人G-CSF转基因鼠的稳定遗传与表达

利用人粒细胞集落刺激因子（Ｇ－ＣＳＦ）基因组基因作为目的片段,将其受控于２．６ｋｂ的小鼠乳清酸蛋白（ＷＡＰ）基因的调控区下,通过显微注射法获得了两只整合有人Ｇ－ＣＳＦ转基因小鼠,通过繁殖建立了稳定的转基因系．一些表型参数测定表明转基因鼠与正常鼠无明显差别．通过ＲＴ－ＰＣＲ及Ｓｏｕｔｈｅｒｎｂｌｏｔ检测,在乳腺表达出人Ｇ－ＣＳＦ,为乳腺表达外源蛋白质及今后大动物研究奠定了基础．     

Expression of the murine wild-type tyrosinase gene in transgenic rabbits

The tyrosinase gene is known to be essential for melanization and has been shown to rescue pigmentation in albino mice. Previously we have described the strict copy-number-dependent expression of a murine wild-type tyrosinase gene construct over several generations in transgenic mice. In this study, we analysed the same gene construct as a marker gene for the transmission and expression of transgenes in rabbits. Using an albino hybrid strain, we produced transgenic rabbits expressing the murine tyrosinase gene. Strict correlation between integration and expression of the transgene and stable germline transmission of the integrated gene construct according to the Mendelian pattern of inheritance was observed. Thus, breeding control was facilitated by simple phenotypic examination of the transgenic animals. In contrast to mice transgenic for the same gene construct, tyrosinase-transgenic rabbits showed a greater variety in hue, intensity and extent of coat pigmentation, which is caused by the diversity in the loci affecting the melanization. Benefits and limitations of tyrosinase as a marker gene for the detection of homozygous individuals in the albino hybrid strain used are discussed.     

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.     

腺相关病毒介导基因组来源的基因转移与整合

构建一个带β-珠蛋白基因组序列的腺相关病毒载体AV53HS2Δβ2Neo.经包装成重组腺相关病毒后,转导红系细胞.DNA印迹证实包含红系增强子、β-珠蛋白基因和筛选标志基因的前病毒基因组完整整合于红系细胞基因组中.结果说明腺相关病毒载体能介导基因组序列来源的目的基因稳定整合于受体细胞基因组中.     

应用接头PCR克隆慢病毒介导的转基因小鼠整合位点旁侧序列

目的为鉴定慢病毒介导的转基因小鼠中外源基因的整合位点信息,应用接头PCR克隆整合位点旁侧序列。方法小鼠基因组总DNA酶解后与设计的接头片段连接,根据慢病毒的LTR序列设计巢式PCR引物,克隆转基因小鼠整合位点旁侧序列。结果成功克隆到转基因小鼠整合位点的旁侧序列,经过测序定位于小鼠染色体上。结论作为反向PCR的改进,本方法可用于转基因小鼠整合位点旁侧序列的克隆,为分析整合位点与外源基因表达之间的关系等提供了科学依据。     

基因治疗载体及其基因转移技术的关键问题与研究现状

目前使用的基因治疗载体及其基因转移技术中还没有一种能用于临床并永久有效的基因转移技术 .该文分析了直接体内转移、间接体内转移及其非载体法、病毒载体法、非病毒性生物载体法等基因治疗转移技术存在的一些关键问题 ,并探讨了各问题的解决办法或研究策略以及基因治疗载体研究的发展方向     

Low-Dose Sarin Exposure Produces Long Term Changes in Brain Neurochemistry of Mice

Sarin is a toxic organophosphorus (OP) nerve agent that has been reported to cause long-term alterations in behavioral and neuropsychological processes. The present study was designed to investigate the effect of low dose sarin exposure on the monoamine neurotransmitter systems in various brain regions of mice. The rationale was to expand our knowledge about the noncholinergic neurochemical alterations associated with low dose exposure to this cholinesterase inhibitor. We analyzed the levels of monoamines and their metabolites in different brain areas after exposure of male C57BL/6 mice to a subclinical dose of sarin (0.4 LD50). Mice did not show any signs of cholinergic toxicity or pathological changes in brain tissue. At 1, 4 and 8 weeks post-sarin exposure brains were collected for neurochemical analysis. A significant decrease in the dopamine (DA) turnover, as measured by the metabolite to parent ratio, was observed in the frontal cerebral cortex (FC) at all time points tested. DA turnover was significantly increased in the amygdala at 4 weeks but not at 1 or 8 weeks after exposure. The caudate nucleus displayed a decrease in DA turnover at 1 week but no significant change was observed at 4 and 8 weeks suggesting a reversible effect. In addition to this, serotonin (5-HT) levels were transiently altered at various time points in all the brain regions studied (increase in FC, caudate nucleus and decrease in amygdala). Since there were no signs of cholinergic toxicity or cell death after sarin exposure, different non-cholinergic mechanisms may be involved in regulating these effects. Our results demonstrate that non-symptomatic dose of OP nerve agent sarin has potent long-term, region-specific effects on the monoaminergic neurotransmitter systems. Data also suggests differential effects of sarin on the various DA projections. These neurochemical alterations could be associated with long term behavioral and neuropsychological changes associated with low dose OP exposure.     

