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

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

A green fluorescent protein fusion strategy for monitoring the expression, cellular location, and separation of biologically active organophosphorus hydrolase

 Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of the gene for green fluorescent protein (GFP) to the 5′ end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP. Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations with inexpensive instrumentation based on detecting green fluorescence. Received: 27 April 1999 / Received last revision: 18 October 1999 / Accepted: 1 November 1999     

Transient expression and stable transformation of soybean using the jellyfish green fluorescent protein

 Embryogenic soybean [Glycine max (L.) Merrill.] suspension cultures were bombarded with five different gene constructions encoding the jellyfish (Aequorea victoria) green fluorescent protein (GFP). These constructions had altered codon usage compared to the native GFP gene and mutations that increased the solubility of the protein and/or altered the native chromophore. All of the constructions produced green fluorescence in soybean cultures upon blue light excitation, although a soluble modified red-shifted GFP (smRS-GFP) was the easiest to detect based on the brightness and number of foci produced. Expression of smRS-GFP was visible as early as 1.5 h after bombardment, with peak expression at approximately 6.5 h. Large numbers of smRS-GFP-expressing areas were visible for 48 h postbombardment and declined rapidly thereafter. Stably transformed cultures and plants exhibited variation in the intensity and location of GFP expression. PCR and Southern hybridization analyses confirmed the presence of introduced GFP genes in stably transformed cultures. Received: 23 September 1998 / Revision received: 4 January 1999 / Accepted: 15 January 1999     

Homogeneous tetracycline‐regulatable gene expression in mammalian fibroblasts

The expression of transfected genes in mammalian cells is rapidly repressed by epigenetic mechanisms such that, within a matter of weeks, only a fraction of the cells in most clonal populations still exhibit detectable expression. This problem can become prohibitive when one wants to express two ectopically introduced genes, as is necessary to establish cell lines that harbor genes regulated by the tetracycline‐controlled transactivators. We describe an approach to establish Chinese hamster ovary (CHO) cell lines that stably induce a tet‐responsive reporter gene in all cells of a transfected clonal population. Screening of more than 100 colonies resulting from a standard co‐transfection of the tetracycline transactivator (tTA) with a green fluorescent protein (GFP) reporter plasmid failed to identify a single colony that could induce GFP in more than 20% of cells. The presence of chromatin insulator sequences, previously shown to protect some transfected genes from epigenetic silencing, moderately improved stability but was not sufficient to produce homogeneous transformants. However, when cell lines were first established in which selection could be maintained either for the expression of tTA activity (co‐transfection with a tTA‐responsive selectable marker) or the presence of tTA mRNA (bicistronic message encoding a selectable marker), these cell lines could be subsequently transfected with the GFP reporter construct, and nearly 10% of the resulting colonies exhibited stable homogeneous tet‐responsive GFP expression in 100% of the expanded clonal cell population. J. Cell. Biochem. 76:280–289, 1999. © 1999 Wiley‐Liss, Inc.     

Expression of Green Fluorescent Protein Impairs the Force-Generating Ability of Isolated Rat Ventricular Cardiomyocytes

Green fluorescent protein (GFP) is widely used as a biologically inert expression marker for studying the effects of transgene expression in heart tissue, but its influence on the contractile function of cardiomyocytes has not yet been fully evaluated. We measured the contractile function of isolated rat ventricular myocytes before and after infection with a recombinant adenovirus expressing GFP (Adv-GFP). Myocytes infected with a non-transgene-containing adenovirus (Adv-Null) or uninfected myocytes (UI) served as controls. Using a carbon-fiber-based force-length measurement system for single cardiomyocytes, we evaluated the contractile function over a wide range of loading conditions including the shortening fraction (%FS) and maximal shortening velocity (Vmax) under the unloaded condition, and isometric force. At 24 hours after infection, nearly 80% of the Adv-GFP-infected myocytes expressed GFP. We found that the %FS and Vmax did not differ among the three groups, however, the isometric force showed a mild, but significant, decrease only in Adv-GFP myocytes (Adv-GFP: 29.1 ± 4.0 mN/mm²; Adv-Null: 42.8 ± 6.2 mN/mm²; UI: 47.1 ± 4.8 mN/mm²; p = 0.03). An evaluation of the contractile function of isolated cardiomyocytes under high load conditions revealed impaired isometric contractility by GFP expression. Adv-GFP expression may not be an ideal control for specific gene expression experiments in myocardial tissue.     

