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Green fluorescent protein marks skeletal muscle in murine cell lines and zebrafish
Abstract
The green fluorescent protein (GFP) acts as a vital dye upon the absorption of blue light. When the gfp gene is expressed in bacteria, flies or nematodes, green fluorescence can be directly observed in the living organism. We inserted the cDNA encoding this 238-amino-acid (aa) jellyfish protein into an expression vector containing the rat myosin light-chain enhancer (MLC-GFP) to evaluate its ability to serve as a muscle-specific marker. Transiently, as well as stably, transfected C2C12 cell lines produced high levels of GFP distributed homogeneously throughout the cytoplasm and was not toxic through several cell passages. Expression of MLC-GFP was strictly muscle-specific, since Cos 7 fibroblasts transfected with MLC-GFP did not fluoresce. When GFP and beta Gal markers were compared, the GFP signal was visible in the cytoplasm of the living cell, whereas visualization of beta Gal required fixation and resulted in deformation of the cells. When the MLC-GFP construct was injected into zebrafish embryos, muscle-specific gfp expression was apparent within 24 h of development. gfp expression was never observed in non-muscle tissues using the MLC-GFP construct. Transgenic fish continued to express high levels of gfp in skeletal muscle at 1.5 months, demonstrating that GFP is an effective marker of muscle cells in vivo.
... While CAT and lacZ have been widely used as markers in fish, the recently described GFP (Chalfie et al., 1994) has been tested only in zebrafish as a convenient live staining marker (Amsterdam et al., 1995; Moss et al., 1996). We found that when the GFP gene was injected into S. aurata embryos, green fluorescence was easily detectable and appeared to have no adverse effects on embryonic development despite widespread expression (data not shown). ...
... According to these authors, this aspect should be taken into consideration in quantifying expression levels in embryos with yolk sac that contain extraembryonic tissues. While CAT and lacZ have been widely used as markers in fish, the recently described GFP (Chalfie et al., 1994) has been tested only in zebrafish as a convenient live staining marker (Amsterdam et al., 1995; Moss et al., 1996). We found that when the GFP gene was injected into S. aurata embryos, green fluorescence was easily detectable and appeared to have no adverse effects on embryonic development despite widespread expression (data not shown). ...
DNA transfer techniques allow genetic manipulation of commercial fish. However, marine species have received little attention because of their difficult zootechnical requirements. The seabream (Sparus aurata) has become one of the most important species in the aquaculture of Mediterranean countries, and the development of suitable DNA transfer procedures represents a main step in its genetic improvement. To assess the response of the seabream to exogenous DNA, naturally fertilized eggs were injected with the plasmids pCMV-CAT, pCMVTklacZ, and pEGFP-N1, in supercoiled and linearized forms. Embryo and larval survival, DNA fate, and reporter gene expression were analyzed during early development. The survival results indicate that microinjection is an effective transfer method in spite of the unfavorable conditions. Linearized plasmids were more efficiently polymerized than supercoiled ones; however, no significant differences were detected either in their persistence or in their expression levels. Reporter gene expression was initiated after mid-blastula transition. The duration of transient expression varied between the promoter-gene combinations, and no integration of transgenes into fish chromosomes was detected. Results suggest that the main factor affecting the persistence and expression of DNA seems to be related to developmental processes. Among the markers used, CAT proved to be the most sensitive, but GFP had obvious methodologic advantages over the spatial marker lacZ. The usefulness of GFP for diagnosis of transgenesis is enhanced by the transparency of embryos and larvae in S. aurata.
... This has advantages compared to the traditional ~-Gal reporter gene studies where visualization requires fixation. GFP is an ideal candidate for a reporter gene in zebra fish where the external development of transparent embryos allows detailed studies throughout embryogenesis (Gong, et al., 2002;Moss, et al., 1996). ...
p> To fill gaps in our knowledge about the functional roles of posterior Hoxc genes, in this thesis I set out to identify and characterise cis-regulatory elements contributing to their regulation.
The starting point was a l0kb region of DNA local to the Hoxc11 transcription unit, presumed to harbour enhancer elements based on transgenic reporter gene evidence. In order to identify these elements, phylogenetic footprinting was performed initially utilizing genomic sequence comparisons between mouse and human sequences. Due to the slowly evolving nature of the Hox clusters, extremely stringent parameters for the sequence comparisons had to be developed in order to avoid false positive Conserved Noncoding Sequence elements (CSEs), while still filtering out CSEs with regulatory function (avoidance of false negatives). Accordingly, the cut-off criteria were set at 97% similarity over a 100bp window of comparison, and three CSEs where identified in the local Hoxc11 region, namely R1, R2, and R3.
A series of Hoxcll/1acZ reporter gene deletion constructs was designed where deletion breakpoints were based on positions of R1, R2, and R3. In vivo recombination was used to create these deletions with pinpoint accuracy. However, none of these Hoxc11 constructs showed appreciable activity in a number of cell lines despite confirmation of expression for the endogenous homolog. While this raised the possibility of an incompatibility between the Hoxc11 promoter and the local elements, a series of constructs was launched to investigate enhancer activity per se of elements R1, R2, and R3 by combining them with a heterologous promoter and luciferase reporter. This demonstrated that element R3 could significantly enhance transcription from the minimal SV40 promoter in CaCo-2 cells, and to a lesser degree in HeLa cells. </p
... 104 These make it easy to distinguish muscle cells from other cells. 105 With all these benefits, the expansive zebrafish toolkit has the potential to simplify and accelerate research and development and to facilitate eventual production of cell-based meat for consumption. ...
The global demand for fish is rising and projected to increase for years to come. However, there is uncertainty whether this increased demand can be met by the conventional approaches of capture fisheries and fish farming because of wild stock depletion, natural resource requirements, and environmental impact concerns. One proposed complementary solution is to manufacture the same meat directly from fish cells, as cell-based fish. More than 30 ventures are competing to commercialize cell-based meat broadly, but the field lacks a foundation of shared scientific knowledge, which threatens to delay progress. Here, we recommend taking a research-focused, more open and collaborative approach to cell-based fish meat development that targets lean fish and an unlikely but very attractive candidate for accelerating research and development, the zebrafish. Although substantial work lies ahead, cell-based meat technology could prove to be a more efficient, less resource-intensive method of producing lean fish meat.
