Transient Expression Of Green Fluorescent Protein Research Articles

Effects of different light conditions on transient expression and biomass in Nicotiana benthamiana leaves.

In the process of the production of recombinant proteins by using an Agrobacterium-mediated transient gene expression system, the effectiveness of the control of light conditions pre- and post-agroinfiltration on efficiency of transient expression is worth being evaluated. In this study, Nicotiana benthamiana plants were used as a bioreactor to investigate the effects of different light conditions pre- and post-agroinfiltration on the transient expression of green fluorescent protein (GFP). The results showed that the plants grown under light condition for 5 weeks had the highest level of transient expression among those grown for 4-8 weeks. In the pre-agroinfiltration, the level of transient expression of GFP was obviously decreased by the increase in light intensity or by the shortening of the photoperiod. Although the shortening of the photoperiod post-agroinfiltration also decreased the level of transient expression, moderate light intensity post-agroinfiltration was needed for higher level of transient expression efficiency. However, there was no strong correlation between the transient expression efficiency and plant growth. The results suggested that light condition was an important factor affecting the level of transient expression in plants. Hence, light conditions should be optimized to obtain higher productivity of recombinant protein from transient expression systems.

Overexpression of a PIP1 Gene from Salicornia bigelovii in Tobacco Plants Improves Their Drought Tolerance

Aquaporin (AQP) proteins can transport water and other small molecules through cellular membranes and are one of the first targets of stress-induced signaling in plants. A number of AQP genes have been identified from glycophytes, and their functions have been studied. However, the reports on AQPs from halophytes and their precise role in abiotic stress response are still rare. In this study, we have identified a PIP1 subgroup AQP gene, designated SbPIP1, from the euhalophyte Salicornia bigelovii and characterized it by overexpressing in tobacco plants. SbPIP1 transcript was induced by cold, but suppressed by NaCl and polyethylene glycol (PEG). Transient expression of GFP (green fluorescent protein)-SbPIP fusion protein indicated its localization in the plasma membrane. Overexpression of SbPIP1 in tobacco (Nicotiana tabacum) plants increased their drought tolerance. Leaf protoplasts from transgenic tobacco plants absorbed water more quickly than those from wild type (WT) plants when they were put into hypotonic solution. In addition, the transgenic tobacco plants possessed higher relative water content (RWC) and proline content, but lower levels of malondialdehyde (MDA) and less ion leakage (IL) when compared with WT under the treatment of the different concentrations of PEG. Taken together, our results demonstrate that heterologous expression of SbPIP1 in tobacco plants confers them drought stress tolerance by reducing membrane injury and increasing the ability to retain water.

Transient expression of green fluorescent protein in parasitic dodder as a tool for studying of cytoskeleton

AbstractDodder (Cuscuta) species cause severe agricultural damage in many countries throughout the world. To establish strategies for control of its growth and spreading it is important to study its life cycle and survival strategies. For these efforts genetic modification would represent a powerful tool. Here we report on Agrobacteriummediated transformation of dodder using green fluorescent protein (GFP) fused to actin-binding protein as a vital marker. Since the shoot of germinating C. europaea contains a functional apical meristem and grows quickly comparing to the root-like structure, the shoot apex was used here as explant. The transgene expression was only transient, nevertheless it enabled to detect allocation of actin filaments and studying the cytoskeleton organization in dodder shoot apex. Transient expression of GFP appears to be a suitable method for studying Cuscuta development through cytoskeleton organisation that is presently largely unexplored.

Large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L.