Construction of an engineered yeast with glucose-inducible emission of green fluorescence from the cell surface

An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface, selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including nutrient concentrations in the media, through the emission of fluorescence. Received: 23 August 1999 / Received revision: 16 November 1999 / Accepted: 29 November 1999     

Polyplex‐mediated gene transfer into human retinal pigment epithelial cells in vitro

The human retinal pigment epithelium (RPE) is a potential target tissue for directed transfer of candidate genes to treat age‐related macular degeneration (AMD). The RPE is uniquely suited to gene therapy protocols that use liposome‐mediated DNA transfer because of its high intrinsic phagocytic function in vivo. In these studies, we examined the efficacy of human RPE cell uptake and expression of the green fluorescent protein (GFP) and neomycin resistance marker genes by polyplex‐mediated gene transfer in vitro. The effects of varying DNA and polyplex concentration and ratios on GFP transgene expression were examined. A narrow range of experimental conditions were found to maximize transgene expression; most important were the DNA concentration and the DNA:polyplex ratio. The transfection efficiency for human RPE cells was reproducibly 20\% in vitro by this method and reached a maximum level of expression after 48 h. There was a rapid decline in gene expression over 2 weeks following polyplex‐mediated gene transfer, but stable integration does occur at low frequencies with and without selection. J. Cell. Biochem. 76:153–160, 1999. © 1999 Wiley‐Liss, Inc.     

Promoters derived from banana bunchy top virus DNA-1 to −5 direct vascular-associated expression in transgenic banana (Musa spp.)

The intergenic regions of banana bunchy top virus (BBTV) DNA-1 to -5 were fused to the green fluorescent protein (GFP) and uidA reporter genes and assessed for promoter activity in transgenic banana (Musa spp. cv. Bluggoe). Promoter activity associated with the BBTV-derived promoters was transgene dependent with greatest activity observed using the GFP reporter. The BBTV promoters (BT1 to BT5) directed expression primarily in vascular-associated cells, although levels of activity varied between individual promoters. Promoters BT4 and BT5 directed the highest levels of GFP expression, while activity from BT1, BT2 and BT3 promoters was considerably weaker. Intron-mediated enhancement, using the maize polyubiquitin 1 (ubi1) intron, generated a significant increase in GUS expression directed by the BBTV promoters in transgenic plants. Received: 17 May 1999 / Revision received: 3 November 1999 / Accepted: 4 November 1999     

Use of the green fluorescent protein as an educational tool

The green fluorescent protein (GFP) is a bioluminescent protein that can be expressed and easily detected as a fully fluorescent protein in both bacterial and eukaryotic cells. These properties, along with its ability to withstand exposure to denaturants, organic solvents, high temperature and a wide pH range, make GFP an ideal educational tool. To that end, two GFP-based laboratory modules are described that can be used to teach recombinant DNA and protein purification techniques to high school and undergraduate college students. Journal of Industrial Microbiology & Biotechnology (2000) 24, 323–326. Received 02 April 1999/ Accepted in revised form 20 November 1999     

GFP as a Genetic Marker Scorable Throughout the Life Cycle of Transgenic Zebra Fish

A fish expression vector, FRM, was constructed by fusing the carp β-actin promoter and first intron to the ocean pout antifreeze protein terminator and putative boundary element. Mutant forms of the green fluorescent protein (GFP) were engineered into this vector, and the resultant series of vectors, FRMwg, FRM3wg (green GFP), and FRM2bl (blue GFP), were used to make transgenic zebra fish. After microinjection of either supercoiled or linearized DNA into one-celled eggs, GFP-expressing cells could be monitored by fluorescence microscopy commencing with the midblastula transition and continuing through embryogenesis. From adult fish, which retained scorable GFP either as patches or as a uniform fluorescence, 11 green and 1 blue GFP-expressing lines of zebra fish have been established. Expression of GFP was nearly ubiquitous and similar among all of these lines. Embryonic expression could be scored at 15 to 30 hours postfertilization and was seen throughout the embryo with the exceptions of the yolk, red blood cells, and in some lines, portions of the head. Adult expression was in a majority of tissues (e.g., muscle, brain, intestine, and heart, but not red blood cells). The notable difference between lines was that fluorescent eggs were scorable in seven of the lines. Adult homozygotes from a different subset of eight lines could be selected by the relative intensity of the GFP marking when compared with that in sibling heterozygotes. All 12 lines contain apparent single locus, multicopy, tandem integrations (1.5–100 copies per cell) of the transgenic DNA. The fish expression vector FRM could be used to drive nearly ubiquitous and strong expression of gene products other than GFP. The GFP expression vectors, FRMwg, FRM2wg, FRM3wg, and FRM2bl, may be useful for optimization of transgenesis and as a comarker. GFP-expressing zebra fish lines could facilitate experimental analysis of chimerism and in vivo gene targeting. Received May 18, 1999; accepted August 26, 1999.     

