Green Fluorescent Protein Marker Research Articles

Antifungal and Root Surface Colonization Properties of GFP-Tagged Paenibacillus brasilensis PB177

This study evaluates the potential of Paenibacillus brasilensis strain PB177 to inhibit phytopathogenic fungi commonly causing maize diseases and to colonize maize plants. In vitro assays demonstrated antagonistic activity against the fungal pathogens, Fusarium moniliforme and Diplodia macrospora. The PB177 strain was tagged with the gfp gene, encoding the green fluorescent protein (GFP) and GFP-tagged bacteria were detected attached to maize roots by stereo- and confocal microscopy. The GFP-tagged bacteria were also used to treat maize seeds before challenging the seeds with two phytopathogenic fungi. The results demonstrated that the bacterial cells are mobilized to the maize roots in the presence of the fungal pathogens. The ability of P. brasilensis PB177 to inhibit fungal growth in vitro and its capability of colonization of maize roots in vivo suggest a potential application of this strain as a biological control agent. This is the first report on the successful introduction of the GFP marker gene into a P. brasilensis strain, enabling the direct observation of these promising plant growth promoting bacteria on maize roots in situ.

Identification of a mitochondrial ATP synthase small subunit gene (RMtATP6) expressed in response to salts and osmotic stresses in rice (Oryza sativa L.)

Large areas of northern China have alkaline soil due to the accumulation of sodium carbonates (NaHCO3, Na2CO3). To understand better how plants can tolerate alkaline soil, a cDNA library was prepared from rice (Oryza sativa L.) roots grown in the presence of NaHCO3 stress. A cDNA clone isolated from this library was identified by a homology search as a mitochondrial ATP synthase 6 kDa subunit gene (RMtATP6; GenBank accession nos AB055076, BAB21526). In transformed yeast and tobacco protoplasts, the RMtATP6 protein was localized in mitochondria using the green fluorescent protein (GFP) marker. Analysis of RMtATP6 mRNA levels suggested that the expression of this gene was induced by stress from sodium carbonates and other sodium salts. Transgenic tobacco overexpressing the RMtATP6 gene had greater tolerance to salt stress at the seedling stage than untransformed tobacco. Among the other genes for F1F0-ATPase of rice, some were found to be up-regulated by some environmental stresses and some were not. These data suggest that the RMtATP6 protein acts as a subunit of ATP synthase, and is expressed in response to stress from several salts, with the other genes coding for the subunits of the same ATP-synthase.

Use of a green fluorescent protein marker for studying splash dispersal of sporangia of Phytophthora infestans

The purpose of this study was to develop a new technique to evaluate the number of spores incorporated in splash droplets by the use of an engineered fluorescent pathogen strain and image analysis hardware and software. The inoculum source consisted of a tomato leaflet infected with a Phytophthora infestans strain transformed with the gene encoding the green fluorescent protein (GFP). Splash droplets formed after impact of incident drops on sporulating lesions were collected on microscope slides located at different distances from the source. Each slide was examined using a fluorescent microscope to visualize the GFP expressing sporangia. Photographs were taken and assessed using image processing to count the sporangia incorporated in each droplet. Data analysis confirmed characteristics of splash dispersal shown using other methods. The use of fluorescent sporangia also facilitates the selective detection and counting of viable (living) sporangia, and is a tool that can be use in the study of splash dispersed diseases.

The engraftment of transplanted bone marrow-derived cells into the olfactory epithelium

To investigate whether bone marrow cells migrate and are engrafted into the olfactory epithelium and differentiate into olfactory neurons, bone marrow cells of green fluorescence protein (GFP) mice were transplanted into lethally irradiated recipient mice. Immunohistochemical staining was performed to evaluate the engraftment of donor bone marrow cells into the olfactory epithelium. Immunostaining for GFP was found initially in the olfactory epithelium 2 weeks after bone marrow reconstruction. The percentage of GFP positive cells increased up to 12 months after bone marrow reconstruction. Double staining for GFP and olfactory marker protein showed that a population of the GFP-positive cells had characteristics of olfactory neurons. These results demonstrate that bone marrow cells can be engrafted in the olfactory epithelium and then differentiate into olfactory neuron cells.

