Transfection of bone marrow mesenchymal stem cells using green fluorescence protein labeled hVEGF165 recombinant plasmid mediated by liposome

Objective:To study the role of bone marrow mesenchymal stem cells(BMSCs)in construction of vascularized engineered tissue.Methods:hVEGF165 was amplified via RT-PCR before recombinant with pShuttle-green fluorescence protein;green fluorescent protein(GFP)-CMV.Then the recombinant shuttle plasmid was transfected into BMSCs with Lipofectamine^(TM)2000 for packaging and amplifying.hVTGF165 mRNA expression in BMSCs cells was tested.Results:The sequence of hVEGFI65 in pShutlle-GFP-hVFGF165 plasmid was confirimed by double-enzyme cleavage method and sequencing.hVECF165 was highly expressed in BMSCs.Conclusions:The GFP/hVECF165 recombinant plasmid vector was constructed successfully and expressed effectively in host cells,which may be helpful for discussing the possibility of the application of VEGF165-BMSCs in tissue engineering and ischemic disease cure.

Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein （GFP） was expressed in C. reinhardtii under the control of promoters： RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae： RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ. In addition, a threefold increase of GFP in Tran-Ⅱ was induced by heat shock at 40℃. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

Transient expression of the enhanced green fluorescent protein(egfp) gene in Sargassum horneri

Sargassum horneri is a macroalga widespread in North Asia-Pacific region, and these years its bloom has caused huge damage to the environment and the economic in China. To make up the blank on genetic engineering research, a transient transformation system for the multicellular marine brown alga S . horneri was established in this research. The algae used in this research were collected from the Yellow Sea of China and verified as a same species S . horneri with analysis of molecular markers. The S . horneri parietal leaves were transformed with the enhanced green fluorescent gene as the reporter by micro-particle bombardment. The results show that green fluorescent protein (GFP) is an eff ective transgene reporter for S . horneri and that particle bombardment is a suitable method for transformation of S . horneri . Through selection of four diff erent promoters for EGFP and six groups’ bombardment characters, the highest transformation efficiency approximately 1.31% was got with the vector pEGFP-N1 at bombardment characters 900 spi and 6 cm distance. This research paves a way for the further research and application of S . horneri .

Constructing retroviral vector carrying green fluorescent protein(GFP)and investigating the expression of GFP in primary rat myoblast

Objective： To construct green fluorescent protein（GFP） retroviral vector（pLgXSN）, and to investigate the expression of GFP in primary rat myoblast. Methods： GFP cDNA was subcloned into the plasmid pLgXSN, and the recombinant vector was transfected into packaging cell PT67. G418 was used to select positive colony. Myoblasts were infected by a high-titer viral supernatant. The recombinant retroviral plasmid vector was identified by restriction endonuclease analysis and DNA sequence analysis. Confocal microscopy and flow cytometry were used to detect the expression of GFP. Results： The GFP cDNA sequence was identical to that of GenBank. Recombinant retroviral plasmid vector pLgGFPSN was constructed successfully. The titer of the packaged recombinant retrovirus was 1×10^6 cfu/ml. Bright green fluorescence of the transfected cells was observed under confocal microscope 48 h after transfection. The transfection rate was 33%. The effective expression of GFP in myoblast infected by recombinant retrovirus lasted for 6 weeks. Conclusion： GFP gene could be effectively and stably expressed in myoblast, which suggests that GFP could act as a marker for studies on myoblast.

Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells

Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa.To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter,the recombinant baculovirus,which contained the GCRVs8 and eGFP(enhanced green fluorescence protein) genes,was constructed by using the Bac-to-Bac insect expression system.In this study,the whole GCRVs8 and eGFP genes,amplified by PCR,were constructed into a pFastBacDual vector under polyhedron(PH) and p10 promoters,respectively.The constructed dual recombinant plasmid(pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid(AcGCRVs8/eGFP) by transposition.Finally,the recombinant bacluovirus(vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells.The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection,and gradually enhanced and extended around 5 days culture in P1(Passage1) stock.The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus(BV) stock.Additionally,PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus.Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.