Transient expression of foreign genes by Agrobacterium infiltration is a versatile technique that can be used as a rapid tool for functional protein production in plants. A reproducible protocol of large-scale production of foreign proteins via the novel plant transient expression system in Pisum sativum L. was established in our study. Non-detached plants from soil-independent culture were used as the target organ, and vacuum infiltrating mediated by Agrobacterium tumefaciens harboring green fluorescent protein (GFP) gene was performed. Step-by-step optimization was performed and showed that the quality of plant material as well as agro-infiltration conditions were the major factors influencing the gene expression. Monitoring the transient GFP expression daily, the highest expression level was achieved on the 8th day post-infiltration. Evidence of anti-acidic fibroblast growth factor-single chain variable fragment (anti-aFGF-scFv) gene expression in pea seedling was also achieved using agro-mediated vacuum infiltration system. Our work proves that the system is suitable for the largescale production of pharmaceutical proteins. The in planta infiltration system described here provides a powerful tool to explore easily gene expression in Pisum sativum L. avoiding tissue culture steps and the labor-intensive generation of transgenic plants.

Generation of BAC transgenic tadpoles enabling live imaging of motoneurons by using the urotensin II-related peptide (ust2b) gene as a driver.

Xenopus is an excellent tetrapod model for studying normal and pathological motoneuron ontogeny due to its developmental morpho-physiological advantages. In mammals, the urotensin II-related peptide (UTS2B) gene is primarily expressed in motoneurons of the brainstem and the spinal cord. Here, we show that this expression pattern was conserved in Xenopus and established during the early embryonic development, starting at the early tailbud stage. In late tadpole stage, uts2b mRNA was detected both in the hindbrain and in the spinal cord. Spinal uts2b+ cells were identified as axial motoneurons. In adult, however, the uts2b expression was only detected in the hindbrain. We assessed the ability of the uts2b promoter to drive the expression of a fluorescent reporter in motoneurons by recombineering a green fluorescent protein (GFP) into a bacterial artificial chromosome (BAC) clone containing the entire X. tropicalis uts2b locus. After injection of this construction in one-cell stage embryos, a transient GFP expression was observed in the spinal cord of about a quarter of the resulting animals from the early tailbud stage and up to juveniles. The GFP expression pattern was globally consistent with that of the endogenous uts2b in the spinal cord but no fluorescence was observed in the brainstem. A combination of histological and electrophysiological approaches was employed to further characterize the GFP+ cells in the larvae. More than 98% of the GFP+ cells expressed choline acetyltransferase, while their projections were co-localized with α-bungarotoxin labeling. When tail myotomes were injected with rhodamine dextran amine crystals, numerous double-stained GFP+ cells were observed. In addition, intracellular electrophysiological recordings of GFP+ neurons revealed locomotion-related rhythmic discharge patterns during fictive swimming. Taken together our results provide evidence that uts2b is an appropriate driver to express reporter genes in larval motoneurons of the Xenopus spinal cord.

Optimization of particle bombardment parameters for DNA delivery into the male flowers of banana

The possibility of increasing the efficiency of banana transformation was investigated by particle bombardment of the male flowers of banana plants for constitutive expression of gfp gene. The effects of particle bombardment parameters, such as acceleration pressure, bombardment distance, chamber vacuum pressure, gold microcarrier size, gold quantity, DNA quantity, number of bombardments and pre-culture were examined. Single cauliflower-like bodies (CLBs) clusters, induced from meristemic parts of Musa sapientum cv. Nangka (AAB) male flowers, were bombarded by pCambia1304 plasmid carrying gfp gene driven by the CaMV 35S promoter. Optimal transient expression of green-fluorescent protein (GFP) was obtained when the three-day old cultured tissues were bombarded two times at 1100 psi helium pressure. However, the highest GFP expression was observed when 9 cm was applied as bombardment distance with 28 mmHg chamber vacuum pressure. Gold particle with 1 μm diameter at 60 μg/μL concentrations coated with 1.5 μg/μL of DNA have been used as the optimum bombardment parameter since GFP expression was significantly different compared to other conditions. Application of optimized condition proved effective for the generation of stable transgenic banana plants. PCR and southern blot analyses confirmed the presence and integration of gfp gene in genomic DNA of transformed plants. Transformation frequency achieved with the optimized protocol was 7.5% which was significantly higher than the conventional protocol.

In planta agro-infiltration system for transient gene expression in grapevine (Vitis spp.)