A Rab-E GTPase mutant acts downstream of the Rab-D subclass in biosynthetic membrane traffic to the plasma membrane in tobacco leaf epidermis.

The function of the Rab-E subclass of plant Rab GTPases in membrane traffic was investigated using a dominant-inhibitory mutant (RAB-E1(d)[NI]) of Arabidopsis thaliana RAB-E1(d) and in vivo imaging approaches that have been used to characterize similar mutants in the plant Rab-D2 and Rab-F2 subclasses. RAB-E1(d)[NI] inhibited the transport of a secreted green fluorescent protein marker, secGFP, but in contrast with dominant-inhibitory RAB-D2 or RAB-F2 mutants, it did not affect the transport of Golgi or vacuolar markers. Quantitative imaging revealed that RAB-E1(d)[NI] caused less intracellular secGFP accumulation than RAB-D2(a)[NI], a dominant-inhibitory mutant of a member of the Arabidopsis Rab-D2 subclass. Furthermore, whereas RAB-D2(a)[NI] caused secGFP to accumulate exclusively in the endoplasmic reticulum, RAB-E1(d)[NI] caused secGFP to accumulate additionally in the Golgi apparatus and a prevacuolar compartment that could be labeled by FM4-64 and yellow fluorescent protein (YFP)-tagged Arabidopsis RAB-F2(b). Using the vacuolar protease inhibitor E64-d, it was shown that some secGFP was transported to the vacuole in control cells and in the presence of RAB-E1(d)[NI]. Consistent with the hypothesis that secGFP carries a weak vacuolar-sorting determinant, it was shown that a secreted form of DsRed reaches the apoplast without appearing in the prevacuolar compartment. When fused to RAB-E1(d), YFP was targeted specifically to the Golgi via a saturable nucleotide- and prenylation-dependent mechanism but was never observed on the prevacuolar compartment. We propose that RAB-E1(d)[NI] inhibits the secretory pathway at or after the Golgi, causing an accumulation of secGFP in the upstream compartments and an increase in the quantity of secGFP that enters the vacuolar pathway.

Enhancer trapping with a red fluorescent protein reporter in Drosophila

Enhancer trapping allows the detection of genomic transcriptional enhancers and provides a rich source of cell- and region-specific markers in Drosophila. We report here the development of a P-element-based enhancer-trap vector with a nuclear red fluorescent protein (DsRed) reporter for enhancer detection. We demonstrate that this vector can be used for a standard enhancer-trap screen as well as for targeted replacement of previously characterized P-element insertions. We isolated DsRed insertion strains of hedgehog, patched, apterous, teashirt, and dachshund that label specific regions of imaginal discs. The time required for red fluorescence maturation in the eye imaginal disc was estimated to be 22.5 hr. The DsRed markers can be combined with green fluorescent protein markers for double labeling of living Drosophila tissues.

Somatic hybrid vigor in Citrus: Direct evidence from protoplast fusion of an embryogenic callus line with a transgenic mesophyll parent expressing the GFP gene

Previous experience with citrus somatic fusion suggests that hybrid cells are generally more vigorous and have higher capacity for embryogenesis, compared to unfused cells from the embryogenic callus parent. However, this observation has never been validated due to the unavailability of an appropriate experimental design. Citrus mesophyll protoplasts never divide and regenerate into plants. Herein, mesophyll protoplasts of transgenic Valencia orange ( Citrus sinensis) which expressed the green fluorescent protein (GFP) gene were fused with protoplasts isolated from an embryogenic callus line of Shekwasha mandarin ( C. reticulata) to visually screen somatic hybrids. At the multi-cell cluster stage, the regenerated callus lines expressing GFP were morphologically identical to the callus parent regenerate. All hybrid callus lines expressing the GFP developed into embryoids, while most callus parent regenerates still remained in a callus stage. Embryoids with strong or weak GFP expression were verified to be diploid cybrid and tetraploid somatic hybrid embryoids, respectively, by flow cytometry, simple sequence repeat (SSR) and chloroplast SSR analyses. Diploid cybrid globular embryoids appeared light green in color and had a tendency to enter subsequent developmental stages earlier than the dark green tetraploids; however, GFP plants were regenerated from both sources. Thus, expression of the GFP marker gene was effectively used to visually screen somatic hybrids, and confirmed our previous observation that somatic hybrid tissue has a regeneration advantage. The intensity of GFP fluorescence and the color difference of regenerated embryoids was also an early indicator of ploidy level (based on visual GFP observation and confirmed by flow cytometry analysis), and could serve as a tool to study the regeneration mechanism of diploid cybrids via symmetric fusion in citrus and other higher plants.

