Neuroprotective Effects of (-)-Epigallocatechin-3-gallate in a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

The purpose of this study is to evaluate neuroprotective effects of (-)-Epigallocatechin-3-gallate (EGCG) in a transgenic mouse model of Amyotrophic lateral sclerosis (ALS). SOD1-G93A transgenic mice and wild-type mice were randomly divided into EGCG-treated groups (10 mg/kg, p.o) and vehicle-treated control groups. Rotarod measurement was performed to assess the motor function of mice starting at the age of 70 days. Nissl staining to examine the number of motor neurons and CD11b immunohistochemical staining to evaluate activation of microglia in the lumbar spinal cords were conducted at the age of 120 days. In addition, for further observation of regulation of cell signaling pathways by EGCG, we used immunohistochemical analysis for nuclear factor kappa B (NF-κB) and cleaved caspase-3 as well as western blot analysis to determine the expression of nitric oxide synthase (iNOS) and NF-κB in the spinal cord. This study demonstrated that oral administration of EGCG beginning from a pre-symptomatic stage significantly delayed the onset of disease, and extended life span. Furthermore, EGCG-treated transgenic mice showed increased number of motor neurons, diminished microglial activation, reduced immunohistochemical reaction of NF-κB and cleaved caspase-3 as well as reduced protein level of iNOS and NF-κB in the spinal cords. In conclusion, this study provides further evidences that EGCG has multifunctional therapeutic effects in the mouse model of ALS.     

Dicer1转基因小鼠模型的建立

目的建立Dicer1转基因小鼠模型。方法构建pcDNA3.1-Dicer1转基因构件,经酶切、纯化后通过显微注射方法导入BDF1小鼠受精卵原核并移植到同期受孕的ICR受体母鼠输卵管内。出生后仔鼠用PCR和Southern方法检测鼠尾DNA鉴定基因型,通过免疫组化检测Dicer1基因表达。结果显微注射172枚卵,移植119枚卵于3只受体输卵管中,2只怀孕,共产仔15只,经PCR检测获得6只阳性鼠,Southern检测6只均为阳性。对Southern检测阳性转基因小鼠子代进行RT-PCR检测和免疫组化分析证明Dicer1基因在肝脏、肾脏、肺内均有表达。对腹腔肿胀的转基因阳性1号鼠解剖发现肝脏、脾脏明显增大,胚胎发育异常。结论成功建立Dicer1基因表达的转基因小鼠模型,该模型为进一步研究DICER1基因功能及miRNA的表达及功能等奠定基础。     

The Effects of Astilbin on Cognitive Impairments in a Transgenic Mouse Model of Alzheimer’s Disease

Bioflavonoids are being utilised as neuroprotectants in the treatment of various neurological disorders, including Alzheimer’s disease (AD). Astilbin, a bioflavanoid, has been reported to have potent neuroprotective effects, but its preventive effects on amyloid-β (Aβ)-induced, Alzheimer’s disease-related, cognitive impairment, and the underlying mechanisms of these effects have not been well characterised. Five-month-old APPswe/PS1dE9 transgenic mice were randomly assigned to a vehicle group and two astilbin (either 20 or 40 mg/kg per day, intraperitoneally) groups. After 8 weeks of treatment, we observed beneficial effects of astilbin (40 mg/kg per day), including lessening learning and memory deficits and reducing plaque burden and Aβ levels. Furthermore, the expressions of both the cAMP responsive element-binding protein (CREB) and brain-derived neurotrophic factor (BDNF) were significantly increased and the disturbance of AKT/GSK-3β signalling pathway was markedly ameliorated in the hippocampus of astilbin-treated (40 mg/kg per day) group. Our data suggest that astilbin might be a potential therapeutic agent against AD.     

慢病毒介导的下丘脑中过表达miR-505小鼠模型的构建

目的:本文用慢病毒定点注射的方法构建了在下丘脑中过表达mi R-505的小鼠模型,并利用荧光原位杂交方法在冰冻切片组织上快速检测mi RNAs,以确认慢病毒载体介导的mi R-505在丘脑中的表达能力。方法:实验小鼠在脑立体定位仪下定位到下丘脑位置,采用原位注射的方式进行慢病毒注射,注射后采用实时荧光定量RCR和应用了LNA探针和TSA系统的FISH(fluorescence in situ hybridization)技术,完成在慢病毒介导的mi R-505过表达老鼠下丘脑区域细胞中的mi R-505检测和示踪。结果:mi R-505慢病毒注射未成年小鼠下丘脑区5、10、20和40天后,均可检测到mi R-505在下丘脑区域的表达,且实验结果表明在慢病毒介导的过表达小鼠下丘脑注射部位,mi R-505表达量有明显的提高。结论:利用慢病毒注射未成年小鼠下丘脑脑区的方法,成功的建立了下丘脑中过表达mi R-505的小鼠模型,使用LNA标记探针的FISH方法探索mi RNA表达规律较稳定,且重复率高。     

