Nerve growth factor promoter activity revealed in mice expressing enhanced green fluorescent protein

Nerve growth factor (NGF) and its precursor proNGF are perhaps the best described growth factors of the mammalian nervous system. There remains, however, a paucity of information regarding the precise cellular sites of proNGF/NGF synthesis. Here we report the generation of transgenic mice in which the NGF promoter controls the ectopic synthesis of enhanced green fluorescent protein (EGFP). These transgenic mice provide an unprecedented resolution of both neural cells (e.g., neocortical and hippocampal neurons) and non-neural cells (e.g., renal interstitial cells and thymic reticular cells) that display NGF promoter activity from postnatal development to adulthood. Moreover, the transgene is inducible by injury. At 2 days after sciatic nerve ligation, a robust population of EGFP-positive cells is seen in the proximal nerve stump. These transgenic mice offer novel insights into the cellular sites of NGF promoter activity and can be used as models for investigating the regulation of proNGF/NGF expression after injury.     

Sweat gland innervation is pioneered by sympathetic neurons expressing a cholinergic/noradrenergic co-phenotype in the mouse

Classic neurotransmitter phenotypes are generally predetermined and develop as a consequence of target-independent lineage decisions. A unique mode of target-dependent phenotype instruction is the acquisition of the cholinergic phenotype in the peripheral sympathetic nervous system. A body of work suggests that the sweat gland plays an important role to determine the cholinergic phenotype at this target site. A key issue is whether neurons destined to innervate the sweat glands express cholinergic markers before or only after their terminals make target contact. We employed cholinergic-specific over-expression of the vesicular acetylcholine transporter (VAChT) in transgenic mice to overcome sensitivity limits in the detection of initial cholinergic sweat gland innervation. We found that VAChT immunoreactive nerve terminals were present around the sweat gland anlage already from the earliest postnatal stages on, coincident selectively at this sympathetic target with tyrosine hydroxylase-positive fibers. Our results provide a new mechanistic model for sympathetic neuron-target interaction during development, with initial selection by the target of pioneering nerve terminals expressing a cholinergic phenotype, and subsequent stabilization of this phenotype during development.     

Sustained hippocampal neurogenesis in females is amplified in P66Shc−/− mice: An animal model of healthy aging

Aging is accompanied by poor learning and memory abilities and by decreased hippocampal neurogenesis, a process that is also modulated by oxidative stress (OS). P66^Shc has recently emerged as a novel mammalian gerontogene able to affect healthspan during aging. Deletion of this gene in mice leads to reduced OS accompanied by decreased incidence of age‐related pathologies and reduced signs of behavioral aging. We hypothesized that p66^Shc?/? mutants might show increased neurogenesis in the hippocampus, a brain region involved in learning and memory processes. To this aim, granule cell number, proliferation, neuronal differentiation, and cell death were assessed in the hippocampus in senescent p66^Shc?/? [knock out (KO)] and p66^Shc+/+ [wild type (WT)] male and female mice. Spatial learning abilities and spontaneous activity were also investigated in a multifunctional behavioral system—IntelliCages. The behavioral analysis revealed that females in general perform better in spatial learning tasks, with genotype effects being apparent in the activity pattern only. Likewise, all females showed increased neuronal differentiation, whereas increased proliferation was found only in those belonging to the p66^Shc?/? genotype, indicating that they might be protected from precursor cell loss. The number of dying cells was not affected by genotype or sex; however, all KO mice showed less granule cells than WT. Overall, our data suggest that hippocampal function is protected in the female gender at older age, an effect amplified by reduced OS in the p66^Shc?/? mutant. © 2012 Wiley Periodicals, Inc.     

斑马鱼炎症模型及其在中药抗炎领域的应用

中药抗炎在近年来已成为一个重要的研究领域,中药制剂的抗炎临床研究已经取得了一定进展,但具体活性成分及作用机制仍需进一步明确。斑马鱼由于其独特的生物学优势,目前已被广泛应用于中药抗炎活性的筛选。综合近年来国内外有关斑马鱼模型在中药抗炎领域的报道,从已构建的斑马鱼炎症模型、斑马鱼模型中的炎症介质、斑马鱼模型在中药抗炎药物筛选中的应用3个方面对斑马鱼炎症模型及其在中药抗炎领域的研究进展进行综述,以期为模式生物斑马鱼在中药抗炎领域的研究应用提供新思路,为抗炎中药的开发与利用提供借鉴参考。     

Discrimination between maintenance- and differentiation-inducing signals during initial and intermediate stages of positive selection