Comparison of the Action of Transient and Continuous PTH on Primary Osteoblast Cultures Expressing Differentiation Stage-Specific GFP

Primary calvarial osteoblast cultures derived from type I collagen promoter-GFP reporter transgenic mice were used to examine progression of the osteoblast lineage. This system was validated by assessing the effect of PTH on osteoblast growth in real time. The anabolic effect of PTH seemed to be the result of enhanced osteoblast differentiation rather than expansion of a progenitor population. Activation of green fluorescent protein (GFP) marker genes driven by Col1a1 promoter fragments has been associated with the level of osteoblast differentiation. GFP-marked cultures provide an approach to continuously monitor the level of osteoblast differentiation in real time without the termination of cultures. Neonatal calvarial cells transgenic for pOBCol2.3GFP and pOBCol3.6GFP were used to establish calvarial osteoblast cultures. Parathyroid hormone (PTH) was added either continuous (days 1-21) or transient (days 1-7) to examine its diverse effect on osteoblast differentiation in cultures for 21 days. Three fluorescent markers were used: (1) pOBCol3.6GFP, which is activated in preosteoblastic cells; (2) pOBCol2.3GFP, which is restricted to differentiated osteoblasts; and (3) xylenol orange (XO), which stains the mineralized nodules. Progression of osteoblast differentiation indicated by fluorescent markers was documented throughout the entire period of culture. Recorded fluorescent images were analyzed in the patterns of expression and quantitated in the area of expression. Continuous PTH blocked osteoblast differentiation, which was evident by the attenuation of pOBCol3.6GFP and an absence of pOBCol2.3GFP. In contrast, transient PTH inhibited the initial osteoblast differentiation but ultimately resulted in a culture with more mineralized nodules and enhanced osteoblast differentiation expressing strong levels of pOBCol3.6GFP and pOBCol2.3GFP. Quantitative analysis showed that transient PTH first decreased then later increased areas of GFP expression and XO staining, which correlated with results of Northern blot and alkaline phosphatase activity. Transient PTH caused a decrease in DNA content during the treatment and after the removal of PTH. GFP-marked cultures combined with fluorescent image analysis have the advantage to assess the effect of PTH on osteoblast differentiation in real time. Results suggest that the anabolic effect of transient PTH is caused by an enhancement in osteoblast differentiation rather than an increase in the population of progenitor cells.

New and highly efficient method for silkworm transgenesis using Autographa californica nucleopolyhedrovirus and piggyBac transposable elements

We have developed a new method for the transgenesis of the silkworm, Bombyx mori. This method couples the use of recombinant baculoviruses with the use of the piggyBac transposable element. One recombinant AcNPV, designated the helper virus, is designed to express the piggyBac transposase under the control of the Drosophila hsp70 promoter. Another recombinant AcNPV encoded the gene to be incorporated into the silkworm genome, in this case a green fluorescent protein (GFP) gene, under the control of B. mori actin A3 promoter and franked by the piggyBac inverted terminal repeats. Preblastoderm eggs were inoculated with a fine needle coated with a mixture of these two recombinant baculoviruses. Most of the inoculated larvae hatched and a high proportion of the newly hatched G0 larvae expressed the GFP marker. Transgenesis was confirmed by Southern blot analysis of G1 insects, sequencing the insertion site junctions isolated by inverse PCR, and the marker segregated in Mendelian fashion, as evidenced by the appearance of green fluorescence in G2 insects. Thus, transgenic silkworms were easily and efficiently obtained using this new method.