潮霉素抗性转基因BDF1小鼠模型的建立

目的建立具有潮霉素（hygromycin）抗性转基因BDF1小鼠,用于制备携有hygromycin抗性筛选标志ES阳性细胞克隆的饲养层。方法通过显微注射的方法,将含有潮霉素B磷酸转移酶基因片段（5.1kb）导入BDF1受精卵雄原核中,共注射169枚受精卵,然后将129枚受精卵细胞植入同期受孕的受体母鼠输卵管内。结果共产生37只转基因小鼠,经PCR和Southern检测获得9只阳性小鼠,对一只子代鼠进行RT-PCR检测证明hyg基因已经在肾、肌肉、脾内表达。结论成功的建立具有潮霉素抗性的BDF1转基因鼠,该模型动物可以为基因敲除研究提供良好的基础条件。     

基于凝血因子IX基因剔除小鼠建立血友病乙转基因动物模型

为在凝血基因IX基因剔除小鼠基础上建立基因组中整合有含特定点突变的人凝血因子IX基因表达载体原转基因小鼠家系,为血友病乙的研究提供更接近临床实际的动物模型。利用体外定点突变技术,构建含有特定点突变的人凝因IX基因表达载体,该载体包括由人凝血因子IX编码区及第一内含子构成的人凝血因子IX基因（hFIXml）、4个拷贝的MCK增强子（MCKe）、鸡β－肌动蛋白启动子（bA）及PolyA,命名为pMe4bAIXml质粒。将其线性化后,用显微注射法注射入817只凝血因子IX基因剔除小鼠受精卵雄原核,再将它们分别回输45只假孕受体母鼠的输卵管中,共产仔69只,存活63只。采用PCR与基因组Southern杂交筛选法鉴定小鼠,证实6只小鼠基因组中整合有含特定点突变的pMe4bAIXml质粒,并对1只小鼠的PCR产物进行测序,证实转基因结构特征符合设计要求。     

Conditional Over-Expression of Estrogen Receptor Alpha in a Transgenic Mouse Model

Attempts to delineate the mechanisms of estrogen action have promoted the creation of several estrogen receptor alpha (ER) mouse models in the past decade. These traditional models are limited by the fact that the receptors are either absent or present throughout all stages of development. The purpose of this work was to develop a conditional transgenic model that would provide an in vivo method of controlling the spatial and temporal regulation of ER expression. The tetracycline responsive system was utilized. Three lines of transgenic mice carrying a transgene composed of the coding sequence for murine ER placed under the regulatory control of a tet operator promoter (tet-op) were generated. These three lines of tet-op-mER mice were each mated to an established line of transgenic mice expressing a tetracycline-dependent transactivator protein (tTA) from the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). Double transgenic MMTV-tTA/tet-op-mER mice were produced. All three lines demonstrated dominant gain of ER shown by RT-PCR, immunoprecipitation, and immunohistochemistry. Transgene-specific ER was expressed in numerous tissues including the mammary gland, salivary gland, testis, seminal vesicle, and epididymis. Expression was silenced by administration of doxycycline in the drinking water. This model can be utilized to evaluate the consequences of ER dominant gain in targeted tissues at specific times during development. In this study dominant gain of ER was associated with a reduction in epididymal/vas deferens and seminal vesicle weights consistent with the proposed action of ER on fluid transport in the male reproductive tract. Combining this model with other dominant gain and gene knockout mouse models will be useful for testing effects of ER action in combination with specific gene products and to evaluate if developmental and stage-specific expression of ER can rescue identified phenotypes in gene knockout mice.     

miR-122过表达转基因小鼠质粒构建及其功能验证

目的:比较两种miR-122转基因小鼠过表达载体构建方法,为建立miR-122过表达转基因小鼠奠定基础。方法:PCR扩增长约291bp的pre-miR-122的序列,分别定向克隆到pBROAD3-GFP载体GFP基因上游内含子或下游3'UTR区域,两种质粒分别转染293T细胞,Q-PCR检测miR-122和GFP的表达水平,并观察GFP绿色荧光。miR-122 sensor reporter是将3个miR-122成熟序列的反义序列串联克隆至psiCHECK2载体luciferase 3'UTR中,然后分别与2种miR-122过表达质粒载体共转染293T细胞,最后检测荧光素酶活性来鉴定miR-122调控功能。结果:2种构建方法的miR-122表达水平都明显增高,而只有插入到GFP基因3'UTR的质粒表达GFP功能正常。结论:构建microRNA过表达载体时,microRNA位于报告基因3'UTR区域不会影响microRNA和报告基因的功能;构建的两种miR-122过表达质粒载体都可应用到转基因小鼠研究中,而将miR-122插入到GFP下游的方法则更利于miR-122的表达。     