... While a popular aquarium fish, it has served for many years as a model organism for vertebrate developmental and genetic studies (Detrich et al. 1999;Westerfield 2000). More recently, zebrafish is finding use in myriad of research areas from neuroscience to oncology as zebrafish has several advantages over mammalian systems: its small size, high fecundity where 200 eggs may be produced by a female fish per day, rapid development of clear embryos, allowing visualization of organ systems with techniques like whole-mount in situ hybridization, green fluorescent protein reporter systems (Amsterdam et al. 1996;Moss et al. 1996;Jowett 1999;Westerfield 2000). In addition to these attributes, numerous, well-defined developmental mutants are available, and gene manipulation by transgenesis, CRISPR or morpholinos is well established (see http:// zfin.org/ for protocols). ...
Increasing organismal complexity during the evolution of life has been attributed to the duplication of genes and entire genomes. More recently, theoretical models have been proposed that postulate the fate of duplicated genes, among them the duplication-degeneration-complementation (DDC) model. In the DDC model, the common fate of a duplicated gene is lost from the genome owing to nonfunctionalization. Duplicated genes are retained in the genome either by subfunctionalization, where the functions of the ancestral gene are sub-divided between the sister duplicate genes, or by neofunctionalization, where one of the duplicate genes acquires a new function. Both processes occur either by loss or gain of regulatory elements in the promoters of duplicated genes. Here, we review the genomic organization, evolution, and transcriptional regulation of the multigene family of intracellular lipid-binding protein (iLBP) genes from teleost fishes. Teleost fishes possess many copies of iLBP genes owing to a whole genome duplication (WGD) early in the teleost fish radiation. Moreover, the retention of duplicated iLBP genes is substantially higher than the retention of all other genes duplicated in the teleost genome. The fatty acid-binding protein genes, a subfamily of the iLBP multigene family in zebrafish, are differentially regulated by peroxisome proliferator-activated receptor (PPAR) isoforms, which may account for the retention of iLBP genes in the zebrafish genome by the process of subfunctionalization of cis-acting regulatory elements in iLBP gene promoters.
... Several examples of GFP expression in nematodes, flies and animal cells (MacGregor & Caskey, 1989;Lin et al., 1994;Wang et al., 1994;Moss et al., 1996;Valdivia & Falkow, 1997) are available in literature, as author has more concern with plants therefore use of GFP in plants is being discussed in details in this review. ...
... Additionally, transgenic fluorescent reporter lines, labeling specific cell types or tissues using various fluorescent protein (FP) derivatives, is revolutionizing our understanding of in vivo cell biology. Initial experiments were directed at expressing green fluorescent protein (GFP)-tagged proteins to label a particular cell type, including different immune cell types allowing live imaging of their function in vivo (Long et al., 1997;Meng et al., 1997;Moss et al., 1996). For example, myeloperoxidase driven GFP (mpx-GFP) labels neutrophils, macrophage-expressed gene 1 (mpeg1)-driven GFP labels all mononuclear phagocytes including microglia, whereas apolipoprotein E (ApoE) more specifically labels microglia (Ellett et al., 2011;Peri and Nusslein-Volhard, 2008;Wittamer et al., 2011). ...
A major question in research on immune responses in the brain is how the timing and nature of these responses influence physiology, pathogenesis or recovery from pathogenic processes. Proper understanding of the immune regulation of the human brain requires a detailed description of the function and activities of the immune cells in the brain. Zebrafish larvae allow long-term, noninvasive imaging inside the brain at high-spatiotemporal resolution using fluorescent transgenic reporters labeling specific cell populations. Together with recent additional technical advances this allows an unprecedented versatility and scope of future studies. Modeling of human physiology and pathology in zebrafish has already yielded relevant insights into cellular dynamics and function that can be translated to the human clinical situation. For instance, in vivo studies in the zebrafish have provided new insight into immune cell dynamics in granuloma formation in tuberculosis and the mechanisms involving treatment resistance. In this review, we highlight recent findings and novel tools paving the way for basic neuroimmunology research in the zebrafish. GLIA 2014.
© 2014 Wiley Periodicals, Inc.
... Several examples of GFP expression in nematodes, flies and animal cells (MacGregor & Caskey, 1989; Lin et al., 1994; Wang et al., 1994; Moss et al., 1996; Valdivia & Falkow, 1997) are available in literature, as author has more concern with plants therefore use of GFP in plants is being discussed in details in this review. Multiple isoforms of GFP with distinct spectral properties are available to track numerous proteins in living cells. ...
The main objective of this study was to find out the extent of female participation in livestock care and management in a salt affected area of Faisalabad. The available studies and general observation of rural family life show that the women in rural areas lack appropriate knowledge regarding feeding, breeding managing and treatment of livestock.
... The GFP was obtained from the jellyfish Aequorea Victoria, and GFP gene was transferred, expressed in a wide range of organisms like mammals (Ludin et al., 1996), fishes (Moss et al., 1996), insects (Wang and Hazelrigg, 1994), plants (Casper and Holt, 1996), yeasts (Niedenthal et al., 1996) and a broad variety of bacteria (Chalfi et al., 1994). GFP has become a popular reporter for gene activity in bacteria. ...
Environmental contamination by heavy metals is a worldwide problem. Therefore, it is necessary to develop sensitive, effective and inexpensive methods, which can efficiently monitor and determine the level of hazardous metals in the environment. Conventional techniques to analyze metals, suffer from the disadvantages of high cost. Alternatively, development of simple system for monitoring heavy metals pollution is therefore needed. The present approach is based on the use of bacteria that are genetically engineered so that a measurable signal is produced when the bacteria are in contact with the bioavailable metal ions. Reporter genes are widely used as genetic tools for quantification and detection of specific cell population, gene expression and constructing whole cell biosensors as specific and sensitive devices for measuring biologically relevant concentrations of pollutants. An attempt has been made to construct the reporter gene enhanced green fluorescent protein and was expressed under the control of cadR gene, responsible for cadmium resistance. Recombinant strain Escherichia coli
cadR30 was used, that carried cadR gene in pET30b expression vector and cloned. Clones confirmed by the expression of enhanced green fluorescent protein was detected under ultraviolet illumination and separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The construction of green fluorescent protein based Escherichia coli bacterial biosensor was developed based on green fluorescent protein expression under the control cadR gene of Pseudomonas aeruginosa BC15. The constructed bacterial biosensor is useful and applicable in determining the availability of heavy metals in soil and wastewater.