Injectable bioartificial myocardial tissue for large-scale intramural cell transfer and functional recovery of injured heart muscle

ObjectivesMost tissue-engineering approaches to restore injured heart muscle result in distortion of left ventricular geometry. In the present study we suggest seeding embryonic stem cells in a liquid matrix for myocardial restoration.MethodsUndifferentiated green fluorescent protein–labeled mouse embryonic stem cells (2 × 106) were seeded in Matrigel (B&D, Bedford, Mass). In a Lewis rat heterotopic heart transplant model an intramural left ventricular pouch was fashioned after ligation of the left anterior descending coronary artery. The liquid mixture (0.125 mL) was injected in the resulting infarcted area within the pouch and solidified within a few minutes after transplantation (37°C). Five recipient groups were formed: transplanted healthy hearts (group I), infarcted control hearts (group II), matrix recipients alone (group III), the study group that received matrix plus cells (group IV), and a group that received embryonic stem cells alone (group V). After echocardiography 2 weeks later, the hearts were harvested and stained for green fluorescent protein and cardiac muscle markers (connexin 43 and α-sarcomeric actin).ResultsThe graft formed a sustained structure within the injured area and prevented ventricular wall thinning. The inoculated cells remained viable and expressed connexin 43 and α-sarcomeric actin. Fractional shortening and regional contractility were better in animals that received bioartificial tissue grafts compared with control animals (infarcted, matrix only, and embryonic stem cells only: group I, 17.0% ± 3.5%; group II, 6.6% ± 2.1%; group III, 10.3% ± 2.2%; group IV, 14.5% ± 2.5%; and group V, 7.8% ± 1.8%).ConclusionsLiquid bioartificial tissue containing embryonic stem cells constitutes a powerful new approach to restoring injured heart muscle without distorting its geometry and structure. Most tissue-engineering approaches to restore injured heart muscle result in distortion of left ventricular geometry. In the present study we suggest seeding embryonic stem cells in a liquid matrix for myocardial restoration. Undifferentiated green fluorescent protein–labeled mouse embryonic stem cells (2 × 106) were seeded in Matrigel (B&D, Bedford, Mass). In a Lewis rat heterotopic heart transplant model an intramural left ventricular pouch was fashioned after ligation of the left anterior descending coronary artery. The liquid mixture (0.125 mL) was injected in the resulting infarcted area within the pouch and solidified within a few minutes after transplantation (37°C). Five recipient groups were formed: transplanted healthy hearts (group I), infarcted control hearts (group II), matrix recipients alone (group III), the study group that received matrix plus cells (group IV), and a group that received embryonic stem cells alone (group V). After echocardiography 2 weeks later, the hearts were harvested and stained for green fluorescent protein and cardiac muscle markers (connexin 43 and α-sarcomeric actin). The graft formed a sustained structure within the injured area and prevented ventricular wall thinning. The inoculated cells remained viable and expressed connexin 43 and α-sarcomeric actin. Fractional shortening and regional contractility were better in animals that received bioartificial tissue grafts compared with control animals (infarcted, matrix only, and embryonic stem cells only: group I, 17.0% ± 3.5%; group II, 6.6% ± 2.1%; group III, 10.3% ± 2.2%; group IV, 14.5% ± 2.5%; and group V, 7.8% ± 1.8%). Liquid bioartificial tissue containing embryonic stem cells constitutes a powerful new approach to restoring injured heart muscle without distorting its geometry and structure.