庚型肝炎病毒转基因小鼠的建立

利用受精卵原核显微注射的方法,产生了含有HGV结构蛋白C、E1、E2及部分非结构蛋白NS2、NS3的转基因小鼠.得到10只founder小鼠,其中有3只founder小鼠与正常小鼠交配后得到了整合有外源基因的F1代阳性小鼠.RT-PCR的结果显示,外源基因可在founder小鼠及F1代小鼠的血液有核细胞及肝细胞内转录;组织病理学检查显示,某些转基因小鼠的肝细胞出现了水样变、脂肪变性及轻微炎性反应等病理学改变,但与同一品系的正常小鼠相比,转基因小鼠血清转氨酶无明显升高.     

庚型肝炎病毒转基因小鼠的建立

利用受精卵原核显微注射的方法,产生了含有ＨＧＶ结构蛋白Ｃ、Ｅ１、Ｅ２及部分非结构蛋白ＮＳ２、ＮＳ３的转基因小鼠。得到１０只ｆｏｕｍｄｅｒ小鼠,其中有３只ｆｏｕｎｄｅｒ小鼠与正常小鼠交配后得到了整合有外源基因的Ｆ１代阳性小鼠。ＲＴ－ＰＣＲ的结果显示,外源基因可在ｆｏｕｎｄｅｒ小鼠及Ｆ１代小鼠的血液有核细胞及肝细胞内转录;组织病理学检查显示,某些转基因小鼠的肝细胞出现了水样变、脂肪变性及轻微炎性反应等     

Carcinogenic Effects in a Phenylketonuria Mouse Model

Phenylketonuria (PKU) is a metabolic disorder caused by impaired phenylalanine hydroxylase (PAH). This condition results in hyperphenylalaninemia and elevated levels of abnormal phenylalanine metabolites, among which is phenylacetic acid/phenylacetate (PA). In recent years, PA and its analogs were found to have anticancer activity against a variety of malignancies suggesting the possibility that PKU may offer protection against cancer through chronically elevated levels of PA. We tested this hypothesis in a genetic mouse model of PKU (PAH^enu2) which has a biochemical profile that closely resembles that of human PKU. Plasma levels of phenylalanine in homozygous (HMZ) PAH^enu2 mice were >12-fold those of heterozygous (HTZ) littermates while tyrosine levels were reduced. Phenylketones, including PA, were also markedly elevated to the range seen in the human disease. Mice were subjected to 7,12 dimethylbenz[a]anthracene (DMBA) carcinogenesis, a model which is sensitive to the anticancer effects of the PA derivative 4-chlorophenylacetate (4-CPA). Tumor induction by DMBA was not significantly different between the HTZ and HMZ mice, either in total tumor development or in the type of cancers that arose. HMZ mice were then treated with 4-CPA as positive controls for the anticancer effects of PA and to evaluate its possible effects on phenylalanine metabolism in PKU mice. 4-CPA had no effect on the plasma concentrations of phenylalanine, phenylketones, or tyrosine. Surprisingly, the HMZ mice treated with 4-CPA developed an unexplained neuromuscular syndrome which precluded its use in these animals as an anticancer agent. Together, these studies support the use of PAH^enu2 mice as a model for studying human PKU. Chronically elevated levels of PA in the PAH^enu2 mice were not protective against cancer.     

NGI重组DNA疫苗对转基因AD小鼠的治疗作用

目的研究NGI对转基因阿尔茨海默病小鼠的治疗作用。方法构建包含小鼠OMgp细胞外8LRP重复序列,人Tenascin-R的EGF-L结构域,以及人Nogo-A的氨基端和66个氨基酸细胞外域的重组质粒,肌肉注射AD小鼠,空载体组和空白对照组为对照组。水迷宫检测小鼠行为学差别,免疫组化检测其病理差别。结果4.5月龄AD小鼠经过水迷宫隐蔽平台实验,2d后疫苗组小鼠潜伏期比对照组缩短30%（P〈0.05）。探索实验显示3月龄疫苗组比空载体组和空白对照组小鼠穿越目标象限的次数分别增加48%和44%（P〈0.05）。结论NGI重组DNA疫苗能够提高转基因AD小鼠的学习与记忆能力。     

Protein Oxidative Damage in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

Abstract: The Gly⁹³→Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1-G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre-motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre-disease), 100 (～50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30-day-old TgN(SOD1-G93A)G1H mice was twice as high as the level found in age-matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1-G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild-type human Cu,Zn-SOD [TgN(SOD1)N29]. The 100–120-day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH-derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn-SOD. Western blot comparison of the extent of Cu,Zn-SOD protein carbonylation revealed that the level in spinal cord samples from 120-day-old TgN(SOD1-G93A)G1H mice was significantly higher than that found in age-matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation-derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn-SOD itself is a key target, which may compromise its antioxidant function.     

