Use of Green Fluorescent Protein in mouse embryos

Green Fluorescent Protein (GFP) has rapidly been established as a versatile and powerful cell marker in many organisms. Initial problems in using it in mammalian cells were solved by introducing mutations to increase its solubility at higher temperatures, such that GFP has now been used as a reporter in both gene expression and cell lineage studies, and to localize proteins within mammalian cells. GFP has two unique advantages: (i) the protein becomes fluorescent in an autocatalytic reaction, so that it can be introduced into any cell type simply as a cDNA or mRNA, or as protein; (ii) it is "bright" enough to be visualized in living cells under conditions that do not cause photodamage to the cells. In this article we outline the ways in which we have used GFP mRNA and cDNA in our studies of mouse cell lineages, and to characterize the behavior of proteins within the embryos.     

mEosFP-based green-to-red photoconvertible subcellular probes for plants

Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists' toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs.     

Universal GFP reporter for the study of vascular development

We report the generation and characterization of transgenic mouse and zebrafish expressing green fluorescent protein (GFP) specifically in vascular endothelial cells in a relatively uniform fashion. These reporter lines exhibit fluorescent vessels in developing embryos and throughout adulthood, allowing visualization of the general vascular patterns with single cell resolution. Furthermore, we show the ability to purify endothelial cells from whole embryos and adult organs by a single step fluorescence activated cell sorting. We expect that these transgenic reporters will be useful tools for imaging vascular morphogenesis, global gene expression profile analysis of endothelial cells, and high throughput screening for vascular mutations.     

Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants

Green fluorescent protein (GFP) has emerged as a powerful new tool in a variety of organisms. An engineered sGFP(S65T) sequence containing optimized codons of highly expressed eukaryotic proteins has provided up to 100-fold brighter fluorescence signals than the original jellyfish GFP sequence in plant and mammalian cells. It would be useful to establish a non-invasive, quantitative detection system which is optimized for S65T-type GFP, one of the brightest chromophore mutants among the various GFPs. We demonstrate here that highly fluorescent transgenic Arabidopsis can be generated, and the fluorescence intensity of whole plants can be measured under non-disruptive, sterile conditions using a quantitative fluorescent imaging system with blue laser excitation. Homozygous plants can be distinguished from heterozygous plants and fully fertile progenies can be obtained from the analyzed plants. In the case of cultured tobacco cells, GFP-positive cells can be quantitatively distinguished from non-transformed cells under non-selective conditions. This system will be useful in applications such as mutant screening, analysis of whole-body phenomena, including gene silencing and quantitative assessments of colonies from microorganisms to cultured eukaryotic cells. To facilitate the elucidation of protein targeting and organelle biogenesis in planta, we also generated transgenic Arabidopsis that stably express the plastid- or mitochondria-targeted sGFP(S65T). Etioplasts in dark-grown cotyledons and mitochondria in dry seed embryos could be visualized for the first time in transgenic Arabidopsis plants under normal growing conditions.     

Three-color imaging using fluorescent proteins in living zebrafish embryos

The zebrafish embryo is especially valuable for cell biological studies because of its optical clarity. In this system, use of an in vivo fluorescent reporter has been limited to green fluorescent protein (GFP). We have examined other fluorescent proteins alone or in conjunction with GFP to investigate their efficacy as markers for multi-labeling purposes in live zebrafish. By injecting plasmid DNA containing fluorescent protein expression cassettes, we generated single-, double-, or triple-labeled embryos using GFP, blue fluorescent protein (BFP, a color-shifted GFP), and red fluorescent protein (DsRed, a wild-type protein structurally related to GFP). Fluorescent imaging demonstrates that GFP and DsRed are highly stable proteins, exhibiting no detectable photoinstability, and a high signal-to-noise ratio. BFP demonstrated detectable photoinstability and a lower signal-to-noise ratio than either GFP or DsRed. Using appropriate filter sets, these fluorescent proteins can be independently detected even when simultaneously expressed in the same cells. Multiple labels in individual zebrafish cells open the door to a number of biological avenues of investigation, including multiple, independent tags of transgenic fish lines, lineage studies of wild-type proteins expressed using polycistronic messages, and the detection of protein-protein interactions at the subcellular level using fluorescent protein fusions.     

