南中国斑节对虾养殖中控制白斑综合症病的理论和策略

White spot syndrome virus (WSSV) is the causative agent of white spot syndrome (WSS) of cultured penaeid shrimps. WSS breaks out and prevails in cultured penaeid shrimps in many countries and regions of the world, especially in southeast Asia. WSSV is the virus most severe damaging to the cultured penaeid shrimps in the world. At the present time, the control of the outbreaks of WSSV will recover and develop the penaeid shrimp cultures in China and even in the world.     

Expression of green fluorescent protein gene with baculovirus vector in insect cells

The green fluorescence of bioluminescent jellyfishAequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10³ dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus-insect cell expression system. Supported by the National Natural Science Foundation of China Hu Jianhong: born in July, 1972, Master graduate student     

绿色荧光蛋白基因转化大岩桐的研究

利用农杆菌介导法 ,用含有绿色荧光蛋白基因的二元双价表达载体pBINm -gfp5 -ER转化大岩桐 ,并得到卡那霉素 (Kanamycin ,Kan)抗性再生植株 .对其进行初步PCR检测 ,结果表明 ,K2 0 0 (含Kan 2 0 0mg/L)培养基上的绿苗中有 3株PCR结果呈阳性 .对PCR阳性的植株进行了点杂交分析 ,均表现出较强的杂交信号 ,这说明外源基因已整合转入到大岩桐基因组中 .在荧光显微镜下观察转基因大岩桐 ,发现部分花、叶细胞均发出一定强度的绿色荧光     

斑节对虾白斑症的发病历程

目的：探讨对虾白斑症的病程及病症特点,方法：对健康斑节对虾进行人工感染,同时用套式ＰＣＲ检测感染后斑节对虾血液中病毒核酸的相对含量,结果：斑节对虾感染白斑症病毒后,依次表现出厌食,不活动、体色变红、甲壳出现白斑,反应迟钝直至侧卧死亡,濒死对虾的典型症状为空胃、红体、甲壳白斑、甲壳下水肿及甲壳易剥离;感染对虾出现拒食后,其血液中白斑症病毒（ＷＳＳＶ）ＤＮＡ的相对含量急剧增加。结论甲过同步不肿和甲壳易     

紫花苜蓿转录因子MsDREB1基因表达产物的亚细胞定位

利用生物信息学方法对紫花苜蓿MsDREBl进行了生物信息学分析．结果表明,该序列舍有AP2典型结构域,在N端存在核定位信号．为进一步验证该基因功能,构建MsDREBl与绿色荧光蛋白（GreenFluorescentProtein,GFP）基因融合的植物表达载体pCAMBIAl302-MsDREBl,再利用基因枪将其转入洋葱表皮细胞,在共聚焦扫描显微镜下观察MsDREBl基因表达产物在洋葱表皮细胞中的亚细胞定位．结果表明,MsDREBl基因表达产物定位于细胞核中,符合DREB家族转录因子特性．     

Silica nanoparticle is a possible safe carrier for gene therapy

In order to develop a safe and effective gene therapy carrier, some toxicological and biodynamical experiments were carried out on silica nanoparticles （SiNPs）. First we prepared SiNPs with appropriate portions of cyclohexane, deionized water and ethyl silicate, and then transfected the modified SiNPs and GFP plasmid DNA complex into the HT1080 cells to test the effectiveness of transfection for gene therapy. At the same time, we injected the SiNPs into a number of mice through tail vein. Then we made the mice crossed to evaluate the acute, long-term and reproductive toxicity. In vivo distribution analysis and pathological examination were made on both adult mice and their offspring. SiNPs were uniform and had an average diameter of 40 nm, and the modified SiNPs carried exogenous DNA molecules into target cells and the transferred GFP fusion gene was effectively expressed in the cells. The SiNPs injected via tail vein were widely distributed in almost all of tissues, and the injected mice had the ability to reproduce normally. The in vivo and in vitro results of this study clearly show that SiNPs can be used as a safe and effective carrier for gene transfection and gene therapy.     