CREB——长时记忆的介导因子

综述了CREB的研究进展和该领域中需深入研究和注意的问题.CREB作为一种转录因子参与短时记忆向长时记忆的转化,它具有激活型和抑制型两种形式,籍此可以更加精细地调节记忆的转化,这在不同种属动物中已经得到证实,且其基因序列存在着高度的保守性.     

人工合成甘蓝型油菜特长角性状的遗传分析

以人工合成甘蓝型油菜特长角品系H218普通甘蓝型油菜中油821和MSP334为材料, 配制成两个杂交组合的各6个世代群体, 即P1、P2、F1、F2、B1、B2, 研究了全果长、果身长、果喙长3个角果性状的遗传特点。结果表明, 两个组合的全果长、果身长、果喙长在F2群体中近乎呈现连续正态分布, 显示为多基因控制的数量性状。两个组合的全果长广义遗传力为65.89%~70.77%、狭义遗传力为44.01%~46.78%, 基因数目均为5对; 果身长广义遗传力为60.14% ~ 63.38%、狭义遗传力为46.89%~47.38%, 基因数目均为6对; 果喙长广义遗传力为26.36% ~ 46.44%, 狭义遗传力为18.08%~37.87%, 基因数目均为2对。基因效应初步分析表明, 两个组合的3个性状基因效应均符合加性-显性二基因互作模型, 存在显著的上位性效应。两个组合的全果长、果身长、果喙长加性效应、显性效应都很重要, 全果长、果身长的加性效应大于显性效应, 果喙长显性效应大于加性效应。     

烟草质体多顺反子定点整合表达载体的构建和转化

构建了烟草质体多顺反子定点整合表达载体pLM4(-psaA-Prrn-RBS-man-RBS-gfp-RBS-aadA-psbA3'-psbC-).用基因枪将该载体轰击烟草叶片5次,用添加了壮观霉素的选择分化培养基筛选,获得质体转基因烟草6株.用PCR、激光扫描、Western blot和RFLP等方法检测都证实多顺反子表达盒中的3个基因甘露聚糖酶基因(man)、绿荧光蛋白基因(gfp)、氨基糖苷3'-腺苷酰基转移酶基因(aadA)已整合到烟草质体基因组中,且均得到表达.     

转基因在玉米中的遗传分离与整合特性的研究

用ＰＣＲ和ＤＮＡ分子杂交方法研究了转基因Ｂｔ在３种不同方法获得的８个转化体后代中的遗传分离、整合性质及其稳定性,结果表明：（１）转基因在大多数转化体后中呈简单的孟德尔遗传;在Ｒ１至Ｒ２代,部分家系中转化体比例偏低,有的发生转基因丢失,但到Ｒ３代以后均趋于正常的孟德尔遗传方式,在群体中固定下来;（２）转基因在不同转化体中的整合类型有一定差异,但整合的位点和拷贝数都较少,且多呈串联或紧密连锁的整合;（     

Curcumin Rescues Aging-Related Loss of Hippocampal Synapse Input Specificity of Long Term Potentiation in Mice

Curcumin has neuroprotective effect and could enhance memory. However, the mechanisms underlying the protection of curcumin on aging-related memory decline are not well understood. In this study, high frequency stimulation (HFS)-induced long term potentiation (LTP) was evaluated by a cellular model of memory formation. A two-input stimulation paradigm was used to record the potentiation as well as synapse input specificity. The data suggested that an N-Methyl-d-aspartate receptors (NMDAR) -dependent LTP was inducible in adult hippocampal slices with a characteristic of synapse input specificity. It also indicated that aging resulted in a reduction in LTP but more importantly a loss of synaptic input specificity. The reason behind the above conclusions is that LTP induction is more dependent on the calcium channel. This is due to a switch of the dependence of LTP induction to voltage-dependent calcium channel (VDCC) compared to NMDA receptors. Curcumin administration recovers input specificity by re-establishing NMDA receptor dependence of induction. In addition, curcumin administration ameliorated aging-related increase of brain thiobarbituric acid-reactive substances and elevated aging-related decrease of glutathione in hippocampus. It is then concluded that curcumin modulates hippocampal redox status and restores aging-related loss of synapse input specificity of HFS-induced LTP by switching VDCC calcium source into NMDA receptor-dependent one.     