... Complementary DNAs encoding redshifted variants of the wild-type GFP have been used for monitoring foreign gene expression, promoter activity, and protein localization in transgenic individuals and in cultured cells. Several studies have shown that variants of GFP cDNA were capable of producing detectable fluorescence products when introduced to zebra fish embryos (Amsterdam et al., 1995(Amsterdam et al., , 1996Moss et al., 1996;Higashijima et al., 1997;Long et al., 1997;Meng et al., 1997) and medaka embryos ( Hamada et al., 1998). Several properties of GFP variants in fish (e.g., their thermosensitivities, half-lives, and toxicities) are still not clear. ...
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.
... Conventional myosin contains two heavy chains (MYH) and four light chains (MYL) ( Rodgers et al. 1987). The C-terminal of each MYH, which defines the maximal shortening velocity of muscle ( Moss et al. 1996), folds into α-helical coiled-coil structure and joins two MYHs together ( Weeds and McLachlan 1974). In contrast, its N-terminal is composed of two regions, the head domain that binds the filamentous actin and hydrolyzes ATP to generate force to "walk" along the filament, and the neck domain that acts as a linker binding two myosin light chains together and as a lever arm for transducing force generated by the head domain ( Ruegg et al. 2002). ...
To identify muscle-related protein isoforms expressed in the white muscle of the mandarin fish Siniperca chuatsi, we analyzed 5,063 high-quality expressed sequence tags (ESTs) from white muscle cDNA library and predicted the integrity of the clusters annotated to these genes and the physiochemical properties of the putative polypeptides with full length. Up to about 33% of total ESTs were annotated to muscle-related proteins: myosin, actin, tropomyosin/troponin complex, parvalbumin, and Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCa). Thirty-two isoforms were identified and more than one isoform existed in each of these proteins. Among these isoforms, 14 putative polypeptides were with full length. In addition, about 2% of total ESTs were significantly homologous to "glue" molecules such as alpha-actinins, myosin-binding proteins, myomesin, tropomodulin, cofilin, profilin, twinfilins, coronin-1, and nebulin, which were required for the integrity and maintenance of the muscle sarcomere. The results demonstrated that multiple isoforms of major muscle-related proteins were expressed in S. chuatsi white muscle. The analysis on these isoforms and other proteins sequences will greatly aid our systematic understanding of the high flexibility of mandarin fish white muscle at molecular level and expand the utility of fish systems as models for the muscle genetic control and function.
... The activity of its product enzyme b-galactosidase is usually assayed in fixed tissues, but can also be used in conjunction with fluorescent substrates to report on gene expression in live embryos [20,34]. Its use for generating stable transgenic lines in zebrafish has been largely superseded by the advent of green fluorescent protein (GFP) and other fluorophores and the demonstration of their successful use in live zebrafish [35][36][37][38][39][40]. The use of fluorescent proteins in conjunction with the detailed imaging possible on live zebrafish embryos has facilitated sophisticated examination of early developmental events. ...
As increasing numbers of vertebrate genomes are sequenced, comparative genomics offers tremendous promise to unveil mechanisms of transcriptional gene regulation on a large scale. However, the challenge of analysing immense amounts of sequence data and relating primary sequence to function is daunting. Several teleost species occupy crucial niches in the world of comparative genomics, as experimental model organisms of wide utility and living roadmaps of molecular evolution. Extant species have evolved after a teleost-specific genome duplication, and offer the opportunity to examine the evolution of thousands of duplicate gene pairs. Transgenesis in zebrafish is being increasingly employed to functionally examine non-coding sequences, from fish and mammals. Here, we discuss current approaches to the study of gene regulation in teleosts, and the promise of future research.
... (ii) Artificial chromosomes have not been used to analyze gene expression in zebrafish. Previously, the GFP reporter gene has been used as part of DNA constructs less than 20 kb to study promoter elements in zebrafish (19,(32)(33)(34)(35). Our results demonstrate that BACs can correctly drive the expression of a reporter gene in living zebrafish embryos. ...
The modification of yeast artificial chromosomes through homologous recombination has become a useful genetic tool for studying gene function and enhancer/promoter activity. However, it is difficult to purify intact yeast artificial chromosome DNA at a concentration sufficient for many applications. Bacterial artificial chromosomes (BACs) are vectors that can accommodate large DNA fragments and can easily be purified as plasmid DNA. We report herein a simple procedure for modifying BACs through homologous recombination using a targeting construct containing properly situated Chi sites. To demonstrate a usage for this technique, we modified BAC clones containing the zebrafish GATA-2 genomic locus by replacing the first coding exon with the green fluorescent protein (GFP) reporter gene. Molecular analyses confirmed that the modification occurred without additional deletions or rearrangements of the BACs. Microinjection demonstrated that GATA-2 expression patterns can be recapitulated in living zebrafish embryos by using these GFP-modified GATA-2 BACs. Embryos microinjected with the modified BAC clones were less mosaic and had improved GFP expression in hematopoietic progenitor cells compared with smaller plasmid constructs. The precise modification of BACs through Chi-stimulated homologous recombination should be useful for studying gene function and regulation in cultured cells or organisms where gene transfer is applicable.
... The cDNA encoding the GFP has been inserted into an expression vector containing the rat myosin light-chain enhancer to evaluate its ability to serve as a muscle-specific marker. Transiently as well as stably, transfected C2C12 cell lines produced high levels of GFP distributed homogeneously throughout the cytoplasm and was not toxic through several cell passages (Moss et al., 1996). ...
Fish metabolism needs special enzymes that have maximum activity at very different conditions than their mammalian counterparts. Due to the differences in activity, these enzymes, especially cold-adapted proteases, could be used advantageously for the production of some foods. In addition to the enzymes, this review describes some other unique fish polypeptides such as antifreeze proteins, fluorescent proteins, antitumor peptides, antibiotics, and hormones, that have already been cloned and used in food processing, genetic engineering, medicine, and aquaculture. Recombinant DNA technology, which allows these biological molecules to be cloned and overexpressed in microorganisms is also described, highlighting innovative applications. The expected impact of cloning fish proteins in different fields of technology is discussed.