Transient expression of foreign genes by Agrobacterium infiltration is a versatile technique that can be used as a rapid tool for functional analysis and gene silencing studies in plants. A reproducible protocol of Agrobacterium-mediated transient gene transfer was developed for gene expression analysis on greenhouse-grown grapevines, as a complementary approach towards functional genomics and alternative to transgenics. Non-detached leaves from green cuttings were used as the target organ and vacuum infiltrated for in planta inoculation with Agrobacterium tumefaciens harboring mgfp5-ER gene construct as visual reporter gene. Step-by-step optimization was performed and showed that the quality of greenhouse material as well as agro-infiltration conditions were the major factors which influenced successful gene expression assays. Following the optimized protocol, up to half of the infiltrated leaf surface displayed green fluorescent foci found in the intercoastal areas. Monitoring of transient Green Fluorescent Protein expression daily achieved for 2 weeks post-infiltration with the highest expression level on day 6. Evidence of GFP silencing in transgenic GFP-expressing grapevine via agro-infiltration was found for the first time. The in planta infiltration system described here provides a powerful tool to explore easily gene function in grapevine avoiding tissue culture steps and the labor-intensive generation of transgenic plants.

Efficient somatic embryogenesis and Agrobacterium-mediated transformation of pothos (Epipremnum aureum) ‘Jade’

Leaf and petiole explants of monocotyledonous pothos (Epipremnum aureum) ‘Jade’ were cultured on Murashige and Skoog basal medium supplemented with N-(2-chloro-4-pyridl)-N′-phenylurea (CPPU) or N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ) with α-naphthalene acetic acid (NAA). Somatic embryos appeared directly from explants after 4–8 weeks of culture; 9.1 μM TDZ with 1.1 μM NAA induced 61.1 % leaf discs and 94.4 % of petiole segments to produce plantlets through embryo conversion. Using this established regeneration method and an enhanced green fluorescent protein (GFP) gene (egfp) as a reporter marker, an Agrobacterium-mediated transformation procedure was developed. Leaf discs and petiole segments were inoculated with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pLC902 that contains novel bi-directional duplex promoters driving the egfp gene and hygromycin phosphotransferase gene (hpt), respectively. The explants were co-cultivated with strain EHA105 for 3, 5, and 7 days, respectively prior to selective culture with 25 mg l−1 hygromycin. A 5-day co-cultivation led to 100 % of leaf discs to show transient GFP expression and 23.8 % of the discs to produce stable GFP-expressing somatic embryos. A 7-day co-cultivation of petiole explants resulted in the corresponding responses at 100 and 14.3 %, respectively. A total of 237 transgenic plants were obtained, and GFP fluorescence was observed in all plant organs. Regular PCR and quantitative real-time PCR analyses confirmed the presence of 1 or 2 copies of the egfp gene in analyzed plants. The highly efficient regeneration and transformation systems established in this study may enable genetic improvement of this vegetatively propagated species through biotechnological means.

Virus-induced gene silencing (VIGS) of genes expressed in root, leaf, and meiotic tissues of wheat

Barley stripe mosaic virus (BSMV)-based virus-induced gene silencing (VIGS) is an effective strategy for rapid functional analysis of genes in wheat leaves, but its utility to transiently express genes, and silencing in other tissues including root, flower, and developing grains, has not been demonstrated in monocots. We monitored green fluorescent protein (GFP) expression to demonstrate the utility of BSMV as a transient expression vector and silenced genes in various wheat tissues to expand VIGS utility to characterize tissue-specific genes. An antisense construct designed for coronatine insensitive1 (COI1) showed an 85% decrease in COI1 transcript level in roots accompanied by a 26% reduction in root length. Similarly, silencing of seed-specific granule-bound starch synthase by antisense and hairpin constructs resulted in up to 82% reduction in amylose content of the developing grains. VIGS of meiosis-specific genes demonstrated by silencing wheat homologue of disrupted meiosis cDNA1 (DMC1) by an antisense construct resulted in a 75-80% reduction in DMC1 transcript level accompanied by an average of 37.2 univalents at metaphase I. The virus-based transient GFP expression was observed in the leaf, phloem, and root cortex at 10-17days post-inoculation. A novel observation was made that 8-11% of the first selfed generation progeny showed VIGS inheritance and that this proportion increased to 53-72% in the second and to 90-100% in the third generations. No viral symptoms were observed in the progeny, making it possible to study agronomic traits by VIGS. VIGS inheritance is particularly useful to study genes expressing during seed germination or other stages of early plant growth.