As well as signaling through the αβ T cell receptor complex, positive selection of immature CD4⁺ 8⁺ thymocytes involves additional ill-defined accessory interactions provided by thymic epithelial cells. Here, we have isolated CD4⁺ 8⁺ thymocytes at a pre-positive selection stage of development (TCR^? CD69^? 4⁺ 8⁺ cells), or after initiation of positive selection (CD69⁺ 4⁺ 8⁺ cells), from mice where the normal lifespan of thymocytes is extended by the presence of a bcl-2 transgene, to allow us to discriminate between requirements for maintenance and differentiation signals during positive selection. We find that MHC class II⁺ thymic epithelial cells drive positive selection of TCR^? CD69^? 4⁺ 8⁺ bcl-2 tg thymocytes to the CD4⁺ and CD8⁺ stage, while no such mature subsets are observed when thymocytes are cultured alone or with major histocompatibility complex (MHC) class II⁺ salivary epithelial cells. However, CD4⁺ 8⁺ cells remain in such cultures in considerable numbers, and retain the potential for positive selection if re-cultured with thymic epithelium, suggesting that thymic epithelial cells provide specific differentiation-inducing signals for positive selection. In contrast, intermediate CD69⁺ 4⁺ 8⁺ thymocytes show some capacity for phenotypic conversion in the absence of thymic stromal cells although strikingly the single-positive CD4⁺ and CD8⁺ cells generated are not functionally competent. Finally, we show that prior culture of thymic epithelial cells under monolayer conditions abrogates their ability to support the initiation of positive selection, suggesting that the epithelial cell molecules necessary for the provision of differentiation signals during positive selection are down-regulated under such conditions.     

Highly sensitive and species-specific assay for quantification of human transgene expression levels

During the past few years great efforts have been made to construct and to test human factor VIII (hFVIII) and IX (hFIX) vectors suitable for haemophilia gene therapy in vivo. However, little is known about the molecular mechanisms of persistence and shut-off of transgene expression in the target organs after gene transfer using recombinant adenoviral vectors. To evaluate low transgene mRNA levels in different tissues, especially at long times after the gene transfer, the common northern blot method is often not sensitive enough. For this reason we developed a new, highly sensitive and species-specific method for hFIX mRNA quantification and employed it in mice treated with an adenoviral vector (Ad5CMVFIX) expressing human FIX. In addition to its very high sensitivity (lowest detection level=1 fg RNA), the method was shown to be strictly species-specific, since hFIX mRNA signals were never detected in untreated mice. In a long-term study of 18 vector-treated mice we compared the human FIX:Ag levels in the mouse plasma, the human FIX mRNA levels and human FIX vector DNA concentrations in the mouse liver. We found that a slow but continuous decrease of hFIX:Ag levels in mouse plasma was associated with a corresponding decrease of hFIX mRNA levels in the liver. However, the Ad5CMVFIX vector DNA levels did not decrease to a comparable degree, suggesting that the decrease of human FIX:Ag levels in mouse plasma is, to a significant extent, also caused by CMV promotor shut-off and only to a minor degree by loss of vector DNA.     

The butadiene metabolite, 1,2:3,4-diepoxybutane,induces micronuclei but is only weakly mutagenic at lacI in the Big Blue® Rat2 lacI transgenic cell line

1,3-Butadiene (BD) is a genotoxic carcinogen that is bioactivated to at least two mutagenic metabolites, 1,2-epoxybutene (EB) and 1,2:3,4-diepoxybutane (DEB). We investigated the mutagenicity and induction of micronuclei by DEB in vitro in Rat2 lambda/lacI transgenic fibroblasts (Big Blue® Rat2 cells, Stratagene, LaJolla, CA). Assays for mutagenicity and micronuclei induction were carried out at concentrations of 0, 2, 5, or 10 μM DEB for 24 hours. Exposure of cells to these concentrations of DEB resulted in approximately 100, 50, and 10% survival, respectively, compared with media controls. In independent replicate experiments, no statistically significant increase in lacI mutant frequency was observed in Rat2 cells at any of the DEB exposure concentrations when compared tomedia or solvent controls. However, regressionanalyses indicated a trend toward increasing mutant frequency with increasing DEB exposure concentration. Experiments to examine the induction of micronuclei by DEB revealed a concentration-dependent increase in micronuclei in Rat2 cells following exposure to DEB. These results indicate that DEB induces micronuclei in the absence of detectable gene mutation at lacI in Big Blue Rat2 cells. The induction ofmicronuclei but only weak mutagenicity at the lacI transgene is likely due to the poor recovery of deletions using this lambda shuttle vector system, demonstrating the need to investigate multiple endpoints of genotoxicity when considering the mutational activity of a compound. Environ. Mol. Mutagen. 31:32–40, 1998. © 1998 Wiley-Liss, Inc.     

