GFP在绿色荧光裸鼠不同组织中的表达及分析

目的研究外源绿色荧光蛋白（green fluorescent protein,简称GFP）基因在BALB/c绿色荧光裸鼠主要器官组织中的表达及其差异。方法小动物成像系统和RT-PCR方法检测GFP的组织分布以及荧光表达水平情况。结果经活体荧光影像系统观察及PCR方法检测发现GFP可以在裸鼠多个器官组织中表达,其中在胰腺、心脏、全脑、皮肤、睾丸中表达量较高。结论外源绿色荧光蛋白可以在模型动物体内成功表达且稳定遗传,其中在胰腺组织中高表达。     

Abrus abrin derived peptides induce apoptosis by targeting mitochondria in HeLa cells

In our previous study, Abrus abrin derived peptide fraction (ABP) with molecular weight in range of 600-1500 Da was shown to have potent antitumor activity in Dalton's lymphoma (DL) tumor bearing mice. The purpose of this study was to elucidate the mechanism of mitochondrial apoptosis induced by the peptide fraction. ABP was found to have selective antiproliferative activity (10 ng-100 ng/ml) on several tumor cell lines in vitro without having any cytotoxic effect on normal cell lines with a dose of 1000 ng/ml. Analysis of the growth inhibitory mechanism in HeLa cells revealed DNA fragmentation with appearance of the sub G₀/G₁ peak indicative of apoptosis. Further investigation results showed that the apoptotic machinery of HeLa induced by ABP was associated with the release of reactive oxygen species, a drop in mitochondrial transmembrane potential, upregulation of Bax, downregulation of Bcl-2, and activation of caspase-3. The peptide fraction was found to target mitochondria of HeLa cells as observed by confocal microscopy. This peptide fraction offers a source of mitochondria penetrating peptides which might have therapeutic induction of apoptosis in cancer cells.     

Isolation of peroxisome-defective CHO mutant cells using green fluorescent protein

The authors constructed a recombinant green fluorescent protein (GFP) (PTS-GFP), which carries peroxisome targeting signal (PTS1 or PTS2) as an additional sequence, by polymerase chain reaction. The gene encoding for the recombinant GFP was constructed into an eukaryotic expression vector, and stable transformant of CHO cell expressing PTS-GFP was isolated, following the transfection of the plasmid encoding for the GFP. Each expressed PTS-GFP appeared to be localized in peroxisomes, because the GFP was observed in cellular structures, as was catalase. The observation proposed a visual screening procedure for isolating peroxisome-defective mutant. Following an enrichment of mutant cells by use of 9-(1′-pyrene)nonanol/ultraviolet irradiation (P9OH/UV) method, five peroxisome-defective mutants were isolated by pursuing the fluorescent signals from GFP. Two mutants (SK24 and SK32) were isolated from CHO cells expressing PTS1-GFP, and three mutants (PT13, PT32, and PT54) were isolated from cells expressing PTS2-GFP. Four mutants, except for PT13, showed cytosolic distributions of both PTS-GFP and catalase. On the other hand, mutant PT13 showed a cytosolic distribution on PTS2-GFP, but a peroxisomal distribution on catalase. Cell fusion analysis between SK24 mutant and other mutants indicated that PT54 mutant is in the same complementation group (CG) as SK24, but that SK32, PT13, and PT32 mutants are in different complementation group(s) from SK24.     

微流控芯片监测绿色荧光蛋白在枯草芽孢杆菌中的表达

目前主要使用激光共聚焦扫描显微镜观察绿色荧光蛋白的表达,但需要昂贵的仪器并耗费大量时间。本研究开发了一种新型激光诱导的微流芯片检测系统来监测绿色荧光蛋白在枯草芽孢杆菌中的表达。该系统主要由激光装置、光路系统、微流控芯片、光电倍增管和计算机处理系统等5部分组成。对该系统的测试结果显示,随着诱导强度的增强监测信号峰也随之增强,并且与激光共聚焦显微镜观察的结果一致。利用该芯片系统能够快速准确地筛选和鉴定用绿色荧光蛋白作为标记的细胞克隆,可以替代PCR鉴定方法。但该系统仅仅能够监测表达强度,不能够满足蛋白定位等高水平研究,因此,该系统适合应用于环境的微生物监测、药物筛选和其他无需观察蛋白定位等研究。     

Testicular somatic cells in the stony coral Euphyllia ancora express an endogenous green fluorescent protein