转基因小鼠中外源基因遗传及表达稳定性的研究

挑选两个乳汁中人凝血因子IX（hFIX）表达量相差较大的转基因小鼠家系,分别用PCR、Southern blot、FISH和ELISA对两个家系中的小鼠进行检测。结果显示后代小鼠的转基因阳性率为50%左右;外源基因的整合是完整的,没有发现可见的丢失现象;家系中的各个小鼠表达量有差异,FIX-33家系中hFIX在乳汁中的表达量为（43.32±5.41）?g/mL;FIX-124家系中hFIX在乳汁中的表达量是（1.16±0.45）?g/mL。而两个家系之间的表达量则差异极为显著(P<0.01)。这表明原代转基因小鼠的遗传及表达特性可以得到稳定的传递。     

突破复杂性状多基因转化技术壁垒,首创胚乳花青素高积累的水稻新种质

随着转基因技术的日趋成熟,利用生物工程手段加快改良作物农艺性状,已经越来越显示出其巨大的应用潜力。在改良多基因调控的复杂农艺性状方面,单基因转化收效甚微,而长期以来多基因转化不仅受限于技术因素,而且在协调表达调控、代谢及修饰等一系列相关基因方面更是难于突破。近期,我国科学家首次利用自创的多基因垛叠表达系统,成功在水稻(Oryza sativa)胚乳中合成了具有抗氧化活性的花青素,在复杂性状多基因转化领域取得了突破性进展。     

精子介导法建立sFat-1转基因小鼠模型

利用精子介导的基因转移(Sperm-mediated gene transfer,SMGT)方法,将sFat-1 DNA片段直接和小鼠精子孵育,再通过体外受精、胚胎移植等技术建立sFat-1转基因小鼠模型。经PCR检测,16只后代中发现2只阳性个体,Southern blot进一步鉴定的结果显示有2只后代整合了sFat-1基因,成功建立sFat-1转基因小鼠模型,转基因率为12.5%。本研究运用精子介导的转基因方法将sFat-1基因整合到小鼠基因组中,为进一步研究sFat-1的生物学功能提供了实验动物模型。     

Transgene expression of human PON1 Q in mice protected the liver against CCl4-induced injury

BACKGROUND: Oxidative stress, often in association with decreased antioxidant defenses, plays a pathogenetic role in both initiation and progression of liver injuries, leading to almost all clinical and experimental conditions of chronic liver diseases. Human paraoxonase 1 (hPON1) is a liver-synthesized enzyme possessing antioxidant properties. Here, we investigate the effects of transgene-expressed hPON1 Q on alleviating lipid peroxidation and preventing liver injury in a mouse model. METHODS: The hPON1 Q gene was cloned into pcDNA3.0 plasmid and electro-transferred into mouse skeletal muscle. After CCl4 had been administrated to induce liver injury, mice were monitored for serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and malonyldialdehyde (MDA). The extent of CCl4-induced liver injury was also analyzed through histopathological observations. RESULTS: After gene delivery, hPON1 mRNA expression was detected in mouse muscle and serum PON1 activity was 1.5 times higher than that of the control counterpart. In the PON1 Q gene transferred mice, protection against CCl4-induced liver injury was reflected by significantly decreased serum ALT, AST and MDA levels compared to those in control mice (P < 0.01). Histological observations also revealed that hepatocyte necrosis, hemorrhage, vacuolar change and hydropic degeneration were apparent in control mice after CCl4 administration. In contrast, the damage was significantly prevented (P < 0.01) in the hPON1 Q transferred mice. CONCLUSIONS: Intramuscular electro-transfer of the hPON1 Q gene led to efficient expression of hPON1 in mice. Elevated levels of PON1, by virtue of its potency to alleviate oxidative stress, could protect mice from suffering CCl4-induced liver damage.     

转基因动物整合位点的研究进展

转基因动物整合位点克隆及相关序列特征研究是探索外源基因整合机制和整合稳定性的重要手段。转基因整合位点序列的克隆方法主要有：个体基因组文库筛选法、反向PCR法、热不对称交互式PCR法和质粒回收法。4种方法各有优缺点,可根据不同条件选用。克隆到的整合位点序列表明,整合位点可能存在一定的共同特征。 Integration Sites of Transgenes in Transgenic Animals WU Bo,ZHU Zuo-Yan State Key Laboratory of Freshwater Ecology and Biotechnology,Institute of Hydrobiology, Chinese Academy of Science,Wuhan 430072,China Abstract:To study the mechanism of transgene ' s integration in the host genome,first step is to clone the integration-site sequence.The method employed for this purpose includes selection from genomic pool,IPCR,TAIL-PCR and plasmid rescued.Different method fits different cases depending on how the transgenics have been produced.The data of integration-site sequences showed some similar features existed. Key words：transgene; integration site; clone method