根癌农杆菌介导的GFP在洋葱表皮细胞定位研究

采用根癌农杆菌介导的方法,以受控于CaMV35S启动子的携带有GFP报告基因的双元植物表达载体pCAMBIA1300-35S-GFP转化洋葱表皮细胞.荧光显微镜下观察结果显示,GFP基因在经浸染和共培养后的洋葱表皮细胞中得到了表达,绿色荧光分布在细胞核和细胞质中,为进一步研究新基因的亚细胞定位和瞬时表达奠定了基础.     

带有绿色荧光蛋白基因(gfp)的植物重组表达质粒pBI-GFP的构建

利用 DNA重组技术 ,将经定点突变改造的绿色荧光蛋白基因 ( gfp)克隆到植物表达载体p BI-1 2 1中 ,成功地构建了植物重组表达质粒 p BI-GFP.     

Fluorescence Tracking of Exogenous DNA in Genetic Transformation of the Chinese Oak Silkmoth Antheraea Pernyi via Sperm-Mediated Gene Transfer

Exogenous DNA expressing green fluorescent protein（ GFP） and labeled with fluorescein isothiocyanate（ FITC） was used to transform the Chinese oak silkmoth Antheraea pernyi（ A. pernyi）via sperm-mediated gene transfer（ SMGT）. Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.     

Production of transgenic mice carrying green fluorescence protein gene by a lentiviral vector-mediated approach

A pseudo-lentivirus, which carries green fluorescence protein (GFP) expressing cassette, was injected into the periv itelline space of murine fertilized oocytes before transplanting into the oviducts of the foster mothers. The GFP transgenic pups were then obtained. By PCR amplification, fluorescent microscopy and flow assisted cytometry sorting analysis, we found that the integration rate of the transgene was estimated at above 40% . Real-time PCR analysis indicated that the copy number of the integrated GFP cassette was around 40. Fluorescent in situ hybridization analysis demonstrated that the integration pattern was random but inheritable. The transgenic mice with multi-integration sites and various expression levels possessed a great value in practice as well as research. The approach reported herein provides an efficient way to generate and screen the transgenic mouse strains.     

Effect of HS2 and HS3 elements on erythroid-specific expression in transgenic mice

The expression plasmids CMV/GFP, HS2ALL, HS3ALL and HS23ALL were selected to investigate the effect of HS2 and HS3 element on erythroid-specific expression in transgenic mice. These plasmids were digested with restriction enzymes and purified. And five DNA fragments, CMV/GFP, HS2/GFP, CMV/HS2/GFP, HS23/GFP and HS3/GFP were obtained. After purification, the above DNA fragments were microinjected into the pre-nuclei of the mice fertilized eggs and transgenic mice were generated, with an integration rate of 10.89%. The green fluorescence protein(GFP) expression in many transgenic mouse tissues was determined by FACS analysis. The results showed that the HS2 and 1.7 kb of β-globin gene promoter were sufficient for the erythroid-specific expression of β-globin gene. The GFP expression of different recombinant constructs was also analyzed in blood of all the transgenic mice with FACS. The results indicated that HS2 and HS3 had the same enhancement activity on the regulation of β-globin gene expression. Moreover, these two elements showed a significant synergistic effect on gene expression at the transgenic mouse level, although the GFP expression varied largely among different transgenic mouse litters.     

Summing up the noise in gene networks

Random fluctuations in genetic networks are inevitable as chemical reactions are probabilistic and many genes, RNAs and proteins are present in low numbers per cell. Such 'noise' affects all life processes and has recently been measured using green fluorescent protein (GFP). Two studies show that negative feedback suppresses noise, and three others identify the sources of noise in gene expression. Here I critically analyse these studies and present a simple equation that unifies and extends both the mathematical and biological perspectives.     