In a variety of organisms, adult gonads contain several specialized somatic cells that regulate and support the development of germline cells. In stony corals, the characteristics and functions of gonadal somatic cells remain largely unknown. No molecular markers are currently available that allow for the identification and enrichment of gonadal somatic cells in corals. Here, we showed that the testicular somatic cells of a stony coral, Euphyllia ancora, express an endogenous green fluorescent protein (GFP). Fluorescence microscopy showed that, in contrast to the endogenous expression of the red fluorescent protein of E. ancora ovaries that we have previously reported, the testes displayed a distinct green fluorescence. Molecular identification and spectrum characterization demonstrated that E. ancora testes expressed a GFP (named EaGFP) that is a homolog of the GFP from the jellyfish Aequorea victoria and that possesses an excitation maximum of 506 nm and an emission maximum of 514 nm. Immunohistochemical analyses revealed that the testicular somatic cells, but not the germ cells, expressed EaGFP. EaGFP was enclosed within one or a few granules in the cytoplasm of testicular somatic cells, and the granule number decreased as spermatogenesis proceeded. We also showed that testicular somatic cells could be enriched by using endogenous GFP as an indicator. The present study not only revealed one of the unique cellular characteristics of coral testicular cells but also established a technical basis for more in‐depth investigations of the function of testicular somatic cells in spermatogenesis in future studies.     

Subcellular targeting of green fluorescent protein to plastids in transgenic rice plants provides a high-level expression system

In order to develop a high-level expression system in transgenic rice, we inserted a synthetic gene (sgfp) encoding a modified form of the green fluorescent protein (GFP) into two expression vectors, Act1-sgfp for an untargeted and rbcS-Tp-sgfp for a chloroplast targeted expression. Several fertile transgenic rice plants were produced by the Agrobacterium-mediated method. Confocal microscopic analyses demonstrated that, in cells expressing the Act1-sgfp, GFP fluorescence was localized within the cytoplasm and nucleoplasm whereas, in cells expressing the rbcS-Tp-sgfp fusion gene, the fluorescence was specifically targeted to chloroplasts and non-green plastids. The levels of sgfp expression were about 0.5% of the total soluble protein in mature leaf tissues of the Act1-sgfp transformed lines. In contrast, expression levels were markedly increased in mature leaf tissues of the rbcS-Tp-sgfp transformed lines, yielding about 10% of the total soluble protein. N-terminal sequencing of the localized GFPs revealed that the Tp-GFP fusion protein was correctly processed during import to non-green plastids, as well as to chloroplasts. Thus, our results demonstrate that GFP can be produced at high levels and localized in specific subcellular spaces of transgenic plants, providing a high-level expression system for general use in rice, an agronomically important cereal.     

Metal-enhanced fluorescence of single green fluorescent protein (GFP)

The green fluorescent protein (GFP) has emerged as a powerful reporter molecule for monitoring gene expression, protein localization, and protein-protein interaction. However, the detection of low concentrations of GFPs is limited by the weakness of the fluorescent signal and the low photostability. In this report, we observed the proximity of single GFPs to metallic silver nanoparticles increases its fluorescence intensity approximately 6-fold and decreases the decay time. Single protein molecules on the silvered surfaces emitted 10-fold more photons as compared to glass prior to photobleaching. The photostability of single GFP has increased to some extent. Accordingly, we observed longer duration time and suppressed blinking. The single-molecule lifetime histograms indicate the relatively heterogeneous distributions of protein mutants inside the structure.     

A theoretical study on the optical absorption of green fluorescent protein chromophore in solutions

Ethyl 4-(4-hydroxyphenyl) methylidene- 2-methyl-5-oxoimidazolacetate (HBMIA) is a model chromophore of green fluorescent protein. The electronic structure of HBMIA in aqueous solution phase is studied using a hybrid method of quantum chemistry and statistical mechanics, RISM-SCF-SEDD. The solvatochromic shift is correctly reproduced by the present computations.     

Rescue and preliminary application of a recombinant newcastle disease virus expressing green fluorescent protein gene

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.     

Caspase-3 sensitive signaling in vivo in apoptotic HeLa cells by chemically engineered intramolecular fluorescence resonance energy transfer mutants of green fluorescent protein

Green fluorescent protein (UV5) was re-engineered to remove native cysteine residues, and a new cysteine was introduced near the C-terminus, approximately 20 A from the native fluorophore, for site-specific attachment of chemical fluorophores. The resultant efficient intramolecular FRET quenched GFP emission and gave a new emission band from the conjugated fluorophore. Caspase-3 cleavage of constructs with a caspase-3 sequence near the C-terminus in the sequence between the native fluorophore and the new cysteine, located C-terminal to the caspase site, destroyed the FRET, the emitted color reverting to that of unmodified GFP. This process was demonstrated in vitro with caspase-3 and lysates from cells undergoing apoptosis. Real-time emission changes for the Alexa Fluor 532 conjugate of this GFP, studied quantitatively in vivo for single HeLa cells using the ratios of fluorescence at the red and green maxima by confocal microscopy, showed that caspase-3 action in the cytosol preceded that in the nucleus.     

