绿色荧光蛋白在转基因研究中的应用

绿色荧光蛋白(green fluorescent prote in,GFP)是一种能够自身催化形成生色团并在蓝光或紫外光激发下发出绿色荧光的蛋白。有现代生物学北斗星之美誉的它,在生物学的很多领域都有广泛应用。GFP具有荧光稳定、易于检测、表达调控简单、生物安全性好等优点,在转基因研究中的各个方面均应用颇多。就GFP在转基因研究中的应用特点及应用进展做一综述。     

Oviduct-Specific Enhanced Green Fluorescent Protein Expression in Transgenic Chickens

In this study, we confirmed the ability of the 2-kb promoter fragment of the chicken ovalbumin gene to drive tissue-specific expression of a foreign EGFP gene in chickens. Recombinant lentiviruses containing the EGFP gene were injected into the subgerminal cavity of 539 freshly laid embryos (stage X). Subsequently the embryos were incubated to hatch using phases II and III of the surrogate shell ex vivo culture system. Twenty-four chicks (G0) were hatched and screened for EGFP with PCR. Two chicks were identified as transgenic birds (G1), and these founders were mated with wild-type chickens to generate transgenic progeny. In the generated transgenic hens (G2), EGFP was expressed specifically in the tubular gland of the oviduct. These results show the potential of the chicken ovalbumin promoter for the production of biologically active proteins in egg white.     

用绿色荧光蛋白监测转基因植物中选择标记基因的消除

绿色荧光蛋白(GFP)可直接进行活体观察,它的这个优点可被用于监测转基因植物中选择标记基因的消除。为此,构建了植物表达载体pGNG,将绿色荧光蛋白基因(gfp)和卡那霉素抗性基因表达盒(NosP-nptll-NosT)一起克隆在两个同向的lox位点间,在第一个lox位点上游置有CaMV 35S启动子以驱动GFP表达,第二个lox位点下游置有不含启动子的大肠杆菌β-葡萄糖醛酸酶(GUS)基因。首先在含卡那霉素(Kan)的培养基上筛选出转pGNG的烟草,借助绿色荧光可容易地检出表达GFP的转化体。然后用另一转化载体pCambia1300Cre二次转化表达GFP的转基因植物,利用另一选择标记基因潮霉素抗性基因(hpt)进行筛选,在获得的再生植株中,Cre重组酶的表达消除了转化体中两lox位点间的gfp和nptll。实验结果表明可借助GFP荧光的消失,快速选出nptII被消除的二次转化体,同时GUS(作为目的蛋白) 在CaMV 35S启动子驱动下获得表达。最后利用后代的分离将hpt和cre除去。     

绿色荧光蛋白基因在转基因小鼠体内的表达

建立绿色荧光蛋白（GFP）转基因小鼠,继而传代建系。采用显微注射法,将GFP基因注入FVB／NJ小鼠受精卵原核内,获得子代鼠。分娩后3周剪取仔鼠尾,提取基因组DNA,应用PCR、Southern印迹技术进行整合检测。结共用雌性小鼠200只,注射受精卵1586枚,移植卵数386枚,受体鼠32只,怀孕鼠4只,子代鼠18只,有4只为阳性：取2只首建鼠的胚胎,在荧光显微镜下观察GFP表达明显,表明初步获得了转绿色荧光蛋白基因小鼠,     

Alternating-Site Mechanism of Kinesin-1 Characterized by Single-Molecule FRET Using Fluorescent ATP Analogues

Kinesin-1 motor proteins move along microtubules in repetitive steps of 8 nm at the expense of ATP. To determine nucleotide dwell times during these processive runs, we used a Förster resonance energy transfer method at the single-molecule level that detects nucleotide binding to kinesin motor heads. We show that the fluorescent ATP analog used produces processive motility with kinetic parameters altered <2.5-fold compared with normal ATP. Using our confocal fluorescence kinesin motility assay, we obtained fluorescence intensity time traces that we then analyzed using autocorrelation techniques, yielding a time resolution of ∼1 ms for the intensity fluctuations due to fluorescent nucleotide binding and release. To compare these experimental autocorrelation curves with kinetic models, we used Monte-Carlo simulations. We find that the experimental data can only be described satisfactorily on the basis of models assuming an alternating-site mechanism, thus supporting the view that kinesin's two motor domains hydrolyze ATP and step in a sequential way.     

Generation of Two-color Transgenic Zebrafish Using the Green and Red Fluorescent Protein Reporter Genes gfp and rfp

Two tissue-specific promoters were used to express both green fluorescent protein (GFP) and red fluorescent protein (RFP) in transgenic zebrafish embryos. One promoter (CK), derived from a cytokeratin gene, is active specifically in skin epithelia in embryos, and the other promoter (MLC) from a muscle-specific gene encodes a myosin light chain 2 polypeptide. When the 2 promoters drove the 2 reporter genes to express in the same embryos, both genes were faithfully expressed in the respective tissues, skin or muscle. When the 2 fluorescent proteins were expressed in the same skin or muscle cells under the same promoter, GFP fluorescence appeared earlier than RFP fluorescence in both skin and muscle tissues, probably owing to a higher detection sensitivity of GFP. However, RFP appeared to be more stable as its fluorescence steadily increased during development. Finally, F₁ transgenic offspring were obtained expressing GFP in skin cells under the CK promoter and RFP in muscle cells under the MLC promoter. Our study demonstrates the feasibility of monitoring expression of multiple genes in different tissues in the same transgenic organism.     

