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。     

FACS-optimized mutants of the green fluorescent protein (GFP)

We have constructed a library in Escherichia coli of mutant gfp genes (encoding green fluorescent protein, GFP) expressed from a tightly regulated inducible promoter. We introduced random amino acid (aa) substitutions in the twenty aa flanking the chromophore Ser-Tyr-Gly sequence at aa 65–67. We then used fluorescence-activated cell sorting (FACS) to select variants of GFP that fluoresce between 20-and 35-fold more intensely than wild type (wt), when excited at 488 nm. Sequence analysis reveals three classes of aa substitutions in GFP. All three classes of mutant proteins have highly shifted excitation maxima. In addition, when produced in E. coli, the folding of the mutant proteins is more efficient than folding of wt GFP. These two properties contribute to a greatly increased (100-fold) fluorescence intensity, making the mutants useful for a number of applications.     

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.     

Novel green fluorescent protein (GFP) baculovirus expression vectors

Baculovirus expression vectors were constructed, which contained gfp as a reporter gene. Substitutions in the amino acid sequences were carried out to produce two mutant forms of GFP. One of these mutants produces blue color when excited by ultraviolet (UV) light. The other mutant produces a yellow color that can be visualized in regular daylight. The gene of interest cloned in-frame with the gfp open reading frame allows visualization of the produced GFP-fusion protein using UV light. The presence of an in-frame 6 x His tag between the gfp cDNA and the multiple cloning site allows purification of the fusion protein on a Ni-NTA (nickel-nitrilo-triacetic acid) agarose matrix.     

Welfare assessment in transgenic pigs expressing green fluorescent protein (GFP)

Since large animal transgenesis has been successfully attempted for the first time about 25 years ago, the technology has been applied in various lines of transgenic pigs. Nevertheless one of the concerns with the technology--animal welfare--has not been approached through systematic assessment and statements regarding the welfare of transgenic pigs have been based on anecdotal observations during early stages of transgenic programs. The main aim of the present study was therefore to perform an extensive welfare assessment comparing heterozygous transgenic animals expressing GFP with wildtype animals along various stages of post natal development. The protocol used covered reproductory performance and behaviour in GFP and wildtype sows and general health and development, social behaviour, exploratory behaviour and emotionality in GFP and wildtype littermates from birth until an age of roughly 4 months. The absence of significant differences between GFP and wildtype animals in the parameters observed suggests that the transgenic animals in question are unlikely to suffer from deleterious effects of transgene expression on their welfare and thus support existing anecdotal observations of pigs expressing GFP as healthy. Although the results are not surprising in the light of previous experience, they give a more solid fundament to the evaluation of GFP expression as being relatively non-invasive in pigs. The present study may furthermore serve as starting point for researchers aiming at a systematic characterization of welfare relevant effects in the line of transgenic pigs they are working with.     

Soluble,highly fluorescent variants of green fluorescent protein (GFP) for use in higher plants

Green fluorescent protein (GFP) from Aequorea victoria has rapidly become a standard reporter in many biological systems. However, the use of GFP in higher plants has been limited by aberrant splicing of the corresponding mRNA and by protein insolubility. It has been shown that GFP can be expressed in Arabidopsis thaliana after altering the codon usage in the region that is incorrectly spliced, but the fluorescence signal is weak, possibly due to aggregation of the encoded protein. Through site-directed mutagenesis, we have generated a more soluble version of the codon-modified GFP called soluble-modified GFP (smGFP). The excitation and emission spectra for this protein are nearly identical to wild-type GFP. When introduced into A. thaliana, greater fluorescence was observed compared to the codon-modified GFP, implying that smGFP is brighter because more of it is present in a soluble and functional form. Using the smGFP template, two spectral variants were created, a soluble-modified red-shifted GFP (smRS-GFP) and a soluble-modified blue-fluorescent protein (smBFP). The increased fluorescence output of smGFP will further the use of this reporter in higher plants. In addition, the distinct spectral characters of smRS-GFP and smBFP should allow for dual monitoring of gene expression, protein localization, and detection of in vivo protein-protein interactions.     