The developmental fate of green fluorescent mouse embryonic germ cells in chimeric embryos

Primordial germ cells (PGCs),as precursors of mammalian germ lineage,have been gaining more attention as a new resource of pluripotent stem cells,which bring a great possibility to study developmental events of germ cell in vitro and at animal level.EG4 cells derived from 10.5 days post coitum (dpc) PGCs of 129/svJ strain mouse were established and maintained in an undifferentiated state.With an attempt to study the differentiation capability of EG4 cells with a reporter protein:green fluorescence protein,and the possible application of EG4 cells in the research of germ cell development,we have generated several EG4-GFP cell lines expressing enhanced green fluorescence protein (EGFP) and still maintaining typical characteristics of pluripotent stem cells.Then,the differentiation of EG4-GFP cells in vitro as well as their developmental fate in chimeric embryos which were produced by aggregating EG4-GFP cells to 8-cell stage embryos were studied.The results showed that EG4 cells carrying green fluorescence have a potential use in the research of germ cell development and other related studies.     

Kinase activity and subcellular distribution of a chimeric green fluorescent protein-tagged Janus kinase 2

Summary Janus kinase 2 (JAK2) is an essential intracellular signal transducer for numerous cytokines and hormones. To examine how JAK2 structural modifications can affect cellular physiology, we created expression vectors for chimeric proteins containing an enhanced green fluorescent protein (EGFP) fused to rat JAK2 (EGFP/rJAK2), and a kinase-inactive variant, EGFP/rJAK2(K882E). The properties of EGFP/rJAK2 were examined following transient transfection of COS-7 cells. EGFP/rJAK2 was expressed throughout the cell, and was found in subcellular membrane, cytosolic and nuclear fractions. Interestingly, EGFP/rJAK2 phosphorylated other proteins in situ without additional cytokine stimulation. Furthermore, despite a much higher level of tyrosine phosphorylation arising from in situ autophosphorylation, the in vitro radiolabelling autokinase activity of EGFP/rJAK2 was significantly less than that of the endogenous JAK2. These results reveal a technical limitation of the application of the “conventional” in vitro radiolabelling autokinase assay to hyperphosphorylated forms of the enzyme and illustrate the potential weaknesses in individual assays commonly used to determine JAK2’s enzymatic activity and subcellular distribution. We also suggest that the EGFP/rJAK2 model can be very useful in studying JAK2-related cancers, because its ubiquitous distribution and abnormal constitutive hyperphosphorylation may distinguish it from the cytokine-regulated, membrane-proximal form of JAK2 associated with normal physiology.     

High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy

Combination of green fluorescent protein (GFP) and two-photon excitation fluorescence microscopy (TPE) has been used increasingly to study dynamic biochemical events within living cells, sometimes even in vivo. However, the high photon flux required in TPE may lead to higher-order photobleaching within the focal volume, which would introduce misinterpretation about the fine biochemical events. Here we first studied the high-order photobleaching rate of GFP inside live cells by measuring the dependence of the photobleaching rate on the excitation power. The photobleaching rate under one- and two-photon excitation increased with 1-power and 4-power of the incident intensity, respectively, implying the excitation photons might interact with excited fluorophore molecules and increase the probability of photobleaching. These results suggest that in applications where two-photon imaging of GFP is used to study dynamic molecular process, photobleaching may ruin the imaging results and attention should be paid in interpreting the imaging results.     

Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non‐invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH‐sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH‐sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole‐root tissues of A. thaliana is reported. The utility of pH‐sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.     

Impact of embryonic expression of enhanced green fluorescent protein on early mouse development

The impact of embryonic enhanced green fluorescent protein (EGFP)-expression on development is not clear. In this study, we comprehensively assessed EGFP-expression pattern and its effect on early mouse development, following pronuclear-microinjection of the EGFP-transgene, containing chicken-beta-actin promoter and cytomegalovirus enhancer. Preimplantation embryos exhibited differential EGFP-expression patterns. While blastocyst development of non-expressing embryos was 77.3+/-1.8%, that of expressing embryos was only 43.9+/-1.6% (P<0.0001). Developmental competence of embryos negatively correlated (r=-0.99) with the levels of EGFP-expression. Faint-, moderate-, and intense-expressing embryos developed to 83.1+/-5.3%, 50+/-5%, and 9.5+/-3.9% blastocysts, respectively (P<0.002). Interestingly, blastocysts expressing faint-moderate levels of EGFP were developmentally competent through the post-implantation period and delivered viable transgenic 'green' mice, following embryo transfer. These results indicate that hyper-expression of EGFP affects preimplantation development and faint-moderate level of its expression is compatible with normal embryogenesis in the mouse.     