An efficient gene transfer method mediated by ultrasound and microbubbles into the kidney

BACKGROUND: Safety issues are of paramount importance in clinical human gene therapy. From this point of view, it would be better to develop a novel non-viral efficient gene transfer method. Recently, it was reported that ultrasound exposure could induce cell membrane permeabilization and enhance gene expression. METHODS: In this study, we examined the potential of ultrasound for gene transfer into the kidney. First, we transfected rat left kidney with luciferase plasmid mixed with microbubbles, Optison, to optimize the conditions (duration of ultrasound and concentration of Optison). Then, 4, 7, 14 and 21 days after gene transfer, luciferase activity was measured. Next, localization of gene expression was assessed by measuring luciferase activity and green fluorescent protein (GFP) expression. Expression of GFP plasmid was examined under a fluorescence microscope at 4 and 14 days after gene transfer. Finally, to examine the side effects of this gene transfer method, biochemical assays for aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (Cre) were performed. RESULTS: Optison and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 70-80% of total glomeruli could be transfected. Also, a significant dose-dependent effect of Optison was observed as assessed by luciferase assay (Optison 25%: 12.5 x 10(5) relative light units (RLU)/g tissue; 50%: 31.3 x 10(5) RLU/g tissue; 100%: 57.9 x 10(5) RLU/g tissue). GFP expression could be observed in glomeruli, tubules and interstitial area. Results of blood tests did not change significantly after gene transfer. CONCLUSIONS: Overall, an ultrasound-mediated gene transfer method with Optison enhanced the efficiency of gene transfer and expression in the rat kidney. This novel non-viral method may be useful for gene therapy for renal disease.     

Transformation of floral organs with GFP in Medicago truncatula

 A high frequency of embryogenesis and transformation from all parts of flowers of two lines of Medicago truncatula R-108–1 and Jemalong J5 were obtained. Using this flower system, we obtained transgenic plants expressing promoter-uidA gene fusions as well as the gfp living cell color reporter gene. Moreover, this method allows us to save time and to use a smaller greenhouse surface for the culture of donor plants. Southern hybridization showed that the internal gfp fragment had the expected size and the number of T-DNA copies integrated in the plant genome varied between one and three. These data suggest that the presence of the GFP protein has no toxic effects, since no rearrangement of the gfp reporter gene was detected in the regenerated plants. Received: 25 May 1999 / Revision received: 2 August 1999 / Accepted: 2 August 1999     

pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves

We have constructed a matched set of binary vectors designated pGD, pGDG and pGDR for the expression and co-localization of native proteins and GFP or DsRed fusions in large numbers of plant cells. The utility of these vectors following agroinfiltration into leaves has been demonstrated with four genes from Sonchus yellow net virus, a plant nucleorhabdovirus, and with a nucleolar marker protein. Of the three SYNV proteins tested, sc4 gave identical localization patterns at the cell wall and nucleus when fused to GFP or DsRed. However, some differences in expression patterns were observed depending on whether DsRed or GFP was the fusion partner. In this regard, the DsRed:P fusion showed a similar pattern of localization to GFP:P, but localized foci appeared in the nucleus and near the periphery of the nucleus. Nevertheless, the viral nucleocapsid protein, expressed as a GFP:N fusion, co-localized with DsRed:P in a subnuclear locale in agreement with our previous observations (Goodin et al., 2001). This locale appears to be distinct from the nucleolus as indicated by co-expression of the N protein, DsRed:P and a nucleolar marker AtFib1 fused to GFP. The SYNV M protein, which is believed to be particularly prone to oligomerization, was detectable only as a GFP fusion. Our results indicate that agroinfiltration with bacteria containing the pGD vectors is extremely useful for transient expression of several proteins in a high proportion of the cells of Nicotiana benthamiana leaves. The GFP and DsRed elements incorporated into the pGD system should greatly increase the ease of visualizing co-localization and interactions of proteins in a variety of experimental dicotyledonous hosts.     