Stability of green fluorescent protein (GFP) in chlorine solutions of varying pH

Green fluorescent protein (GFP) is an excellent biosensor as a result of its ability to be easily monitored in a wide variety of applications. Enzymes and proteins have been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. The purpose of this work was to study GFP stability in chlorinated water for injection (WFI) and chlorinated buffered solutions at various pH ranges, with and without agitation, to evaluate the exposure time required for chlorine to decrease 90% of its fluorescence intensity (decimal reduction time, D-value, min, 25 degrees C). Fluorescence intensity (Ex/Emmax = 394/509 nm) was measured immediately after the addition of GFP (8.0-9.0 microg/mL) into buffered or unbuffered chlorine solutions with or without constant stirring. With solutions constantly stirred, GFP fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH = 7.15 +/- 0.08), GFP retained its structure between 52 and 94 ppm, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. The recovery of GFP fluorescence intensity due to renaturation was observed between 30 and 100 ppm chlorine in WFI (final pH = 11.01 +/- 0.23) without stirring. Stirring enhanced the contact between GFP and chlorine throughout the assay and provided a more accurate D-value evaluation. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness.     

Construction of green fluorescent protein (GFP)-marked strains of Bradyrhizobium for ecological studies

AIM: To introduce the gfp gene encoding green fluorescent protein (GFP) into bradyrhizobia for their identification in nodules, soil and carrier-based inoculants. METHODS AND RESULTS: Bradyrhizobium sp. strains M29 and GN7, which nodulate mungbean (Vigna radiata), were conjugated with Escherichia coli S17-1 carrying plasmid EDS 15 (a suicide plasmid carrying a promoterless gfp gene fused with Tn5). The GFP-marked strain expressed the gfp gene from a Bradyrhizobium promoter and gave green fluorescence when observed under an epifluorescent microscope or u.v. transilluminater. All the GFP-marked strains were able to nodulate mungbean and fix nitrogen. The GFP-marked bradyrhizobia were recovered at a frequency of 90-100% and 16-63% from nodules formed under sterilized and unsterilized conditions, respectively. The GFP-marked bradyrhizobia were identified from soil and from charcoal-based inoculants on the basis of green fluorescence. CONCLUSIONS: The GFP-marked Bradyrhizobium was successfully identified on the basis of green fluorescence to study its competition and survival in the soil and in charcoal-based inoculants. SIGNIFICANCE AND IMPACT OF THE STUDY: Introduction of the gfp gene into Bradyrhizobium provides a simple, specific and cost-effective method of strain identification for ecological studies.     

On-line measurement of green fluorescent protein (GFP) fluorescence for the monitoring of recombinant adenovirus production

An on-line fluorescence sensor prototype was constructed to monitor the production of the green fluorescent protein (GFP) by 293S cells infected with a recombinant adenovirus vector (rAdV) containing the GFP gene. Fluorescence was correlated to GFP concentration and therefore to viral protein expression, but not to rAdV production. The sensor signal can also be used to compute the GFP production rate, which predicts well the occurrence of maximum viral titer.     

The green fluorescent protein (GFP) as a vital screenable marker in rice transformation

 An engineered green fluorescent protein (GFP) from the jellyfish Aequora victoria was used to develop a facile and rapid rice transformation system using particle bombardment of immature embryos. The mgfp4 gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright-green fluorescence easily detectable and screenable in rice tissue 12–22 days after bombardment. Visual screening of transformed rice tissue, associated with a low level of antibiotic selection, drastically reduced the quantity of tissue to be handled and the time required for the recovery of transformed plants. GFP expression was observed in primary transformed rice plants (T₀) and their progeny (T₁). We describe various techniques to observe GFP in vitro and in vivo. The advantages of this new screenable marker in rice genetic engineering programmes are discussed. Received: 6 October 1997 / Accepted: 9 October 1997     

Optimization of the green fluorescent protein (GFP) expression from a lactose-inducible promoter in Lactobacillus casei

An expression vector for Lactobacillus casei has been constructed containing the inducible lac promoter and the gene encoding ultraviolet visible green fluorescent protein (GFP(UV)) as reporter. Different conditions to grow L. casei were assayed and fluorescence as well as total protein synthesized were quantified. The maintenance of neutral pH had the greatest incidence on GFP(UV) expression, followed by aeration and a temperature of 30 degrees C. Environmental factors favoring GFP(UV) accumulation did not exactly correlate with those enhancing fluorescence. Therefore, oxygenation, by stirring the culture, had the greatest influence on the proportion of fluorescent protein, which is in accordance with the structural requirements of this protein. The highest yield obtained was 1.3 microg of GFP per mg of total protein, from which 55% was fluorescent.     

Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras

MAP 4 is a ubiquitous microtubule-associated protein thought to play a role in the polymerization and stability of microtubules in interphase and mitotic cells. We have analyzed the behavior of protein domains of MAP 4 in vivo using chimeras constructed from these polypeptides and the green fluorescent protein (GFP). GFP-MAP 4 localizes to microtubules; this is confirmed by colocalization of GFP-MAP 4 with microtubules that have incorporated microinjected rhodamine-tubulin, and by loss of localized fluorescence after treatment of cells with anti-microtubule agents. Different subdomains of MAP 4 have distinct effects on microtubule organization and dynamics. The entire basic domain of MAP 4 reorganizes microtubules into bundles and stabilizes these arrays against depolymerization with nocodazole. Within the basic domain, the PGGG repeats, which are conserved with MAP 2 and tau, have a weak affinity for microtubules and are dispensable for microtubule binding, whereas the MAP 4-unique PSP region can function independently in binding. The projection domain shows no microtubule localization, but does modulate the association of various binding subdomains with microtubules. The acidic carboxy terminus of MAP 4 strongly affects the microtubule binding characteristics of the other domains, despite constituting less than 6% of the protein. These data show that MAP 4 association with microtubules is modulated by sequences both within and outside the basic domain. Further, our work demonstrates that GFP chimeras will allow an in vivo analysis of the effects of MAPs and their variants on microtubule dynamics in real time.     

Escherichia coli expressing a green fluorescent protein (GFP) in Anopheles stephensi: a preliminary model for paratransgenesis

Issues, like emerging insecticide resistance in Anopheles mosquitoes, have led to a breakdown in many vector control programs. In this study, a recombinant Escherichia coli with plasmid expressing a green fluorescent protein (E.coli-GFP) was used as a paratransgenesis model to determine: the possibility of E. coli-GFP trans-stadial transmission. The effect of the water microflora, of bacteria-impregnated sugar solutions, and of blood-feeding on E. coli-GFP colonization and localization within An. stephensi tissues, were studied. The results demonstrated the persistence of E. coli-GFP during molting and metamorphosis events and its trans-stadial transmission. Also the efficacy of bacteria-impregnated sugar solutions for transferring the bacteria to the adult mosquito’s midgut was shown. A blood meal dramatically increased the number of bacteria within 24–48 h post feeding. In addition to fluorescence microscope evaluation, GFP gene PCR amplification showed the presence of the bacteria in the midgut of larvae, pupae, and adults up to 13 days after eclosion. Massive colonization of bacteria was observed in the larvae and in the adult mosquito’s malpighian tubules which may play a role in retaining bacteria in adult mosquitos. The results of this study showed that E. coli could be used as a laboratory model in paratransgenesis studies for the evaluation of various effector molecules as anti-parasite agents for malaria and filariasis.     

Genetic transformation of Arabidopsis lyrata: specific expression of the green fluorescent protein (GFP) in pistil tissues

Arabidopsis thaliana has become widely used as a model system for plant biology. Recent phylogenetic studies led to a severe revision of the systematic relationships across species of the Brassicaceae family. This provided an opportunity to examine close relatives of A. thaliana and to study the function and molecular evolution of genes that play roles in ecology and speciation. In this context, developing tools to genetically transform “non-model plants” appears as a major issue to ascertain gene function. Here, we report a method to transform A. lyrata, one of the closest relatives of A. thaliana.     

Screening by coral green fluorescent protein (GFP)-like chromoproteins supports a role in photoprotection of zooxanthellae

Green fluorescent protein (GFP)-like pigments are responsible for the vivid colouration of many reef-building corals and have been proposed to act as photoprotectants. Their role remains controversial because the functional mechanism has not been elucidated. We provide direct evidence to support a photoprotective role of the non-fluorescent chromoproteins (CPs) that form a biochemically and photophysically distinct group of GFP-like proteins. Based on observations of Acropora nobilis from the Great Barrier Reef, we explored the photoprotective role of CPs by analysing five coral species under controlled conditions. In vitro and in hospite analyses of chlorophyll excitation demonstrate that screening by CPs leads to a reduction in chlorophyll excitation corresponding to the spectral properties of the specific CPs present in the coral tissues. Between 562 and 586 nm, the CPs maximal absorption range, there was an up to 50 % reduction of chlorophyll excitation. The screening was consistent for established and regenerating tissue and amongst symbiont clades A, C and D. Moreover, among two differently pigmented morphs of Acropora valida grown under identical light conditions and hosting subclade type C3 symbionts, high CP expression correlated with reduced photodamage under acute light stress.