Single-Molecule Microscopy Reveals Membrane Microdomain Organization of Cells in a Living Vertebrate

It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.     

绿色荧光蛋白——照亮生命科学的一盏明灯

绿色荧光蛋白的发现及应用具有划时代的重要意义,它不仅为当代生物学研究提供了极为实用的基本研究手段,并且在此基础上改造发展和发现了一系列荧光蛋白,拓展了应用范围。这使得对微观生物学的研究也可以进入一个时空结合,研究鲜活动态过程的新时代。本文主要回顾总结了绿色荧光蛋白的发现、优化改造及其应用。     

GFP expression in Hydra: lessons from the particle gun

The cnidarian Hydra is an important model organism to study pattern formation and tem cell differentiation. In the past, however, it has been difficult to study gene function in Hydra because the animals have hot been accessible to gene transfection studies, we have now developed a method to transiently express GFP-tagged proteins in Hydra using a green fluorescent protein (GFP) expression plasmid under the control of the Hydra actin promoter and a particle gun to introduce it into Hydra cell nuclei. We achieve strong transient GFP expression in a small but reproducible number of epithelial and interstitial cells. Implications for the use of this method to carry out single cell assays with GFP-tagged Hydra proteins are discussed.     

From fixed to FRAP: measuring protein mobility and activity in living cells

Experiments with fluorescence recovery after photobleaching (FRAP) started 30 years ago to visualize the lateral mobility and dynamics of fluorescent proteins in living cells. Its popularity increased when non-invasive fluorescent tagging became possible with the green fluorescent protein (GFP). Many researchers use GFP to study the localization of fusion proteins in fixed or living cells, but the same fluorescent proteins can also be used to study protein mobility in living cells. Here we review the potential of FRAP to study protein dynamics and activity within a single living cell. These measurements can be made with most standard confocal laser-scanning microscopes equipped with photobleaching protocols.     

转基因DT40细胞导入早期鸡胚后的分布及绿色荧光蛋白表达的研究

目的为了研究经过基因修饰的体细胞导入到禽类胚胎以后,供体细胞及外源基因是否能在受体胚胎中成活并且外源基因是否可以长期表达。方法筛选得到稳定整合绿色荧光蛋白基因的鸡DT40细胞作为外源蛋白的运载工具,通过血管微注射的方法将其导入到于38.5℃温度条件下孵化65～70 h的鸡胚中,并将操作后的鸡胚在原孵化条件下继续孵化。在孵化的不同时期取移植了DT40细胞的嵌合体胚胎在荧光显微镜下观察荧光细胞的存活与分布情况。并通过PCR以及免疫组织化学方法检测供体细胞在受体中的位置以及绿色荧光蛋白的表达情况。结果荧光标记的DT40细胞可以存活于受体不同的组织器官中,包括：脑、心脏、肝脏等。导入胚胎的整合外源基因的DT40细胞可以存活到胚胎出雏之前,并且外源基因能够正常表达。结论可以通过此方法将外源基因导入到受体中,并使目的蛋白在受体胚胎中持续表达,为胚胎期导入外源蛋白诱导免疫耐受的研究以及将转基因细胞移植到动物体内生产目的蛋白的研究提供科学依据和技术平台。     

A new configuration of the Zeiss LSM 510 for simultaneous optical separation of green and red fluorescent protein pairs.