SHORT REPORT: FLUORESCENT LEISHMANIA: APPLICATION TO ANTI-LEISHMANIAL DRUG TESTING

Classic techniques for detecting the susceptibility of Leishmania to different drugs are time-consuming, laborious, and require the use of macrophages. The use of flow cytometry for monitoring Leishmania susceptibility to drugs is beginning to be implemented. Using green fluorescent protein (GFP), we have improved and simplified the screening procedure. We introduced a GFP marker into field strains of Leishmania causing kala-azar (visceral leishmaniasis) and explored the suitability of transgenic L. donovani promastigotes that constitutively express GFP in their cytoplasm as target cells for in vitro screening of anti-leishmanial drugs.

Injectable bioartificial myocardial tissue for large-scale intramural cell transfer and functional recovery of injured heart muscle

Most tissue-engineering approaches to restore injured heart muscle result in distortion of left ventricular geometry. In the present study we suggest seeding embryonic stem cells in a liquid matrix for myocardial restoration. Undifferentiated green fluorescent protein-labeled mouse embryonic stem cells (2 x 10 6 ) were seeded in Matrigel (B&D, Bedford, Mass). In a Lewis rat heterotopic heart transplant model an intramural left ventricular pouch was fashioned after ligation of the left anterior descending coronary artery. The liquid mixture (0.125 mL) was injected in the resulting infarcted area within the pouch and solidified within a few minutes after transplantation (37 degrees C). Five recipient groups were formed: transplanted healthy hearts (group I), infarcted control hearts (group II), matrix recipients alone (group III), the study group that received matrix plus cells (group IV), and a group that received embryonic stem cells alone (group V). After echocardiography 2 weeks later, the hearts were harvested and stained for green fluorescent protein and cardiac muscle markers (connexin 43 and alpha-sarcomeric actin). The graft formed a sustained structure within the injured area and prevented ventricular wall thinning. The inoculated cells remained viable and expressed connexin 43 and alpha-sarcomeric actin. Fractional shortening and regional contractility were better in animals that received bioartificial tissue grafts compared with control animals (infarcted, matrix only, and embryonic stem cells only: group I, 17.0% +/- 3.5%; group II, 6.6% +/- 2.1%; group III, 10.3% +/- 2.2%; group IV, 14.5% +/- 2.5%; and group V, 7.8% +/- 1.8%). Liquid bioartificial tissue containing embryonic stem cells constitutes a powerful new approach to restoring injured heart muscle without distorting its geometry and structure.

Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies

Genetic transformation has been achieved for several citrus genotypes. However, regeneration of escapes at high frequency is a major problem, making the available procedures rather inefficient. Attempts to improve selection by increasing the concentration of kanamycin, used as the selective agent, or substituting it by geneticin have been unsuccessful. Here, we have critically assessed the actual frequency and origin of escapes in citrus by using visual screening with β-glucuronidase (gusA) and green fluorescent protein (gfp) markers, by studying the persistence of engineered Agrobacterium in the explants, and by characterising through Southern blot analysis all the regenerants obtained under kanamycin selection. Our results show that inefficient selection could be attributed to the protection of the non-transformed cells from the selective agent by the surrounding transformed cells, and to the persistence of kanamycin-resistance Agrobacterium in explant tissues over long periods of time after co-cultivation. This also explained the high frequency (12%) of chimeric shoots that were commonly recovered. High frequency regeneration of chimeras that resulted from the fusion of different transformation events is reported for the first time. On the other hand, molecular analysis of all the regenerants reveals that transformation frequency is underestimated when based on the expression of a screenable marker gene, and that low expressors and silenced lines could account for at least 25% of those plants considered escapes based on selectable and screenable marker analysis. Consequences of these results at the practical level are also discussed.

Identification of the adult human hemangioblast.