Visualization of plant viral suppressor silencing activity in intact leaf lamina by quantitative fluorescent imaging

BackgroundTransient expression of proteins in plants has become a favoured method over the production of stably transformed plants because, in addition to enabling high protein yields, it is both fast and easy to apply. An enhancement of transient protein expression can be achieved by plant virus-encoded RNA silencing suppressor proteins. Since viral suppressor proteins differ in their efficiency to enhance transient protein expression in plants, we developed a whole-leaf green fluorescent protein (GFP)-based imaging assay to quantitatively assess suppressor protein activity.ResultsIn a transient GFP-expression assay using wild-type and GFP-transgenic N. benthamiana, addition of the plant viral suppressors Beet mild yellowing virus (BMYV-IPP) P0 or Plum pox virus (PPV) HC-Pro was shown to increase fluorescent protein expression 3-4-fold, 7 days post inoculation (dpi) when compared to control plants. In contrast, in agroinfiltrated patches without suppressor activity, near complete silencing of the GFP transgene was observed in the transgenic N. benthamiana at 21 dpi. Both co-infiltrated suppressors significantly enhanced GFP expression over time, with HC-Pro co-infiltrations leading to higher short term GFP fluorescence (at 7 dpi) and P0 giving higher long term GFP fluorescence (at 21 dpi). Additionally, in contrast to HC-Pro co-infiltrations, an area of complete GFP silencing was observed at the edge of P0 co-infiltrated areas.ConclusionsFluorescence imaging of whole intact leaves proved to be an easy and effective method for spatially and quantitatively observing viral suppressor efficiency in plants. This suppressor assay demonstrates that plant viral suppressors greatly enhanced transient GFP expression, with P0 showing a more prolonged suppressor activity over time than HC-Pro. Both suppressors could prove to be ideal candidates for enhancing target protein expression in plants.

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of Pelargonium × hortorum, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium x hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-microF capacitor in a 250-V cm(-1) electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33-36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 microS cm(-1) allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l(-1) kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l(-1) kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.

Transient transfection of Cryptosporidium parvum using green fluorescent protein (GFP) as a marker

Cryptosporidium parvum is a protozoan parasite that infects a variety of mammals. The parasite has been shown to harbor a dsRNA virus (CPV) and in the present study, we have developed a CPV transient transfection system for this parasite by using green fluorescent protein (GFP) to replace the partial gene encoding region of the larger dsRNA (CPV-L) and the smaller dsRNA (CPV-S) virus. Two viral RNA-mediated transfection vectors: pCPVL-GFP and pCPVS-GFP were successfully constructed and both in vitro transcripts were electroporated into oocysts and sporozoites. Transient expression of GFP was detected in C. parvum oocysts and excysted sporozoites by fluorescence microscopy and by RT-PCR detection of GFP mRNA and antisense RNA in transfected C. parvum oocysts. Our study provides a new approach for studying gene expression and regulation in C. parvum and will hopefully lead to the construction of a stable CPV transfection system in the future.