The power and simplicity of genetically encoded fluorophores (fluorescent proteins, FPs) have drawn many molecular biologists to light microscopy. First generation FPs suffered from overlapping excitation and emission spectra, which limited their use together in pairs (Patterson et al., J Cell Sci 2001;114 (Part 5):837-838). Image acquisition and processing techniques, collectively known as linear unmixing, have been developed to separate overlapping fluorescence signals encountered in the imaging of FP pairs and also in FRET. These specialized techniques are not without their potential drawbacks, including limitations on sensitivity and time-resolution for live cell imaging, and the risk of artifact in the hands of nonspecialists. With the advent of a new generation of red-shifted FPs (Shaner et al., Nat Biotechnol 2004;22:1567-1572; Verkhusha and Lukyanov, Nat Biotechnol 2004;22:289-296) careful selection of excitation sources and emission filters obviate the need for linear unmixing when simple two channel imaging of FPs is required. Here we introduce a new configuration of the Zeiss LSM 510 laser scanning confocal microscope, optimized for live cell imaging of green fluorescent protein (GFP) together with spectral variants such as mRFP1 and mCherry using standard photo-multipliers. A 2 mW, 594 nm HeNe laser was chosen as the excitation source for the red FP. This wavelength efficiently excites the aforementioned red variants without limiting the detection range of GFP emission during simultaneous two-channel imaging. Compared to excitation of GFP and mCherry at 488 and 543 nm, excitation at 488 and 594 nm approximately doubles the sensitivity of GFP detection and eliminates bleed-through of GFP into the mCherry channel. However, sensitivity of mCherry detection is decreased by 30%, suggesting the need for red FPs having longer emission peaks. Practical advantages to the simultaneous optical separation of FPs with nonoverlapping emission spectra include simplicity, robustness, reduced risk of artifact, and increased sensitivity during live cell imaging.     

Photomodulatable fluorescent proteins for imaging cell dynamics and cell fate

An organism arises from the coordinate generation of different cell types and the stereotypical organization of these cells into tissues and organs. Even so, the dynamic behaviors, as well as the ultimate fates, of cells driving the morphogenesis of an organism, or even an individual organ, remain largely unknown. Continued innovations in optical imaging modalities, along with the discovery and evolution of improved genetically-encoded fluorescent protein reporters in combination with model organism, stem cell and tissue engineering paradigms are providing the means to investigate these unresolved questions. The emergence of fluorescent proteins whose spectral properties can be photomodulated is one of the most significant new developments in the field of cell biology where they are primarily used for studying protein dynamics in cells. Likewise, the use of photomodulatable fluorescent proteins holds great promise for use in developmental biology. Photomodulatable fluorescent proteins also represent attractive and emergent tools for studying cell dynamics in complex populations by facilitating the labeling and tracking of individual or defined groups of cells. Here, we review the currently available photomodulatable fluorescent proteins and their application in model organisms. We also discuss prospects for their use in mice, and by extension in embryonic stem cell and tissue engineering paradigms.Key words: fluorescent protein, photomodulation, photoactivation, photoconversion, mouse, live imaging, embryonic development, organogenesis, GFP, PA-GFP, PS-CFP, Kaede, KikGR     

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.     

Plant-adapted green fluorescent protein is a versatile vital reporter for gene expression, protein localization and mitosis in the filamentous fungus, Aspergillus nidulans

Green fluorescent protein (GFP) is a useful reporter to follow the in vivo behaviour of proteins, but the wild-type gfp gene does not function in many organisms, including many plants and filamentous fungi. We show that codon-modified forms of gfp , produced for use in plants, function effectively in Aspergillus nidulans both as gene expression reporters and as vital reporters for protein location. To demonstrate the use of these modified gfp s as reporter genes we have used fluorescence to follow ethanol-induced GFP expression from the alcA promoter. Translational fusions with the modified gfp were used to follow protein location in living cells; plant ER-retention signals targeted GFP to the endoplasmic reticulum, whereas fusion to the GAL4 DNA-binding domain targeted it to the nucleus. Nuclear-targeted GFP allowed real-time observation of nuclear movement and division. These modified gfp genes should provide useful markers to follow gene expression, organelle behaviour and protein trafficking in real time.     