... The zebrafish chihuahua (chi) mutant shows skeletal dysplasia resembling osteogenesis imperfecta in man (159). Molecular and cellular development of skeletal muscle, and neuromuscular function in normal and abnormal zebrafish are well studied, although not yet fully understood (39, 73, 74, 200, 224, 229, 236, 363, 376). Mutant screens have identified over 50 genes essential for normal somite development (136, 137, 486). ...
The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1-2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology.
... In these assays, dozens of fish are injected with a given plasmid in order to generate an "expression map" by pooling expression information from the mosaic embryos. This method has been used successfully to analyze cis-acting promoter/enhancer elements that contribute to tissue-specific, speciesspecific, or developmental time-specific gene expression (Chen et al., 1998(Chen et al., , 2001Du and Dienhart, 2001;Hieber et al., 1998;Higashijima et al., 2000;Huang et al., 2001;Ju et al., 1999;Marsh-Armstrong et al., 1995;Meng et al., 1997Meng et al., , 1999bMoav et al., 1993;Moss et al., 1996;Motoike et al., 2000;Muller et al., 1999;Park et al., 2000;Picker et al., 2002;Reinhard et al., 1994;Udvadia et al., 2001;Westerfield et al., 1992). ...
The recent explosion of transgenic zebrafish lines in the literature demonstrates the value of this model system for detailed in vivo analysis of gene regulation and morphogenetic movements. The optical clarity and rapid early development of zebrafish provides the ability to follow these events as they occur in live, developing embryos. This article will review the development of transgenic technology in zebrafish as well as the current and future uses of transgenic zebrafish to explore the dynamic environment of the developing vertebrate embryo.
... The GFP was obtained from the jellyfish Aequorea Victoria, and GFP gene was transferred, expressed in a wide range of organisms like mammals (Ludin et al., 1996), fishes (Moss et al., 1996), insects (Wang and Hazelrigg, 1994), plants (Casper and Holt, 1996), yeasts (Niedenthal et al., 1996) and a broad variety of bacteria (Chalfi et al., 1994). GFP has become a popular reporter for gene activity in bacteria. ...
The aim of the present study was to investigate sexual behavior in population of psychiatric patients affected by schizophrenia, schizoaffective disorder or bipolar disorder by means of an ad hoc questionnaire designed to explore the three phases of the sexual response: desire (or interest), arousal, and performance. The study assessed patients' attitude toward sexuality, several aspects of their sexual behavior, including patients' awareness of the risk of sexually transmitted diseases (STD), contraceptive strategy preferred by patients, and sexual effects of psychotropic medication. Patients reported a high frequency of sexual dysfunction, in particular, hyposexuality. Schizophrenia diagnosis and female gender were associated with lower levels of sexual performance. The impact of psychotropic drugs on patients' sexuality was significant, with both positive and negative effects. Although 65.8% of patients reported to be concerned about the risk of contracting infections during sexual intercourse, most of them engaged in sexual behavior at high risk for acquisition and transmission of STD. Patients' compliance with contraceptive measures was poor.
... .(Long et al., 1997;; Pownall et al., 1998; Moss et al., 1996; Higashijima et al., 1997; Meng et al., 1997; Long et al., 1997; Jessen et al., 1999). Moderate to low levels of GFP can be detected if the specimen has low levels of autofluorescence, the signal is not too deep in a tissue, and the tissue is Wiley STM / Editor: Book Title, Chapter ?? / Authors?? / filename: ch??.doc page 4 transparent. ...
IntroductionTypes of Transgenics VertebratesIssues Affecting the Successful Use of GFP in Transgenic VertebratesMethods for Creating Transgenics in Different Vertebrate SpeciesUses of GFP Transgenics in VertebratesConclusion
References
... One of the key technologies that makes many embryonic development studies possible is the production of transformed embryos and adult animals. Transformation with GFP encoding constructs generated green embryos in mice (27,189,190), rats (191,192), cows (102,164,191,193), monkeys (29,194,195), porcine (165), Drosophila (39,95,196), mosquito (197), silkworm (96,135), Xenopus (45,91), C. elegans (65,198), medakafish (46,199), Zebrafish (34,200,201), rainbow trout (41,63), sea urchin (80), and the ascidian Ciona intestinalis (202). The most common way of producing transformed animals is to inject restriction enzymelinearized plasmid DNA directly into the pronuclei of one-cell stage embryos (54). ...
An Erratum has been published for this article in Luminescence (2003) 18(4) 243
During the past 5 years, green fluorescent protein (GFP) has become one of the most widely used in vivo protein markers for studying a number of different molecular processes during development, such as promoter activation, gene expression, protein trafficking and cell lineage determination. GFP fluorescence allows observation of dynamic developmental processes in real time, in both transiently and stably transformed cells, as well as in live embryos. In this review, we include the most up-to-date use of GFP during embryonic development and point out the unique contribution of GFP visualization, which resulted in novel discoveries. Copyright
The transgenic technology has been widely used in biotechnology, from generation of genetically modified (GM) foods to production of pharmaceutical proteins. Although fast-growing transgenic fish using exogeneous growth hormone gene have been successfully produced, the marketing of such GM food fish remains to face an uphill battle. In the present study, we attempted to use the transgenic technology to develop novel varieties of ornamental fish. So far, we have succeeded in generation of several colourful transgenic zebrafish by transferring a jellyfish green fluorescent protein (gfp) gene and an anemone red fluorescent protein (rfp) gene. These transgenic zebrafish displayed green, red, yellow or orange fluorescent colors under both daylight and uv light. Thus, our work demonstrated the feasibility to generate novel varieties of ornamental fish by the transgenic techniques. Currently we are conducting mating behaviour experiments using these transgenic zebrafish. We are also aiming at development of biomonitoring transgenic fish by using some inducible gene promoters that can respond certain environmental pollutants.