Development of a Functional Antibody by Using a Green Fluorescent Protein Frame as the Template

Single-chain variable fragment (scFv) antibodies are widely used as diagnostic and therapeutic agents or biosensors for a majority of human disease. However, the limitations of the present scFv antibody in terms of stability, solubility, and affinity are challenging to produce by traditional antibody screening and expression formats. We describe here a feasible strategy for creating the green fluorescent protein (GFP)-based antibody. Complementarity-determining region 3 (CDR3), which retains the antigen binding activity, was introduced into the structural loops of superfolder GFP, and the result showed that CDR3-inserted GFP displayed almost the same fluorescence intensity as wild-type GFP, and the purified proteins of CDR3 insertion showed the similar binding activity to antigen as the corresponding scFv. Among of all of the CDRs, CDR3s are responsible for antigen recognition, and only the CDR3a insertion is the best format for producing GFP-based antibody binding to specific antigen. The wide versatility of this system was further verified by introducing CDR3 from other scFvs into loop 9 of GFP. We developed a feasible method for rapidly and effectively producing a high-affinity GFP-based antibody by inserting CDR3s into GFP loops. Further, the affinity can be enhanced by specific amino acids scanning and site-directed mutagenesis. Notably, this method had better versatility for creating antibodies to various antigens using GFP as the scaffold, suggesting that a GFP-based antibody with high affinity and specificity may be useful for disease diagnosis and therapy.     

Production of Transgenic Korean Native Cattle Expressing Enhanced Green Fluorescent Protein Using a FIV-Based Lentiviral Vector Injected into MII Oocytes

The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins.Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer,but these approaches have been largely ineffective;however,a third approach, lentivirus-mediated transgenesis,has successfully produced transgenic livestock.In this study,we generated transgenic(TG) Korean native cattle using perivitelline space injection of viral vectors,which expressed enhanced green fluorescent protein(EGFP) systemically. Two different types of lentiviral vectors derived from feline immunodeficiency virus(FIV) and human immunodeficiency virus(HIV) carrying EGFP were injected into the perivitelline space of MII oocytes.EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group(47.5%±2.2%v.s.22.9%±2.9%).Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers.Ten heifers were successfully impregnated and delivered 10 healthy calves.All of these calves expressed EGFP as detected by in vivo imaging,PCR and Southern blotting.In addition,we established an EGFP-expressing cell line from TG calves,which was followed by nuclear transfer(NT).Recloned 8-cell embryos also expressed EGFP,and there were no differences in the rates of fusion,cleavage and development between cells derived from TG and non-TG calves,which were subsequently used for NT.These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle.Moreover,our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells.     

Green Fluorescent Protein As a Cell-Labeling Tool and a Reporter of Gene Expression in Transgenic Rainbow Trout

Green fluorescent protein (GFP) has been used as an indicator of transgene expression in living cells and organisms. For testing the utility of GFP in rainbow trout, we microinjected fertilized eggs with four types of supercoiled constructs containing two variants of GFP complementary DNA (S65T and EGFP), driven by two ubiquitous regulatory elements, human cytomegalovirus immediate early enhancer-promoter (CMV) and Xenopus laevis elongation factor 1α enhancer-promoter (EF1). Green fluorescence was first observed at 3 days postfertilization, when the embryo was in the mid-blastula stage. Fluorescence could be detected mosaically in various types of embryonic cells and tissues of swim-up fry. Both the percentage of fluorescent cells and the fluorescence intensity of GFP-expressing cells on blastoderms, measured with a microscopic photometry system, were highest in CMV-EGFP-microinjected embryos. We conclude that GFP is capable of producing detectable fluorescence in rainbow trout, and can be a powerful tool as a cell marker and reporter gene for cold-water fish, and that analysis of GFP expression in living cells is useful for characterizing the activity of cis-elements in vivo. Received December 21, 1998; accepted March 31, 1999.     

SYBR Green实时荧光PCR高通量检测转基因大豆油

采用SYBR Green实时荧光PCR技术,建立了食用大豆油转基因成分的检测方法.根据转基因大豆中内源参照基因lectin和外源基因35S启动子、NoS终止子和ep4 epsps基因,设计特异性引物,在Roche荧光PCR仪上进行实时荧光PCR扩增.荧光曲线表明,SYBR Green实时荧光PCR可特异性地检测大豆油中的转基因成分,方法准确、快速,并运用熔解曲线进行产物分析,验证了试验结果的特异性和准确性,检测方法灵敏度高.     