Inhibitory neuron‐specific Cre‐dependent red fluorescent labeling using VGAT BAC‐based transgenic mouse lines with identified transgene integration sites

Inhibitory neurons are crucial for shaping and regulating the dynamics of the entire network, and disturbances in these neurons contribute to brain disorders. Despite the recent progress in genetic labeling techniques, the heterogeneity of inhibitory neurons requires the development of highly characterized tools that allow accurate, convenient, and versatile visualization of inhibitory neurons in the mouse brain. Here, we report a novel genetic technique to visualize the vast majority and/or sparse subsets of inhibitory neurons in the mouse brain without using techniques that require advanced skills. We developed several lines of Cre‐dependent tdTomato reporter mice based on the vesicular GABA transporter (VGAT)‐BAC, named VGAT‐stop‐tdTomato mice. The most useful line (line #54) was selected for further analysis based on two characteristics: the inhibitory neuron‐specificity of tdTomato expression and the transgene integration site, which confers efficient breeding and fewer adverse effects resulting from transgene integration‐related genomic disruption. Robust and inhibitory neuron‐specific expression of tdTomato was observed in a wide range of developmental and cellular contexts. By breeding the VGAT‐stop‐tdTomato mouse (line #54) with a novel Cre driver mouse line, Galntl4‐CreER, sparse labeling of inhibitory neurons was achieved following tamoxifen administration. Furthermore, another interesting line (line #58) was generated through the unexpected integration of the transgene into the X‐chromosome and will be used to map X‐chromosome inactivation of inhibitory neurons. Taken together, our studies provide new, well‐characterized tools with which multiple aspects of inhibitory neurons can be studied in the mouse.     

液压转基因技术用于脂肪肝大鼠的肝脏转基因实验

目的 探讨液压转基因技术（HDT）应用于大鼠脂肪肝转基因的条件、方法。 方法 以不同速度将不同体积、浓度的绿色荧光蛋白基因质粒pEGFP-C1一次性注射到脂肪肝大鼠尾静脉,于注射后不同时间取大鼠（各4只）各肝叶制备冷冻切片,在波长488nm的荧光显微镜下观察、计数各肝叶绿色荧光蛋白阳性细胞。 结果 在pEGFP-C1浓度为33mg/L、注射速度为2ml/s、注射液体积为大鼠体重的8.5%条件下,注射质粒后6h,各肝叶绿色荧光蛋白阳性细胞比例最高,其中,蒂状叶的绿色荧光蛋白阳性细胞约占18%,左叶约占14%、中叶约占12.5%、右叶约占10%,尾状叶约占8%。转基因后24h绿色荧光蛋白的表达量逐渐减少,至72h时各肝叶均难以检出绿色荧光蛋白阳性细胞。 结论 大鼠尾静脉液压转基因技术可用于脂肪肝大鼠的肝脏转基因研究,转基因的适宜条件为：质粒溶液浓度33mg/L,注射量占大鼠体重的8.5%,注射速度为2ml/s,观察转基因效果的适宜时间是转基因后6～24h。     

Colon-specific tumorigenesis in mice driven by Cre-mediated inactivation of Apc and activation of mutant Kras

Several genetically engineered mouse (GEM) models of colorectal cancer have been developed and are a mainstay in our efforts to identify means of preventing and treating this disease. Many of these models involve a germline disruption of the adenomatous polyposis coli (Apc) tumor suppressor gene and share the limitation that the great preponderance of tumors appear in the small rather than large intestine. In recent years efforts have been made to increase the similarity of these models to human sporadic colorectal cancer by disrupting Apc in a tissue-specific fashion using the Cre-Lox system so that the genetic aberrations are confined to the colonic epithelium. These models have shown great promise but reproducible and high penetrance colon-specific tumorigenesis has not yet been achieved without invasive techniques to introduce the Cre enzyme. We therefore sought to create a new model with high penetrance colon-specific tumorigenesis but without the need for exogenous Cre administration. We utilized existing mice possessing a conditional knock out for the Apc gene and a latent activated Kras allele and crossed them with mice expressing Cre recombinase solely in the large intestine. Using this approach we generated mice that developed 1–9 colonic adenomas per mouse (average 4.3) but without any tumors in the small intestine or cecum. No invasive tumors were observed. Despite the apparent lack of invasion, the geographical correctness, complete penetrance and intermediate tumor burden make this model a promising addition to our toolkit for the study of colorectal cancer treatment and prevention.