The cytoskeleton is a dynamic structure comprised of at least three distinct cellular filament systems: microfilaments, intermediate filaments, and microtubules. Microfilaments are composed of assembled globular actin monomers that form a filamentous system involved in the maintenance of cell shape and polarity. Intermediate filaments are made of fibrous proteins including vimentin, cytokeratin, nuclear lamins, and neurofilament proteins that assemble into fibers providing mechanical stability to animal cells. Microtubules are formed by the assembly of tubulin (α and β subunits) producing long, rigid polymers which govern the location of membrane-bounded organelles and other cellular components. While each filament system has specific cellular functions, an emerging view of the cytoskeleton is that actin, intermediate filaments and microtubule networks function in concert to provide the cell with stability, polarity and organization.
Use of green fluorescent protein (gfp) as a reporter gene is a powerful approach for the investigation of tissue-specific gene expression and cellular localization of proteins because the fluorescence of its protein product can be conveniently detected in living cells without supplementing a substrate. The approach is particularly useful in zebrafish because of the transparency and external development of their embryos. In the past few years, using several zebrafish tissue-specific promoters, we have developed several stable lines of gfp transgenic zebrafish that display green fluorescence in different tissues; these include five transgenic lines under an epidermis-specific keratin8 (krt8) promoter, two transgenic lines under a fast skeletal muscle-specific promoter from the myosin light polypeptide 2 (mylz2) gene, and five transgenic lines under an elastaseA (elaA) promoter that is specifically expressed in pancreatic exocrine cells. In all cases, transgenic GFP is faithfully expressed according to the specificity of the promoters used. These gfp transgenic lines are useful for recapitulation of a gene expression program, investigation of tissue and organ development, cell sorting for in vitro cell culture, and construction of cell type-specific cDNA library. Recently, by using two tissue-specific promoters linked to two different reporter genes, gfp and rfp (red fluorescent protein), we have generated two-color transgenic zebrafish that express GFP in skin epidermis and RFP in fast skeletal muscle. Currently, we are also developing gfp transgenic fish for biomonitoring using selected inducible gene promoters that can respond to heavy metals and estrogenic compounds. Thus, generation of living color transgenic zebrafish will have important applications in biotechnology as well as in developmental biology.
The use of a non-toxic visual marker is advantageous, for discerning transgenic cells and removing untransformed or non-expressing cells, tissues or organs, over genes required substrates and cofactors for their expression to report. The green fluorescent protein (gfp) of the jellyfish, Aequorea victoria, has recently been used as a reporter gene in plants and animals. In plants it has extensively been used in plant transformation experiments, localization of proteins to organelles and to study gene expression. Visual marker like FLARES (Khan & Maliga, 1999) made it possible to differentiate transformed from untransformed plastids resulted in chimeras in leaves of monocot as well as dicot plants. Unlike tobacco and Arabidopsis, plant regeneration from leaves in monocots (cereals) is not possible, therefore, it is difficult to make transformants homoplasmic. To make rice transformants homoplasmic fluorescent marker like FLARES is required which enables selection of fluorescent cells on the medium. Moreover, fluorescent antibiotic resistance marker will enable extension of plastid transformation to other cereals, where plastid transformation is not associated with a readily identifiable phenotype. Furthermore, GFP has multiple isoforms with distinct spectral properties could be useful to track numerous proteins in living cells.
Abstract Mycobacterium chelonae is widespread in aquatic environments and can cause mycobacteriosis with low virulence in zebrafish. The risk of infection in zebrafish is exacerbated in closed-recirculating aquatic systems where rapidly growing mycobacteria can live on biofilms, as well as in zebrafish tissues. We have discovered a method of identifying and visualizing M. chelonae infections in living zebrafish using endogenous autofluorescence. Infected larvae are easily identified and can be excluded from experimental results. Because infection may reduce fertility in zebrafish, the visualization of active infection in contaminated eggs of transparent casper females simplifies screening. Transparent fish are also particularly useful as sentinels that can be examined periodically for the presence of autofluorescence, which can then be tested directly for M. chelonae.
This chapter describes the procedures used to generate transgenic zebrafish by microinjection of plasmid DNA. Transgenic zebrafish generated using plasmid DNA constructs are used for a variety of genetic analyses. The chapter discusses the generation of transgenic zebrafish containing the green fluorescent protein (GFP) reporter gene ligated to 5.6 kb of zebrafish GATA-1 promoter sequence. These fish are used to obtain pure populations of hematopoietic progenitor cells by isolating fluorescent—GFP-expressing hematopoietic cells—from 16-hr-old embryos, using fluorescence-activated cell sorting. These cells can be used to perform differential display polymerase chain reaction (PCR) to identify genes uniquely expressed in the earliest hematopoietic progenitor cells. In addition to generating transgenic zebrafish, transient gene expression assays can be performed in living zebrafish embryos after microinjection of plasmid DNA. In contrast to in situ hybridization, these assays provide three-dimensional information of gene expression patterns in real time because living embryos can be observed for several days. A plasmid construct containing GFT ligated to zebrafish GATA-2 promoter sequences is used to identify a regulatory element responsible for GATA-2 expression in the central nervous system.