Inhibition of mTOR Reduces Anal Carcinogenesis in Transgenic Mouse Model

The molecular mechanism of human anal squamous cell carcinoma (ASCC) is unclear, and the accumulating evidence indicate association of ASCC with the activation of the Akt/mTOR pathway. Here we describe a mouse model with spontaneous anal squamous cell cancer, wherein a combined deletion of Tgfbr1 and Pten in stratified squamous epithelia was induced using inducible K14-Cre. Histopathologic analyses confirmed that 33.3% of the mice showed increased susceptibility to ASCC and precancerous lesions. Biomarker analyses demonstrated that the activation of the Akt pathway in ASCC of the Tgfbr1 and Pten double knockout (2cKO) mouse was similar to that observed in human anal cancer. Chemopreventive experiments using mTOR inhibitor-rapamycin treatment significantly delayed the onset of the ASCC tumors and reduced the tumor burden in 2cKO mice by decreasing the phosphorylation of Akt and S6. This is the first conditional knockout mouse model used for investigating the contributions of viral and cellular factors in anal carcinogenesis without carcinogen-mediated induction, and it would provide a platform for assessing new therapeutic modalities for treating and/or preventing this type of cancer.     

microRNA转基因小鼠在心脏发育研究中的应用

microRNA（miRNA）参与调控胚胎心脏的发育,在心脏形态发生、心肌细胞生长及分化过程中发挥着极其重要的作用。通过转基因技术可以实现特异miRNA在心肌组织的过表达与敲除,据此建立的心肌特异性miRNA转基因小鼠模型可以在整体水平揭示miRNA心脏方面的功能。近年,以miRNA为研究对象的心肌特异性转基因小鼠模型数量不断增加。     

Overexpression of the Fibroblast Growth Factor Receptor 1 (FGFR1) in a Model of Spinal Cord Injury in Rats

Spinal cord injury (SCI) is a severe condition that affects many people and results in high health care costs. Therefore, it is essential to find new targets for treatment. The fibroblast growth factor receptor 1 (FGFR1) signalling pathway has a history of being explored for SCI treatment. Several groups have examined the effect of high availability of different FGFR1 ligands at the injury site and reported corticospinal tract (CST) regeneration as well as improved motor functions. In this study, we investigated overexpression of the FGFR1 in rat corticospinal neurons in vivo after injury (unilateral pyramidotomy) and in cerebellar granule neurons (CGNs) in vitro. We show that overexpression of FGFR1 using AAV1 intracortical injections did not increase sprouting of the treated corticospinal tract and did not improve dexterity or walking in a rat model of SCI. Furthermore, we show that overexpression of FGFR1 in vitro resulted in decreased neurite outgrowth compared to control. Thus, our results suggest that the FGFR1 is not a suitable therapeutic target after SCI.     

Characterization of Gastric and Neuronal Histaminergic Populations Using a Transgenic Mouse Model

Histamine is a potent biogenic amine that mediates numerous physiological processes throughout the body, including digestion, sleep, and immunity. It is synthesized by gastric enterochromaffin-like cells, a specific set of hypothalamic neurons, as well as a subset of white blood cells, including mast cells. Much remains to be learned about these varied histamine-producing cell populations. Here, we report the validation of a transgenic mouse line in which Cre recombinase expression has been targeted to cells expressing histidine decarboxylase (HDC), which catalyzes the rate-limiting step in the synthesis of histamine. This was achieved by crossing the HDC-Cre mouse line with Rosa26-tdTomato reporter mice, thus resulting in the expression of the fluorescent Tomato (Tmt) signal in cells containing Cre recombinase activity. As expected, the Tmt signal co-localized with HDC-immunoreactivity within the gastric mucosa and gastric submucosa and also within the tuberomamillary nucleus of the brain. HDC expression within Tmt-positive gastric cells was further confirmed by quantitative PCR analysis of mRNA isolated from highly purified populations of Tmt-positive cells obtained by fluorescent activated cell sorting (FACS). HDC expression within these FACS-separated cells was found to coincide with other markers of both ECL cells and mast cells. Gastrin expression was co-localized with HDC expression in a subset of histaminergic gastric mucosal cells. We suggest that these transgenic mice will facilitate future studies aimed at investigating the function of histamine-producing cells.