Recent studies show that human CD133(+) (previously known as AC133(+)) cells from mobilized peripheral blood consist of stem cells with either hematopoietic or endothelial potential. To test whether this population also contains individual precursors with both capacities, the defining characteristics of the elusive adult hemangioblast, we developed a culture system that allows single-cell analyses of differentiation. In the presence of vascular endothelial growth factor (VEGF), stem cell growth factor (SCGF), and FLT-3 ligand, CD133(+)-enriched cells were first expanded and the amplified cells were transduced with a vector encoding an enhanced green fluorescent protein (EGFP) marker gene. Single EGFP(+) cells were then cocultured with corresponding non-transduced cells from the same experiment, yielding 50-100 marked cells in 8% of the wells after 2 weeks. The resultant cells were divided and differentiated with either granulocyte colony-stimulating factor (G-CSF) or with SCGF and VEGF. These culture conditions resulted in the formation of neutrophil or endothelial cells, respectively, as identified morphologically and by phenotypic staining. Dual differentiation of EGFP(+) cells could be observed in one-quarter of clones from single-seeded cells, suggesting that 2% of EGFP(+) cells were in fact human hemangioblasts. These cells could be expanded for at least 28 days without losing this dual capacity. Hence, this culture system may be of clinical relevance in the development of cellular therapies for disorders involving hematopoiesis and the vascular system. In addition, our results provide important information related to the development of the vasculature and the potential role of hemangioblasts in vasculogenesis in adult humans.

Development of a transformation system for the nematophagous fungus Pochonia chlamydosporia

The nematophagous fungus Pochonia chlamydosporia is a potential biocontrol agent against root knot and cyst nematodes. Genetic transformation of the fungus to introduce visual marker genes, novel traits, or changes in expression levels of endogenous genes, would greatly enhance understanding of its behaviour on nematode-infested roots and of its interactions with other soil and rhizosphere microorganisms. A transformation system for the introduction of novel genes into P. chlamydosporia has been developed. Methods to generate protoplasts, introduce DNA and regenerate transformed viable fungal mycelium have been optimised, using plasmids carrying the green fluorescent protein marker gene gfp and the hygromycin resistance gene hph. Cultures of P. chlamydosporia were resistant to high levels of a range of fungal inhibitors, including hygromycin, that are commonly used with dominant selectable marker genes in the transformation of other fungi. However, regenerating protoplasts transformed with hph could be selected by their ability to grow through an agar overlay containing 1 mg ml(-1) hygromycin. Green fluorescence was observed in protoplasts and regenerating mycelium after transformation with gfp, but the GFP phenotype was lost on subculture. Maintenance of introduced genes was not stable, and during subculture, PCR assays indicated that the transformants lost both hph and gfp. When these genes were introduced on the same plasmid, segregation of hph and gfp was observed prior to their loss. It was unclear whether the introduced plasmids were able to replicate autonomously in P. chlamydosporia, or if they integrated transiently into the fungal genome. Possible reasons for the instability of the transformants are discussed.

Identification of new members of Fertilisation Independent Seed Polycomb Group pathway involved in the control of seed development in Arabidopsis thaliana.

In higher plants, double fertilisation initiates seed development. One sperm cell fuses with the egg cell and gives rise to the embryo, the second sperm cell fuses with the central cell and gives rise to the endosperm. The endosperm develops as a syncytium with the gradual organisation of domains along an anteroposterior axis defined by the position of the embryo at the anterior pole and by the attachment to the placenta at the posterior pole. We report that ontogenesis of the posterior pole in Arabidopsis thaliana involves oriented migration of nuclei in the syncytium. We show that this migration is impaired in mutants of the three founding members of the FERTILIZATION INDEPENDENT SEED (FIS) class, MEDEA (MEA), FIS2 and FERTILIZATION INDEPENDENT ENDOSPERM (FIE). A screen based on a green fluorescent protein (GFP) reporter line allowed us to identify two new loci in the FIS pathway, medicis and borgia. We have cloned the MEDICIS gene and show that it encodes the Arabidopsis homologue of the yeast WD40 domain protein MULTICOPY SUPRESSOR OF IRA (MSI1). The mutations at the new fis loci cause the same cellular defects in endosperm development as other fis mutations, including parthenogenetic development, absence of cellularisation, ectopic development of posterior structures and overexpression of the GFP marker.