Exposure and Bioaccumulation of Dioxin and Related Compounds by FishDioxin Toxicity in Juvenile and Adult FishDioxin Toxicity in Embryonic and Larval FishAhR Signal Transduction Pathway in FishMechanisms of Dioxin Toxicity in FishFuture DirectionsAcknowledgmentsReferences
Transgenic fish are produced by the artificial transfer of rearranged genes into newly fertilized eggs. Currently microinjection is the preferred method, although the integration rates of transgenes are generally low. A number of fusion genes, containing retrovirus sequences which direct integration, have been developed to enhance integration of transgenes. Mass gene transfer methods are also being developed. These include lipofection, particle bombardment, and electroporation of embryos and sperm cells. These methods are potentially useful for marine organisms such as crustaceans and molluscs as well as fish. In contrast to microinjection, which treats single cells individually, these methods can transfer genes into a large number of eggs at once. There is some evidence to indicate successful integration and expression of transgenes transferred by the electroporation of embryos and sperm cells. Germline transmission of transgenes has been observed through mating studies, and in some cases the progeny express the new phenotype consistently. However, germline transmission does not necessarily confirm stable integration of the transgene. There is evidence that transgenes may exist extrachromosomally. Transgenic fish are viewed as a useful model for the study of complex biological phenomena such as growth and differentiation, and as a fast track to the production of broodstock for the aquaculture industry. Current research focuses on the elucidation of the mechanisms controlling the regulation of gene expression. The use of transgenic fish for the isolation of developmental genes has just begun. Applications of transgenesis to broodstock development have been focused on the development of fish with accelerated growth, tolerance to low temperature, and disease resistance. However, before the release of transgenic fish into the environment, the possible impact on the environment must be assessed. There must be safeguards to protect the genetic diversities of the natural populations, and to conserve the natural habitats
The zebrafish has been the model of choice amongst developmental biologists for many years. This small freshwater species offers many advantages to the study of organ and tissue development that are not provided by other model systems. Against this background, modern molecular genetic approaches are being applied to expand the physical and genetic mapping of the zebrafish genome. These approaches complement the large-scale mutagenic screens that have led to the isolation of mutant phenotypes. Some of the phenotypes have been found to resemble human disease states, while mapping and sequencing data have revealed zebrafish genes with significant homology to human disease-causing genes. It is the realization that the zebrafish offers an amenable system for understanding disease, as opposed to development, that underpins this review. The adventitious identification of disease phenotypes amongst zebrafish mutants and the important area of deliberate disease modelling using transgenesis and gene targeting should lead to a better application of the zebrafish as a vertebrate model of human diseases.
Lysosomal storage disorders are amenable to treatment by enzyme replacement. Genetic modification of muscle via direct injection of expression vectors might represent an alternative method of providing the defective enzymes, if adequate and long-lasting expression levels can be achieved in muscle. We have used the C2C12 mouse myogenic cell line to study the effect of combination of muscle-specific regulatory elements on the expression of the human lysosomal enzyme alpha-galactosidase (alpha-gal). In differentiated myotubes, a construct containing the myosin light chain 1/3 enhancer in combination with the human cytomegalovirus promoter resulted in higher expression than constructs combining the same enhancer with the rabbit beta-myosin heavy chain promoter, or containing the CMV promoter only. Increased enzymatic activity was detectable both in cell extracts and in supernatants. Furthermore, human fibroblasts deficient in alpha-gal were able to take up the enzyme from medium conditioned by transfected myoblasts. This did not occur in the presence of mannose-6-phosphate which indicates that the uptake was via mannose-6-phosphate receptors. To our knowledge, this is the first report in which a correctly processed form of human alpha-gal was expressed and secreted from differentiated muscle cells. Direct injection of a plasmid expression vector into mouse tibialis anterior muscle showed significantly increased levels of alpha-gal 7 days after injection.
Use of the green fluorescent protein (Gfp) from the jellyfish Aequorea victoria is a powerful method for nondestructive in situ monitoring, since expression of green fluorescence does not require any substrate addition. To expand the use of Gfp as a reporter protein, new variants have been constructed by the addition of short peptide sequences to the C-terminal end of intact Gfp. This rendered the Gfp susceptible to the action of indigenous housekeeping proteases, resulting in protein variants with half-lives ranging from 40 min to a few hours when synthesized in Escherichia coli and Pseudomonas putida. The new Gfp variants should be useful for in situ studies of temporal gene expression.
We report the first use of green fluorescent protein (GFP) for mutation detection. We have constructed a plasmid-based bacterial system whereby mutated cells fluoresce and non-mutated cells do not fluoresce. Fluorescence is monitored using a simple hand-help UV lamp; no additional cofactors or manipulations are necessary. To develop a reversion system, we introduced a +1 DNA frameshift mutation in the coding region of GFP and the resulting protein is not fluorescent in Escherichia coli. Treatment of bacteria containing the +1 frameshift vector with ICR-191 yields fluorescent colonies, indicating that reversion to the wild-type sequence has occurred. Site-directed mutagenesis was used to insert an additional cytosine into a native CCC sequence in the coding region of GFP in plasmid pBAD-GFPuv, expanding the sequence to CCCC. A dose-related increase in fluorescent colonies was observed when the bacteria were treated with ICR-191, an agent that induces primarily frameshift mutations. The highest dose of ICR-191 tested, 16 microg/ml, produced a mutant fraction of 16 x 10(-5) and 8.8 x 10(-5) in duplicate experiments. The reversion system did not respond to MNNG, an agent that produces mainly single-base substitutions. To develop a forward system, we used GFP under the control of the arabinose PBAD promoter; in the absence of arabinose, GFP expression is repressed and no fluorescent colonies are observed. When cells were treated with MNNG or ENNG, a dose-dependent increase in fluorescent colonies was observed, indicating that mutations had occurred in the arabinose control region that de-repressed the promoter. Treating bacteria with 100 microg/ml MNNG induced mutant fractions as high as 82 x 10(-5) and 40 x 10-5 in duplicate experiments. Treating bacteria with 150 microg/ml ENNG induced a mutant fraction of 2.1 x 10(-5) in a single experiment.
We have designed a new approach to the direct cloning and rapid analysis of mammalian enhancer elements by fusing green fluorescent protein and neomycinphosphotransferase under the control of a thymidine kinase minimal promoter. DNA fragments containing known or potential enhancer elements can be inserted into a polylinker upstream of GFPneo and re-isolated from stably transfected cell lines by a direct transgene-specific polymerase chain reaction (PCR), for further analysis. C2C12 muscle cells were transfected with four vectors containing the GFPneo fusion gene regulated by the cytomegalovirus promoter, the myoD distal core enhancer and myoblast- and myotube-specific enhancers from the desmin gene. GFPneo shows robust epifluorescence by microscopy and flow cytometry and retains sufficient neo activity to permit selection of G418-resistant clones. The fluorescence signal pattern of GFPneo expressed under the control of the desmin enhancers mirrors their transcriptional profile during myogenic differentiation. This finding demonstrates the value of GFPneo as a tool to analyse differentiation stage-specific regulatory DNA elements in stably transfected mammalian cell lines. We were able to re-isolate the myoD enhancer mediating GFPneo expression from a stably transfected C2C12 clone by a transgene-specific PCR reaction, demonstrating the feasibility of using this new vector system for the isolation of regulatory sequences.