Transformation of isolated barley (Hordeum vulgareL.) microspores: II. Timing of pretreatment and temperatures relative to results of bombardment

Based on paper I in this series, our goals in this paper were to determine the relationship between prebombardment pretreatments and temperatures, microspore cell cycle when bombarded, and the frequencies of homozygous and hemizygous transgenic progeny in barley (Hordeum vulgare L.). Of the 104 fluorescent plants selected when using the GFP fluorescence transgene, 28 were albino and 76 plants were green. Thirty-one green plants were confirmed to be transgenic; the others were either transient green fluorescent protein expression or selected due to autofluorescence. Of the 31 plants, 23 came from embryos expressing a high level of fluorescence during selection and eight from 51 plants exhibiting a low level of fluorescence. Of the two pretreatments used to induce embryogenesis, 24 of 31 plants were from the cold pretreatment for 21 days (C) versus seven from the 4 day cold plus mannitol pretreatment. Following pretreatment, the microspores were subjected to a high-osmotic period (0.5 mol/L mannitol plus sorbitol) of 4 h prebombardment and 18 h postbombardment at either 25 or 4 degrees C. Of the 31 transgenic plants, 19 were produced following the 25 degrees C 4 h prebombardment. Sixteen of the 19 were doubled haploid plants (seven being homozygous for the transgene) and the other three plants were haploid. Of the remaining 12 plants recovered following the 4 h 4 degrees C prebombardment treatment, nine were haploid and three were doubled haploid plants, two of the latter being homozygous for the transgene. All 12 haploid plants obtained were treated with colchicine and produced homozygous transgenic doubled haploids. Of the two promoters compared, 30 plants had the actin promoter and only one had the 35S promoter. The use of arabinogalactan protein in the culture medium was very beneficial, giving rise to 29 of the 31 plants. The best procedure for obtaining transgenic barley plants from this study was pretreatment C, leaving the cultures at either 4 or 25 degrees C during the 4 h prebombardment high-osmotic period, using the actin promoter and having arabinogalactan protein in the microspore culture medium. With this procedure, the transgenic frequency was improved 8- to10-fold over previous reports on bombardment of microspores. It yielded about one transgenic plant per Petri dish and is comparable with Agrobacterium frequencies on structures derived from microspores.

Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.

Structural basis for RNA‐silencing suppression by Tomato aspermy virus protein 2b

The 2b proteins encoded by cucumovirus act as post-transcriptional gene silencing suppressors to counter host defence during infection. Here we report the crystal structure of Tomato aspermy virus 2b (TAV2b) protein bound to a 19 bp small interfering RNA (siRNA) duplex. TAV2b adopts an all alpha-helix structure and forms a homodimer to measure siRNA duplex in a length-preference mode. TAV2b has a pair of hook-like structures to recognize simultaneously two alpha-helical turns of A-form RNA duplex by fitting its alpha-helix backbone into two adjacent major grooves of siRNA duplex. The conserved pi-stackings between tryptophan and the 5'-terminal base of siRNA duplex from both ends enhance the recognition. TAV2b further oligomerizes to form a dimer of dimers through the conserved leucine-zipper-like motif at its amino-terminal alpha-helix. Biochemical experiments suggest that TAV2b might interfere with the post-transcriptional gene silencing pathway by directly binding to siRNA duplex.

Quantification and extension of transient GFP expression by the co-introduction of a suppressor of silencing

Using particle bombardment, a DNA expression vector containing the green fluorescent protein (GFP) reporter gene was introduced into plant cells. Expression of the GFP gene was transient; resulting in peak GFP Expression about 24 h post introduction and a rapid decline thereafter. This well known decline in gene expression has previously been attributed to pre-integrative DNA events that involved the loss of introduced DNA or cell death. Here, we show that post-transcriptional gene silencing (PTGS) is also involved. Introduction of a GFP expression vector alone resulted in a rapid decline in transient expression after 30 h. However, if GFP was expressed as a translational fusion to the RNA silencing suppressor protein HCPro from tobacco etch potyvirus, transgene expression was extended to well over 100 h. Mutant analyses of HCPro showed that a functional HCPro protein was required for this extension of transient expression. Various deletion and translational fusion analyses confirmed that the C-terminal region of the protein was important for suppressor activity and the entire protein was required for optimal suppression of host silencing. The transient nature of gene expression during particle bombardment appears to result from induction of PTGS, which can be mitigated by the presence of a suppressor of silencing. The use of RNA silencing suppressor proteins may make particle bombardment-mediated transient expression assays more useful for evaluating factors that effect gene expression.