Effect of protein supplementation in potassium simplex optimization medium on preimplantation development of bovine non-transgenic and transgenic cloned embryos

The present study evaluated the effect of protein supplementation in potassium simplex optimization medium (KSOM) on bovine preimplantation embryo development. The in vitro fertilized (IVF) (Experiment 1), non-transgenic (Experiment 2) and transgenic cloned embryos (Experiment 3) were cultured for 192 h in KSOM supplemented with 0.8% BSA (KSOM-BSA), 10% FBS (KSOM-FBS) or 0.01% PVA (KSOM-PVA). Transfected cumulus cells with an expression plasmid for human α1-antitrypsin gene and a green fluorescent protein (GFP) marker were used to produce transgenic cloned embryos. Modified synthetic oviductal fluid (mSOF) supplemented with 0.8% BSA (mSOF-BSA) was used as a control medium. In Experiment 1, cleavage rate was significantly ( P<0.05) lower (69.1%) in IVF embryos cultured in KSOM-FBS than in KSOM-BSA (80.3%). The rate of hatching/hatched blastocyst formation was significantly ( P<0.05) lower in embryos cultured in KSOM-PVA than in KSOM-FBS (2.2% versus 10.8%). Blastocysts cultured in KSOM-FBS contained significantly ( P<0.05) higher numbers of inner cell mass cells (50.4±20.2) than those cultured in mSOF-BSA (36.9±19.2). In Experiment 2, the rate of blastocyst formation was significantly ( P<0.05) lower (20.5%) in embryos cultured in KSOM-PVA than in other culture media (33.3–38.5%). The rate of hatching/hatched blastocysts was significantly ( P<0.05) lower in KSOM-PVA (13.9%) and KSOM-FBS (17.1%) than in KSOM-BSA (30.8%) and mSOF-BSA (33.9%). The numbers of total and trophectoderm cells (104.6±32.2 and 71.7±25.5, respectively) were significantly ( P<0.05) lower in blastocysts cultured in KSOM-PVA than in KSOM-BSA (125.7±39.7 and 91.7±36.2, respectively). In Experiment 3, no significant differences in embryo development, GFP expression and blastocyst cell numbers were observed among the culture groups. In conclusion, the present study demonstrated that KSOM and mSOF supplemented with BSA were equally effective in supporting development of bovine non-transgenic and transgenic cloned embryos. Moreover, different developmental competence in response to protein supplementation of KSOM was observed between bovine non-transgenic and transgenic cloned embryos.

Impact of ecological factors on the initial invasion of Bt transgenes into wild populations of birdseed rape (Brassica rapa)

The inevitable escape of transgenic pollen from cultivated fields will lead to the emergence of transgenic crop-wild plant hybrids in natural patches of wild plants. The fate of these hybrids and that of the transgene depend on their ability to compete with their wild relatives. Here we study ecological factors that may enhance the fitness of genetically modified hybrids relative to wild plants for a Bacillus thuringiensis ( Bt) transgene conferring resistance to insects. Mixed stands of wild plants and first-generation hybrids were grown under different conditions of herbivore pressure and density, with Bt oilseed rape ( Brassica napus) as the crop and B. rapa as the wild recipient. Biomass and fitness components were measured from plant germination to the germination of their offspring. The frequency of transgenic seedlings in the offspring generation was estimated using the green fluorescent protein marker. The biomass of F(1) Bt-transgenic hybrids relative to that of wild-type plants was found to be sensitive to both plant density and herbivore pressure, but herbivore pressure appeared as the major factor enhancing their relative fitnesses. In the absence of herbivore pressure, Bt hybrids produced 6.2-fold fewer seeds than their wild neighbors, and Bt plant frequency fell from 50% to 16% within a single generation. Under high herbivore pressure, Bt hybrids produced 1.4-fold more seeds, and Bt plant frequency was 42% in the offspring generation. We conclude that high-density patches of highly damaged wild plants are the most vulnerable to Bt-transgene invasion. They should be monitored early to detect potential transgene spread.