The coupling of the GFP reporter system with the optical clarity of embryogenesis in model fish such as zebrafish and medaka is beginning to change the picture of transgenic fish study. Since the advent of first GFP transgenic fish in 1995, GFP transgenic fish technology have been quickly employed in many areas such as analyses of gene expression patterns and tissue/organ development, dissection of promoters/enhancers, cell lineage and axonal pathfinding, cellular localization of protein products, chimeric embryo and nuclear transplantation, cell sorting, etc. The GFP transgenic fish also have the potentials in analysis of upstream regulatory factors, mutagenesis screening and characterization, and promoter/enhancer trap. Our own studies indicate that GFP transgenic fish may become a new source of novel variety of ornamental fish. Efforts are also being made in our laboratory to turn GFP transgenic fish into biomonitoring organisms for surveillance of environmental pollution.
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.
The zebrafish has recently emerged as a powerful model for study of the genetics and molecular biology of vertebrate development. Zebrafish reach sexual maturity in ∼2-3 mo, and produce hundreds of embryos in weekly intervals. The embryos develop rapidly and organogenesis occurs within the first 2 d of development in transparent bodies. The availability of thousands of chemically induced mutations (1,2), as well as the results of insertional mutagenesis studies (3–6), provide valuable resources to study genetics of vertebrate development.
Mammalian liver fatty acid binding protein (L-FABP) is a small cytosolic protein in various tissues including liver, small intestine and kidney and is thought to play a crucial role in intracellular fatty acid trafficking and metabolism. To better understand its tissue-specific regulation during zebrafish hepatogenesis, we isolated 5'-flanking sequences of the zebrafish L-FABP gene and used a green fluorescent protein (GFP) transgenic strategy to generate liver-specific transgenic zebrafish. The 2.8-kb 5'-flanking sequence of zebrafish L-FABP gene was sufficient to direct GFP expression in liver primordia, first observed in 2 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. F2 inheritance rates of 42-51% in all the seven transgenic lines were consistent with the ratio of Mendelian segregation. Further, hhex and zXbp-1 morphants displayed a visible liver defect, which suggests that it is possible to establish an in vivo system for screening genes required for liver development.
DNA recombinases show some promise as reporters of pollutants providing that appropriate promoters are used and that the apparent dependence of expression on cell density can be solved. Further work is in progress using different recombinases and other promoters to optimize recombinase expression as well as to test these genetic constructs in contaminated environmental samples such as soil and water. It may be that a graded response reflecting pollutant concentration may not be possible. However, they show great promise for providing definitive detection systems for the presence of a pollutant and may be applicable to address the problem of bioavailability of pollutants in complex environments such as soil.
A recombinant measles virus (MV) expressing red fluorescent protein (MVDsRed1) was used to produce a persistently infected cell line (piNT2-MVDsRed1) from human neural precursor (NT2) cells. A similar cell line (piNT2-MVeGFP) was generated using a virus that expresses enhanced green fluorescent protein. Intracytoplasmic inclusions containing the viral nucleocapsid protein were evident in all cells and viral glycoproteins were present at the cell surface. Nevertheless, the cells did not release infectious virus nor did they fuse to generate syncytia. Uninfected NT2 cells express the MV receptor CD46 uniformly over their surface, whereas CD46 was present in cell surface aggregates in the piNT2 cells. There was no decrease in the overall amount of CD46 in piNT2 compared to NT2 cells. Cell-to-cell fusion was observed when piNT2 cells were overlaid onto confluent monolayers of MV receptor-positive cells, indicating that the viral glycoproteins were correctly folded and processed. Infectious virus was released from the underlying cells, indicating that persistence was not due to gross mutations in the virus genome. Persistently infected cells were superinfected with MV or canine distemper virus and cytopathic effects were not observed. However, mumps virus could readily infect the cells, indicating that superinfection immunity is not caused by general soluble antiviral factors. As MVeGFP and MVDsRed1 are antigenically indistinguishable but phenotypically distinct it was possible to use them to measure the degree of superinfection immunity in the absence of any cytopathic effect. Only small numbers of non-fusing green fluorescent piNT2-MVDsRed1 cells (1 : 300 000) were identified in which superinfecting MVeGFP entered, replicated and expressed its genes.
The zebrafish has emerged as a powerful animal model for human diseases. While it has long informed us about the biology of early development, it has recently come into favor for the investigation of clinically relevant problems. Genes conserved from fish to humans can be rapidly analyzed using the zebrafish embryo in what is essentially a transparent in vivo assay. This unit describes methodologies including genetic screening, targeted knockdowns, ectopic overexpression, and transgenesis.
The rat myosin light chain (MLC)1/3 gene locus contains a potent muscle-specific enhancer, located downstream of the coding region, greater than 24 kilobases away from the MLC1 transcription start site. To assess the role of this enhancer in the activation of MLC expression during development, transgenic mice were generated carrying multiple copies of a MLC1 promoter-chloramphenicol acetyltransferase (CAT) transcription unit linked to a genomic fragment including the enhancer. CAT expression was detected in four mouse lines, up to 1000-fold higher in skeletal muscles than in other tissues. Activation of endogenous MLC1 transcription in these animals 4 days before birth was reflected in the onset of CAT transgene expression. This study identifies the transcriptional control elements necessary to activate the 21-kilobase MLC1/3 locus at the appropriate fetal stage and indicates that the MLC enhancer is sufficient to induce developmentally regulated expression from the MLC1 promoter exclusively in skeletal muscle cells.
Two skeletal myosin light chains, MLC1 and MLC3, are generated from a single gene by transcription from two different promoters and alternate splicing of the pre-mRNAs. To define DNA sequences involved in MLC transcriptional control, we constructed a series of plasmid vectors in which segments of the rat MLC locus were linked to a CAT gene and assayed for expression in muscle and nonmuscle cells. Whereas sequences proximal to the two MLC promoters do not appear to contain tissue-specific regulatory elements, a 0.9-kb DNA segment, located greater than 24 kb downstream of the MLC1 promoter, dramatically increases CAT gene expression in differentiated myotubes but not in undifferentiated myoblasts or nonmuscle cells. The ability of this segment to activate gene expression to high levels, in a distance-, promoter-, position-, and orientation-independent way, defines it as a strong muscle-specific enhancer element.
