment with 4-HPR did not reveal discriminating differences between cell lines derived from primary tumors and bone metastases or control and treatment groups in regard to growth arrest or apoptotic responses. Overall these studies indicate limited anti-tumor and anti-metastatic activity in this highly aggressive in vivo mouse model of prostate cancer, yet 4-HPR treatment significantly suppressed the development of bone metastases in p53 +/− and p53 −/− MPRs revealing a novel and potentially clinically useful activity of this retinoid. This revised version was published online in July 2006 with corrections to the Cover Date.     

Age- and sex-dependent development of adrenocortical hyperactivity in a transgenic mouse model of Alzheimer's disease

In this study, we investigated mice of the TgCRND8 line, an APP transgenic mouse model of Alzheimer's disease (AD), with respect to behavioral, endocrinological, and neuropathological parameters. Our results show that transgenic and wild-type mice did not differ in their general health status, exploratory and anxiety related behavior as well as in the activity of their sympathetic-adrenomedullary system. Significant differences, however, were found regarding body weight, amyloid plaque formation, and the activity of the hypothalamic-pituitary-adrenocortical (HPA) axis. Continuous monitoring of glucocorticoid (GC) concentrations over a period of 120 days, utilizing a noninvasive technique to measure corticosterone metabolites in fecal samples, revealed that transgenic animals showed adrenocortical hyperactivity, starting very early in males (from day 30) and later in females (around day 90). It is hypothesized that these changes in the activity of the HPA axis are linked to amyloid-beta associated pathological alterations in the hippocampus, causing degenerations in the negative feedback regulation of the HPA axis leading to hypersecretion of GC. Thus, the development of adrenocortical hyperactivity might be a key-element in the understanding of AD.     

Trehalose reduces aggregate formation and delays pathology in a transgenic mouse model of oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease that presents in the fifth or sixth decade with dysphagia, ptosis and proximal limb weakness. OPMD is caused by the abnormal expansion of a polyalanine tract within the coding region of polyA binding protein nuclear 1 (PABPN1). The resultant mutant PABPN1 forms aggregates within the nuclei of skeletal muscle fibres. We have previously described a transgenic mouse model of OPMD that recapitulates the human disease and develops progressive muscle weakness accompanied by the formation of aggregates in skeletal muscle nuclei. The chemical chaperone trehalose has been used effectively to alleviate symptoms in a mouse model of Huntington's disease and is thought to elicit its effect by binding and stabilizing partially folded polyglutamine proteins and inhibiting the formation of aggregates. Here, we show that trehalose reduces aggregate formation and toxicity of mutant PABPN1 in cell models. Furthermore, oral administration of trehalose attenuated muscle weakness, reduced aggregate formation and decreased the number of TUNEL-labelled nuclei in skeletal muscle in an OPMD transgenic mouse model. Thus, anti-aggregation therapy may prove effective in the treatment of human OPMD.