绿色荧光蛋白在分子细胞生物学研究中的应用

绿我荧光蛋白具有优良的特性,在蓝光或长紫外光的激发下,不需要任何外源底物或内源辅助因子的参入就能发出绿色荧光,绿色荧光蛋白基因的表达可用来监控活细胞或生物体中基因表达和蛋白质的定位,这是一个革命性的进展,而且,对基因ＤＮＡ序列的改造可能使绿色荧光蛋白的发光特性更加优良,从而共应用范围会更加广泛。     

Transferring gfp gene with ion implantation and transient expression of gfp in liliaceous pollen cells

Liliaceous pollen cells were implanted by 4.0 MeV C2+ ion beam or by 25.0 keV N+ ion beam. Laser confocal scanning microscopy (LCSM) of the implanted intact samples showed that parts of the implanted pollen cells could be stained by propidium iodide (PI). This indicated that energetic ion beam could directly act on cells beneath the pollen coats and made channels for entry of the molecules from outside of the cells. LCSM analysis of green fluorescent protein (GFP) showed that energetic ion beam could mediate transient expression of gfp in treated pollen cells. Compared with 25.0 keV N+ ion beam, implantation of 4.0 MeV C2+ ion beam greatly improved gene transfer efficiency in pollen cells.     

Introduction of exogenous DNA into cotton via the pollen-tube pathway with GFP as a reporter

The green fluorescent protein (GFP) gene from the jellyfishAequorea victoria as a vital reporter for gene expression in plants is considered to have several advantages over other reporter genes. The pBIN35S-mGFP4 plasmid DNA has been introduced into cotton embryos by the pollen-tube pathway method. A transformed seedling has been verified according to its GFP-related fluorescence and Southern blotting analysis. The results provided direct and convincing facts in cytology and molecular biology for the pollen-tube pathway method, an efficient transformation technique used in plants.     

线粒体定位序列介导的绿色荧光蛋白基因在烟草中表达的分析

由于绿色荧光蛋白可在活组织或细胞中直接检出 ,因而近年已在转基因植物的研究中用作报告基因 ,这样可在植物生长的任何阶段进行活体筛选和鉴定。本研究利用线粒体定位序列对改良 gfp基因在转基因烟草中的表达进行了观察 ,结果表明 :将GFP直接在细胞质中大量表达会对植物细胞产生毒性 ,从而影响植物细胞的分化 ,而将其定位在线粒体中 ,则从转化细胞产生植株的频率明显增高。     

菠菜叶绿体基因组定点整合表达载体构建及原核表达

分析菠菜叶绿体基因组全序列,选用了rbcL基因和accD基因的间隔区作为外源基因的定点整合位点,并从菠菜叶绿体基因组中克隆了rbcL基因全长和accD基因的5′端部分,长度分别为1956 bp和1320 bp。以这2个DNA片段作为同源重组片段,以烟草叶绿体基因的启动子Prrn和终止子psbA3′控制外源基因的转录,构建了包含筛选标记基因aadA基因(编码氨基糖苷-3′-腺苷酸转移酶,具有壮观霉素和链霉素抗性)和报告基因GFP(编码绿色荧光蛋白)的菠菜叶绿体基因组定点整合表达载体pRAGA。酶切结果显示构建正确。将该载体转化大肠杆菌,在激光扫描共聚焦显微镜下用488 nm蓝光激发,发现大肠杆菌发出强烈的绿色荧光,而对照菌体没有荧光,表明GFP基因在原核大肠杆菌中已经成功表达。实验结果说明构建的菠菜叶绿体定点整合表达载体pRAGA可以用于菠菜叶绿体转化。     

高效率基因打靶载体的构建及受体成肌细胞的制备

通过建立高效率“双无”（无启动子、 无polyA）打靶载体MSTN-GFP和MSTN neo及新生绵羊和胎儿成肌细胞的培养和鉴定, 优化了培养条件, 改善了细胞状态, 为提高打靶效率及体细胞克隆提供了稳定的核移植供体； 同时无启动子打靶载体的构建也有利于打靶后阳性细胞克隆的分子鉴定.