Localized electroporation: a method for targeting expression of genes in avian embryos

Avian embryos are a popular model for cell and developmental biologists. However, analysis of gene function in living embryos has been hampered by difficulties in targeting the expression of exogenous genes. We have developed a method for localized electroporation that overcomes some of the limitations of current techniques. We use a double-barreled suction electrode, backfilled with a solution containing a plasmid-encoding green fluorescent protein (GFP) and a neurophysiological stimulator to electroporate small populations of cells in living embryos. As many as 600 cells express GFP 24-48 h after electroporation. The number of cells that express GFP depends on the number of trains, the pulse frequency and the voltage. Surface epithelial cells and cells deep to the point of electroporation express GFP. No deformities result from electroporations, and neurons, neural crest, head mesenchyme, lens and otic placode cells have been transfected. This method overcomes some of the disadvantages of viral techniques, lipofection and in vivo electroporation. The method will be useful to biologists interested in tracing cell lineage or making genetic mosaic avian embryos.     

Studying cytoskeletal dynamics in living cells using green fluorescent protein

Microfilaments, intermediate filaments, and microtubules are three major cytoskeletal systems providing cells with stability to maintain proper shape. Although the word “cytoskeleton” implicates rigidity, it is quite dynamic exhibiting constant changes within cells. In addition to providing cell stability, it participates in a variety of essential and dynamic cellular processes including cell migration, cell division, intracellular transport, vesicular trafficking, and organelle morphogenesis. During the past eight years since the green fluorescent protein (GFP) was first used as a marker for the exogenous gene expression, it has been an especially booming era for live cell observations of intracellular movement of many proteins. Because of the dynamic behavior of the cytoskeleton in the cell, GFP has naturally been a vital part of the studies of the cytoskeleton and its associated proteins. In this article, we will describe the advantage of using GFP and how it has been used to study cytoskeletal proteins.     

Generation and characterization of a GFP transgenic rat line for embryological research

Model organisms expressing fluorescent proteins are important tools for research. The present study was performed to generate and characterize a new line of green fluorescent protein (GFP) transgenic rats for use as a model in experimental embryological research. We injected a GFP expression vector into 135 zygotes of the Sprague-Dawley (SD) rat strain. Embryo transfer of 103 surviving embryos resulted in the production of 35 offspring (33.9%) and two of them were transgenic (5.7%). Two transgenic rat lines that ubiquitously express GFP under the control of the cytomegalovirus-enhancer/beta-actin (CAGGS) promoter were generated by breeding. We studied the main embryological parameters of one these GFP transgenic lines. Homozygous GFP-transgenic females have the same ovulation and superovulation rates as wild type (WT) females. Transgenic embryos reached blastocyst stage in vitro and developed in vivo after embryo transfer without decrease in their developmental ability compared to the control group. The genotype of the parents determined the onset of GFP expression in preimplantation embryos. When the GFP gene is derived from the transgenic female parent, fluorescence was detected in oocytes and in embryos of all further stages of development. When the GFP gene is inherited by the transgenic male parent, GFP was only expressed from the blastocyst stage on. GFP-transgenic rats represent a valuable tool to mark embryos for many embryological studies such as transgenesis, gene expression patterns during early development, embryo aggregation for analysis of the distribution of cells in chimeric embryos and nuclear transfer to confirm the origin of the cloned offspring.     

Combinatorial marking of cells and organelles with reconstituted fluorescent proteins

Expression of GFP and other fluorescent proteins depends on cis-regulatory elements. Because these elements rarely direct expression to specific cell types, GFP production cannot always be sufficiently limited. Here we show that reconstitution of GFP, YFP, and CFP previously split into two polypeptides yields fluorescent products when coexpressed in C. elegans. Because this reconstitution involves two components, it can confirm cellular coexpression and identify cells expressing a previously uncharacterized promoter. By choosing promoters whose expression patterns overlap for a single cell type, we can produce animals with fluorescence only in those cells. Furthermore, when one partial GFP polypeptide is fused with a subcellularly localized protein or peptide, this restricted expression leads to the fluorescent marking of cellular components in a subset of cells.