Early Events of Virus-Cell Interaction in an Adenovirus System

The interaction of (32)P-labeled adenovirus type 2 and HeLa or KB cells has been examined during early infection. The kinetics of virus uncoating to deoxyribonuclease-sensitive products, the partial characterization of three such products by gradient centrifugation, and the distribution of these products in the extranuclear and nuclear portions of infected cells are reported. The results are compatible with the following model. Extracellular virus attaches to a receptor on the plasma membrane. The membrane-bound virus has a half-life of less than 15 min and is transformed to a partly uncoated product which is free inside the cell and about half of which rapidly enters the cell nucleus. This is rapidly transformed, in both cytoplasm and nucleus, to a membrane-bound virion "core." The proteins of the bound "core" are then removed from the intact virus deoxyribonucleic acid (DNA). In the nucleus, viral DNA is the main product and there the overall sequence is completed in about 2 hr.     

In Vivo Tracking of Campylobacter jejuni by Using a Novel Recombinant Expressing Green Fluorescent Protein

Campylobacter jejuni is a leading cause of food-borne disease in developed countries. The goal of this study was to develop a plasmid-based reporter system with green fluorescent protein (GFP) to facilitate the study of C. jejuni in a variety of niches. C. jejuni transformants harboring the pMEK91 GFP gene (gfp)-containing vector were readily detectable by both fluorescence microscopy and flow cytometry. Given the ease of detecting these organisms, additional experiments were performed in which BALB/c mice were injected intraperitoneally with C. jejuni harboring the gfp-containing vector. Four hours after injection of the mice, flow cytometry analyses determined that C. jejuni synthesizing GFP were predominantly associated with granulocytes. More specifically, the proportion of CD11b⁺ Gr-1⁺ lavage neutrophils with green fluorescence ranged from 99.7 to 100%, while the proportion of CD11b⁺ Gr-1⁻ lavage macrophages ranged from 77.0 to 80.0%. In contrast, few CD11b⁻ CD45R⁺ B lymphocytes from the lavage of the C. jejuni-injected mice were associated with green-fluorescent C. jejuni (proportions ranged from 0.75 to 0.77%). Cell-free C. jejuni was recovered from tissue homogenates after intraperitoneal injection. Macrorestriction profiling with pulsed-field gel electrophoresis identified a genotypic variant of the C. jejuni F38011 wild-type isolate. In vivo this variant displayed a phenotype identical to that of the wild-type isolate. In summary, we demonstrate that C. jejuni associates with marker-defined cellular subsets in vivo with a novel gfp reporter system and that C. jejuni genotypic variants can be isolated from both in vitro and in vivo systems.     

Analysis of Nuclear Localization Signals Using a Green Fluorescent Protein-Fusion Protein Library

We describe here an efficient method for identifying intracellular localization signals in proteins with stereospecific intracellular localizations in culture cells. The method involves rapid fluorescence screening of cells transfected with a cDNA library in which cDNAs are fused to the gene encoding the Aequorea victoria green fluorescent protein (GFP). We analyzed nuclear localization and nuclear localization signals (NLSs) in a model application of this method. As a result, we identified classical NLSs in 75% of nuclear localized proteins. We identified some novel NLS candidates among the classical NLS-negative sequences whose nuclear localization was also identified in another cell line and with other molecular tag sequences. This method will be useful for identifying intracellular localization signals and for more detailed analysis of intracellular architecture.     

Assessment of Transient Gene Expression in Plant Tissues Using the Green Fluorescent Protein as a Reference

Four different promoters (35S and enhanced 35S of the cauliflower mosaic virus, polyubiquitin of maize and actin1 of rice) were compared in a transient assay using maize leaves and particle bombardment. A gene encoding the jellyfish green fluorescent protein (GFP) driven by the 35S promoter was used as an internal standard to monitor the effectiveness of each bombardment. Normalisation of the transient expression assay using the GFP reference significantly reduced the variability between separate bombardments and allowed for a rapid and accurate evaluation of different promoters in microprojectile-bombarded leaves.     

Early Events in the Interaction of Melanotropin with Cloudman S91 Melanoma Cells

Melanotropin stimulates melanin synthesis in Cloudman S91 melanomacells, the accumulation of melanin being preceded by a numberof events which are understood to various degrees. In this reviewI will discuss the control of melanogenesis by melanotropin;emphasis will be on the early events of the process, althoughother aspects will be discussed. The probes (fluorescent andradioactive labels) used to examine the binding of the hormoneto the cells will be described, as will be the results of initialbinding studies. One of the events immediately following thebinding of the hormone is the activation of adenylate cyclase:the evidence supporting this, plus the role of cyclic AMP instimulating melanin synthesis, will be discussed. Also examinedis the role which internalization of the melanotropin-receptor-adenylatecyclase complex may play in mediating hormone action.