Overexpression of barley hva1 gene in creeping bentgrass for improving drought tolerance

The objectives of this study were to test the feasibility of introducing barley hva1 gene, a LEA3 member, into perennial grass species using the Agrobacterium-mediated transformation technique and to determine whether heterologous expression of hva1 would alleviate water-deficit injury in grass species. Creeping bentgrass (Agrostis stolonifera var. palustris), a drought-intolerant grass species, was transformed transiently or stably using three different promoters in conjunction with the downstream report/target genes. Two abscisic acid (ABA)-inducible promoters, ABA1 and ABA2 derived from ABA-response complex (ABRC3) were used to examine stress-responsive expression of the green fluorescent protein (GFP). Transient expression of GFP demonstrated the inducibility of ABA1 and ABA2 promoters in response to exogenous ABA application. The ABA2 promoter was further studied for stress-responsive expression of hva1 and a maize Ubi-1 promoter was tested for constitutive expression of the gene. In the T(0) generation, the Ubi-1::hva1 transformants displayed variable expression levels of HVA1 protein under normal growth conditions. The hva1 gene in the ABA2::hva1 transformants maintained low expression under well-watered conditions, but was upregulated under water-deficit conditions. The tolerance to water deficit of T(0) transgenic lines was assessed by measuring leaf relative water content and visually rating the severity of leaf wilting during to water stress. Under water-stressed conditions, some transgenic lines maintained high water content in leaves and showed significantly less extent of leaf wilting compared with non-transgenic control plants. These results indicated that the introduction of barley hva1 gene using constitutive or stress-inducible promoters lessened water-deficit injury in creeping bentgrass, suggesting that heterologous expression of LEA3 protein genes may enhance the survival ability of creeping bentgrass in water limiting environments.

Preparation of an Endosperm Protoplasts from a Dwarf Rice Variety and Transient Expression of Green-Fluorescent Protein

Vital protoplasts were isolated from developing dwarf rice endosperms about 10 days post anthesis. A yield of 0.98x105±0.12 protoplasts was obtained from 10 developing grains. These protoplasts were viable for at least three days in the culture medium. Transient expression of the green-fluorescent protein (GFP) was detected after four hours of introduction of the GFP gene by the polyethylene glycol (PEG)-mediated DNA transfer. As expected, the fluorescence of GFP without the targeting sequence was observed diffusely in the cytoplasm. On the other hand, the fluorescence of the fusion protein for rice prolamin followed by the GFP was observed in the small particles and the ER-network. These endosperm protoplasts had no autofluorescence upon excitation with 490 nm (blue) light. The vital protoplasts obtained from this dwarf rice variety are very useful for the physiological, biochemical and molecular biology studies of rice endosperm.

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.

Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape ( Vitis vinifera)

Activity of three constitutive promoters and enhanced derivatives in transgenic grape ( Vitis vinifera L. cv. Thompson Seedless) was characterized using a bifunctional fusion marker containing the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTII) genes. Relative differences in transient GFP expression and stable transformation efficiencies were used to compare promoter activity. Expression patterns in transformed somatic embryos revealed that the ACT2 promoter from Arabidopsis thaliana, previously shown to be a strong constitutive promoter in A. thaliana and other species, failed to promote strong expression in grape. In contrast, a promoter isolated from cassava vein mosaic virus (CsVMV) supported high levels of transgene expression equivalent to those achieved using an enhanced double cauliflower mosaic virus (CaMV) 35S promoter. Duplication of the 5′-upstream enhancer region of the CsVMV promoter further enhanced its ability to increase transgene expression. However, the pattern of transgene expression driven by these two viral promoters was significantly different at the whole plant level. The enhanced double CaMV 35S promoter was highly active in most tissues and organs including roots, mature leaves, shoot apices and lateral buds. In contrast, the CsVMV promoter and its double enhancer derivative induced relatively weak expression in these tissues. Our results suggest that activity of the CsVMV promoter, in contrast to the CaMV 35S promoter, was under developmental regulation in transgenic grape plants as compared with the CaMV 35S promoter.