An assay system to study migratory behavior of cranial neural crest cells in Xenopus

An increasing number of genes are known to show expression in the cranial neural crest area. So far it is very difficult to analyze their effect on neural crest cell migration because of the lack of transplantation techniques. This paper presents a simple method to study the migratory behavior of cranial neural crest cells by homo- and heterotopic transplantations: Green fluorescent protein (GFP) RNA was injected into one blastomere of Xenopus laevis embryos at the 2-cell stage. The cranial neural crest area of stage 14 embryos was transplanted into the head or trunk region of an uninjected host embryo, and the migration was monitored by GFP fluorescence. The transplants were further examined by double immunostaining and confocal microscopy to trace migratory routes inside the embryo, and to exclude contaminations of grafts with foreign tissues. Our results demonstrate that we developed a highly efficient and reproducible technique to study the migratory ability of cranial neural crest cells. It offers the possibility to analyze genes involved in neural crest cell migration by coinjecting their RNA with that of GFP. Received: 28 September 1999 / Accepted: 17 November 1999     

Microinjection of mRNA encoding rat synapsin Ia alters synaptic physiology in identified motoneurons of the crayfish,Procambarus clarkii

Studies of identified neurons have made important contributions to our understanding of cellular neurophysiology. We have developed a technique for modifying gene expression in identified motoneurons of the crayfish Procambarus clarkii in the isolated nervous system as well as in the intact animal through the injection of exogenously synthesized RNAs. mRNA suitable for injection was transcribed in vitro from cDNA templates cloned into a plasmid, pSEM. Initially, mRNAs encoding green fluorescent protein (GFP) and β-galactosidase were injected into the soma of the motor giant neuron (MoG) to determine whether these mRNAs could be successfully translated into protein. Both proteins were expressed. Measurements of GFP fluorescence increase indicated that GFP mRNA was stable and translated into protein for at least 3 days postinjection. We then examined the effects of expression of GFP, AASP-168 (an endogenous crayfish axonal protein), and rat synapsin Ia on MoG synaptic physiology. The mRNA injection procedure did not appear to directly influence synaptic physiology based on the results of the AASP-168 and GFP injections. Injection of mRNA encoding rat synapsin Ia resulted in a significant increase in peak excitatory postsynaptic potential (EPSP) amplitude during repetitive stimulation. These data are consistent with previous studies that have shown that synapsin deficiency reduces synaptic vesicle numbers. The translation of mRNAs with diverse functions and species of origin suggests that this approach will prove useful for studying the function of a wide variety of endogenous and exogenous genes in identified neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 224–236, 1998     

脉冲电泳不同参数对介导GFP基因转化玉米种胚效果的研究

利用脉冲电泳介导绿色荧光蛋白(GFP)基因导入玉米种胚;以GFP基因在种胚中瞬时表达作为外源基因导人种胚细胞的标记,分析了外源．DNA浓度、电泳时间、电压、电流转换时间等脉冲电泳转化参数对种胚发芽率和外源基因导入率的影响。结果表明：脉冲电泳时间对种胚发芽率和外源GFP基因导入率影响最大;通过脉冲电泳可将外源基因导入胚芽细胞,其GFP基因导人种子的频率与各电泳参数均呈二次曲线关系,300μg／ml外源DNA浓度、120min电泳时间、5V电压、2s电流转换时间可作为脉冲电泳介导玉米种胚转化较适宜的参数。     

Low‐intensity microwave irradiation does not substantially alter gene expression in late larval and adult Caenorhabditis elegans

Reports that low‐intensity microwave radiation induces heat‐shock reporter gene expression in the nematode, Caenorhabditis elegans, have recently been reinterpreted as a subtle thermal effect caused by slight heating. This study used a microwave exposure system (1.0 GHz, 0.5 W power input; SAR 0.9–3 mW kg^?1 for 6‐well plates) that minimises temperature differentials between sham and exposed conditions (≤0.1 °C). Parallel measurement and simulation studies of SAR distribution within this exposure system are presented. We compared five Affymetrix gene arrays of pooled triplicate RNA populations from sham‐exposed L4/adult worms against five gene arrays of pooled RNA from microwave‐exposed worms (taken from the same source population in each run). No genes showed consistent expression changes across all five comparisons, and all expression changes appeared modest after normalisation (≤40% up‐ or down‐regulated). The number of statistically significant differences in gene expression (846) was less than the false‐positive rate expected by chance (1131). We conclude that the pattern of gene expression in L4/adult C. elegans is substantially unaffected by low‐intensity microwave radiation; the minor changes observed in this study could well be false positives. As a positive control, we compared RNA samples from N2 worms subjected to a mild heat‐shock treatment (30 °C) against controls at 26 °C (two gene arrays per condition). As expected, heat‐shock genes are strongly up‐regulated at 30 °C, particularly an hsp‐70 family member (C12C8.1) and hsp‐16.2. Under these heat‐shock conditions, we confirmed that an hsp‐16.2::GFP transgene was strongly up‐regulated, whereas two non‐heat‐inducible transgenes (daf‐16::GFP; cyp‐34A9::GFP) showed little change in expression. Bioelectromagnetics 30:602–612, 2009. © 2009 Wiley‐Liss, Inc.     

Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection

The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex-pression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.