We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F1 progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 x 1 hr in 3 mM ENU and 6 x 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F2 screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.
In transgenic mice, muscle-specific regulatory elements from the myosin light chain (MLC) 1/3 locus drive graded expression of a linked CAT reporter gene in selected fast muscles along the anteroposterior axis of the adult animal. The gradient of MLC-CAT transcripts is established early in development, during the generation of somites from the paraxial mesoderm and the activation of myogenic factor gene expression, and is not reflected in the expression of the endogenous MLC1 gene. At later embryonic stages, the gradient of MLC-CAT transcripts persists in intercostal and intervertebral muscles, but is not maintained in other axial muscles. Profiles of CAT transgene activity reveal that the gradient is generated during the maturation of increasingly caudal somites, opposite to the direction of somite development, and is retained in dissociated somite cultures. We propose that coexpression of myogenic factors is necessary but not sufficient to regulate expression of the MLC-CAT transgene, which is responsive to additional positional cues in the embryo.
Many cnidarians utilize green-fluorescent proteins (GFPs) as energy-transfer acceptors in bioluminescence. GFPs fluoresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca(2+)-activated phosphoprotein. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid (aa) residues within the polypeptide. This report describes the cloning and sequencing of both cDNA and genomic clones of GFP from the cnidarian, Aequorea victoria. The gfp10 cDNA encodes a 238-aa-residue polypeptide with a calculated Mr of 26,888. Comparison of A. victoria GFP genomic clones shows three different restriction enzyme patterns which suggests that at least three different genes are present in the A. victoria population at Friday Harbor, Washington. The gfp gene encoded by the lambda GFP2 genomic clone is comprised of at least three exons spread over 2.6 kb. The nucleotide sequences of the cDNA and the gene will aid in the elucidation of structure-function relationships in this unique class of proteins.
Transgenic mice were produced in which expression of the reporter gene chloramphenicol acetyltransferase (CAT) is controlled by regulatory elements of a rodent myosin light chain gene. CAT activity was readily detectable in muscles of these mice but negligible in a variety of nonmuscle tissues. Unexpectedly, levels of CAT expression varied greater than 100-fold from muscle to muscle, forming a gradient in which a muscle's position in the rostrocaudal axis was correlated with its level of CAT enzyme activity and abundance of CAT mRNA. Thus, rostral muscles (innervated by cranial nerves) had the lowest levels of CAT, thoracic muscles had intermediate levels, and caudal muscles (innervated through lumbar and sacral roots) had the highest levels. We established that myosin light chain sequences are responsible for the gradient of CAT expression but observed no strong gradient of endogenous myosin light chain expression. We argue that elements that are silent or masked by other sequences in their native context are revealed in the transgene and that the rostrocaudal gradient of gene expression they produce reveals the existence of a positionally graded endogenous regulator of gene expression. These transgenic mice provide evidence that cells in adult mammals retain "positional information" of a sort hitherto studied largely in embryos. The transgene they express may provide a means for determining how such positional values are generated and maintained.
Subcellular RNA localization in different cell types leads to asymmetric distribution of proteins in these cells. The localization of bicoid (bcd) messenger RNA to the anterior pole of the developing Drosophila oocyte gives rise in embryogenesis to a steep concentration gradient of the bcd protein, a transcription factor that activates expression of zygotic genes needed for anterior development. The exuperantia (exu) gene is necessary for this localization of bcd mRNA. Here we express a chimaeric gene encoding a fusion between the Acquorea victoria green fluorescent protein (GFP) and the exu protein (Exu) in female germ cells, and find that the fusion protein fluoresces strongly in both live and fixed cells during Drosophila oogenesis. The fusion protein rescues an exu null allele, restoring full fertility to females, and is expressed and localized in a temporal and spatial pattern similar to native Exu. The high sensitivity of the GFP tag provides important new details on the subcellular localization of Exu. The fusion protein is found in particles concentrated at ring canals, where transport occurs between nurse cells and the oocyte. Drugs such as colchicine and taxol that affect microtubule stability alter localization of the particles. We propose that the particles are ribonucleoprotein complexes or vesicles which transport bcd mRNA along microtubules and target it to the anterior oocyte cortex.
The zebrafish is rapidly becoming a popular model system for the study of vertebrate development because it is ideal for both
embryological studies and genetic analysis. To determine if a retroviral vector pseudotyped with the envelope glycoprotein
of the vesicular stomatitis virus could infect zebrafish embryos, and in particular, the cells destined to become the germ
line, a pseudotyped virus was injected into blastula-stage zebrafish embryos. Fifty-one embryos were allowed to develop and
eight transmitted proviral DNA to their progeny. Founders were mosaic, but as expected, transgenic F1's transmitted proviral
DNA in a Mendelian fashion to the F2 progeny. Transgenic F1 fish inherited a single integrated provirus, and a single founder
could transmit more than one viral integration to its progeny. These results demonstrate that this pantropic pseudotyped vector,
originally developed for human gene therapy, will make the use of retroviral vectors in zebrafish possible.
Expression of the cDNA for Aequorea green fluorescent protein in E. coli yielded a fused protein with fluorescence excitation and emission spectra virtually identical to those of the native green fluorescent protein. Further, a solution of the protein, when mixed with aequorin and calcium ion, emitted a greenish luminescence characteristic of the in vivo luminescence of the animal, indicating a radiationless energy transfer to the protein.
A complementary DNA for the Aequorea victoria green fluorescent protein (GFP) produces a fluorescent product when expressed in prokaryotic (Escherichia coli) or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and cofactors are not required for this fluorescence, GFP expression can be used to monitor gene expression and protein localization in living organisms.
Expression of the myogenic helix-loop-helix (HLH) protein myogenin in muscle cell precursors within somites and limb buds
is among the earliest events associated with myogenic lineage determination in vertebrates. Mutations in the myogenin promoter
that abolish binding sites for myogenic HLH proteins or myocyte enhancer factor-2 (MEF-2) suppressed transcription of a linked
lacZ transgene in subsets of myogenic precursors in mouse embryos. These results suggest that myogenic HLH proteins and MEF-2
participate in separable regulatory circuits leading to myogenin transcription and provide evidence for positional regulation
of myogenic regulators in the embryo.