High-level secretion of functional green fluorescent protein from transgenic tobacco cell cultures: characterization and sensing

Green fluorescent protein (GFP) is useful for studying protein trafficking in plant cells. This utility could potentially be extended to develop an efficient secretory reporter system or to enable on-line monitoring of secretory recombinant protein production in plant cell cultures. Toward this end, the aim of the present study was to: (1) demonstrate and characterize high levels of secretion of fluorescent GFP from transgenic plant cell culture; and (2) examine the utility of GFP fluorescence for monitoring secreted recombinant protein production. In this study we expressed in tobacco cell cultures a secretory GFP construct made by splicing an Arabidopsis basic chitinase signal sequence to GFP. Typical extracellular GFP accumulation was 12 mg/L after 10 to 12 days of culture. The secreted GFP is functional and it accounts for up to 55% of the total GFP expressed. Findings from culture treatments with brefeldin A suggest that GFP is secreted by the cultured tobacco cells via the classical endoplasmic reticulum-Golgi pathway. Over the course of flask cultures, medium fluorescence increased with the secreted GFP concentrations that were determined using either Western blot or enzyme-linked immunoassay. Real-time monitoring of secreted GFP in plant cell cultures by on-line fluorescence detection was verified in bioreactor cultures in which the on-line culture fluorescence signals showed a linear dependency on the secreted GFP concentrations.     

Ganoderic acid Mf and S induce mitochondria mediated apoptosis in human cervical carcinoma HeLa cells

In this work, the effects of a pair of positional isomer of ganoderic acids (GAs), namely ganoderic acid Mf (GA-Mf) and ganoderic acid S (GA-S) purified from the fermented mycelia of Ganoderma lucidum, on induction of cell apoptosis and the apoptotic pathway in HeLa cells were investigated. The results demonstrate that both isomers decreased cell population growth on various human carcinoma cell lines by MTT assay, while GA-Mf had better selectivity between normal and cancer cells. The flow cytometry analysis indicated that treatment of HeLa cells with GA-S caused cell cycle arrest in the S phase, while GA-Mf caused cell cycle arrest in the G1 phase. Compared with GA-S, GA-Mf had more potent increase in the number of early and late apoptotic cells. Treatment of HeLa cells with each isomer decreased the mitochondria membrane potential and caused the release of cytochrome c from mitochondria into the cytosol. In addition, stimulation of caspase-3 and caspase-9 activity was observed. The Bax/Bcl-2 ratio was also increased in GA-treated HeLa cells. The results demonstrated that both isomers GA-Mf and GA-S induced apoptosis of human HeLa cells through a mitochondria mediated pathway, but they had the different cell cycle arrest specificity. The findings will be helpful to the development of useful cancer chemopreventive compounds from G. lucidum.     

Visualizing the infection process of Xanthomonas campestris in cabbage using green fluorescent protein

The plant pathogen, Xanthomonas campestris NRRL B-1459 was chromosomally tagged with gfp, and the transformant, which was subjected to Southern hybridization showed the presence of gfp in the chromosome. The virulence-related gene of the transformant was not affected by the insertion of gfp. After inoculation into cabbage plants, the infection process was visually studied in planta. Using a fluorescence microscope, the migration and distribution of gfp-labelled bacteria was visualized in real time. As the gfp-labelled cells were easily visualized from the beginning of infection, we observed a time delay of 2 days between distribution of the Xanthomonas cells in cabbage plant and the appearance of visible necrosis.     

Green fluorescent protein-labeled mapping of neural stem cells migrating towards damaged areas in the adult central nervous system

Neural stem cells, which are clonogenic cells with multilineage differentiation properties from regions of the fetal brain, cortex and hippocampus, are currently considered as powerful candidates for cell replacement therapy in neurodegenerative disorders, such as Parkinson's disease. A key issue is whether stem cells can survive, migrate and differentiate following transplantation into the adult CNS. Here, enhanced green fluorescent protein plasmid electroporation-transfected neural stem cells from the fetal cortex were grafted into the striatum of a rat model of Parkinson's disease. We found most of the grafted cells could survive in the adult parkinsonian rat brain and migrated towards damaged areas, while they moved randomly in the normal brain. Several grafted cells differentiated into neurons.