804. Expression of Green Fluorescent Protein Modifies the Antitumor Effect of Oncolytic HSV-1 Vectors

Replication-competent, oncolytic herpes simplex virus type 1 (HSV-1) vectors are useful tools for cancer gene therapy. The virus genome is genetically engineered so that the vector selectively replicates in and destroys tumor cells while sparing the surrounding normal tissues. A marker gene is frequently used in order for replicating virus vectors to be easily detected. Green Fluorescent Protein (GFP) is now a popular marker, because it can be detected in living cells and also be used for selection. MGH-1 is a double-mutated HSV-1 with deletions in both copies of the γ34.5 gene and an insertion of the LacZ gene down-stream of the ICP6 promoter inactivating the ICP6 gene. Using this MGH-1 as a backbone and a bacterial artificial chromosome technology, we newly created multiple types of “armed” oncolytic HSV-1 vectors (vHSV series) in which the ICP6 and LacZ genes were replaced with the ICP6 promoter-driven GFP gene and a transgene cassette driven by the HSV IE4/5 promoter. Prior to testing “armed” oncolytic vectors, we compared the antitumor activity of vHSV-null, the control oncolytic vector expressing only the GFP marker, with that of MGH-1. The replication capability of vHSV-null in cultured Vero cells at a MOI of 0.01 was approximately 10 times lower than that of MGH-1. Despite decreased replication capability, vHSV-null showed slightly faster cytopathic effect than MGH-1 in Neuro2a murine neuroblastoma cells at MOI=0.1. A/J mice bearing bilateral subcutaneous Neuro2a tumors were used for in vivo evaluations. When vectors were inoculated directly into the left tumor at 2×106 plaque-forming units (days 0 and 3), vHSV-null was significantly more effective than MGH-1 for inhibiting the growth of inoculated tumors (p < 0.05). Contralateral non-inoculated tumors also tended to be smaller in vHSV-null-treated animals than MGH-1-treated animals although not statistically significant. Such difference in tumor growth inhibition was not observed in athymic mice, indicating that the greater antitumor activity of vHSV-null in vivo compared with MGH-1 is associated with T cell-mediated immune responses most likely due to expression of GFP. While this possible immunostimulatory effect by GFP may be beneficial to oncolytic virus therapy, a caution is needed to evaluate the antitumor activity of oncolytic HSV-1 vectors when the GFP gene is used as a marker for virus replication.

698. Gene Therapy of Murine GM1 Gangliosidosis by Genetically Modified Bone Marrow Hematopoietic Progenitor Cells

b-galactosidase (b-gal), a lysosomal enzyme involved the removal of b-linked terminal galactosyl residues of many glycoconjugates, is deficient the neurodegenerative lysosomal disorder GM1-gangliosidosis (GM1). GM1-/-mice closely mimic the most fundamental aspects of the neuropathological and neurochemical abnormalities of the human disorder. Bone marrow progenitor cells have been used as a source of corrective protein because of their ability to repopulate the recipients and to supply functional enzyme to different cells by in trans correction. Hematopoietic progenitors transduced with a murine stem cell virus (MSCV)-based bicistronic retroviral vector over-expressing b-gal and the green fluorescent protein (GFP) marker were used for transplantation into sublethally irradiated GM1-/-mice. Transduction efficiency of total BM cells with the MSCV-b-gal ranged from 25–89% prior to the transplantation. b-gal expressing BM-derived cells were detected histologically and enzymatically many tissues including spleen, liver, lungs, intestine and kidney after one, three and six months post transplantation (BMT). GFP-expressing cells of the erythroid, myeloid or lymphoid lineage were identified by FACS analysis of peripheral blood samples, collected at different time points after transplantation. Secondary transplantations demonstrated consistent long-term in vivo b-gal expression all tissues including brain. In addition, the thin layer chromatography of brain lipids showed a reduction the GM1-ganglioside content brainstem and cerebellum after three and six months post BMT. In line with the biochemical and histological findings, different behavioral tests including open field, rotorod and walking pattern, indicated a clear improvement of the neurological function transplanted GM1-/-compared to untreated littermates. Taken together these results have encouraged the use of ex-vivo gene therapy for the treatment of GM1.