Synthesis and characteristic of a novel green fluorescent protein eYGFPuv

Recently, a novel green fluorescent protein eYGFPuv has been identified in the marine organism Chiridius poppei which displays high fluorescence intensity and can be visible by eyes in dark. Although strong green fluorescence was achieved in transgenic petunia, 3 expression cassettes (about 8 kb) complicate its application. In this study, to confirm whether 1 expression cassette could be used as a transgenic marker in prokaryotes and eukaryotes, eYGFPuv was cloned into prokaryotic expression vector pET28α-eYGFPuv- His and plant binary expression vector 35S::eYGFPuv. Compared to EGFP, eYGFPuv protein exhibited stronger dazzling green fluorescence in E. coli under excited light at 365 nm and maintains steadily over a long period of time without degradation. When transiently expressed in tobacco leaves, eYGFPuv protein displayed strong green fluorescence. Moreover, the fluorescence of eYGFPuv protein also could be directly observed in living plant, and thus can be used easily as a marker to screen transformed lines in transgenic research. Overall, compared to previous studies on eYGFPuv tandem repeats, our data confirmed that single eYGFPuv sequence still possesses high fluorescence intensity and quenching resistance. Furthermore, because of small size of expression cassette,it is suitable for efficient transformation in both prokaryotic and eukaryotic organisms.

Jellyfish Green Fluorescent Protein(GFP) as a Reporter for Fusarium gramminearum Development on Wheat

The plasmid pGPDGFP under the control ofpgpdA promotor was used together with vector pAN7-1 containing the hygromycin resistance cassette to co-transform protoplasts of HG1, Fusarium graminearum from Hubei Province, China. Twelve out of 14 hygromycin-resistant transformants showed green signal under the UV light and contained one or several copies ofgfp, as indicated by Southern analysis of genomic DNA digested with different restriction enzymes and hybridized to the gfp probe. A single gfp copy transformant （HG1C5） was selected for further evaluation of 80 Chinese wheat cultivars or advanced lines. The results showed different resistance type to F. graminearum were observed. GFP signals observed in the rachis and adjacent spikes of 70 Chinese wheat lines such as Chuanchongzu 104 indicated both type I （host resistance to the initial infection by the fungus） and type II （resistance to the spread of FHB symptoms within an infected spike） were not observed. While other 10 lines showed type II resistance to F. graminearum with GFP signals only in inoculated spikelets. Development of the mycelium can be intuitively observed and the resistance of wheat to F. graminearum can be identified at 7 days post inoculation （dpi） in this way. The results showed no differences were evaluated between the transformed HG1C5 and the non-transgene artificial inoculation by SAS paired chi-square test and McNernar＇s test （P=-0.0625）.

PERFORMANCE AND PERSISTENCE OF GREEN FLUORESCENT PROTEIN (gfp) MARKED AZOTOBACTER CHROOCOCCUM IN STERILIZED AND UNSTERILIZED WHEAT RHIZOSPHERIC SOIL

The persistence and performance (growth promoting potential) of green fluorescent protein (gfp) marked Azotobacter chroococcum strain ABR 4G were studied in sterilized and unsterilized wheat rhizospheric soil. The gfp was integrated via Tn 5 transposition into A. chroococcum chromosome and the resultant gfp marked colonies were identified by green fluorescent emission under UV light. The gfp was stably maintained in A. chroococcum and the gfp insertion had no apparent adverse effect on the growth promoting properties of the marked soil isolate ABR 4G. The growth promoting properties (nitrogen fixation, ammonia excretion, phosphate solubilization and IAA production) of the parent soil isolate and the gfp marked strain were found to be almost the same. All the quantitative wheat plant traits were significantly influenced by inoculation of A. chroococcum ABR 4G strain in sterilized and unsterilized soil. Inoculated bacterial counts increased gradually in wheat rhizosphere, reached maximum on 60 th d and declined on 80 th d. Fertility levels also affected survival of marked strain and the survival was comparable in sterilized and unsterilized soil. The growth promoting properties were also determined from the marked strain reisolated from wheat rhizosphere in both types of soil. Fig 1, Tab 2, Ref

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase

Transformation of Arabidopsis by Rice OsWRKY78::GFP Fusion Gene and Subcellular Localization of OsWRKY78 Protein

[Objective] The study was to understand the subcellular localization of OsWRKY78 protein in plants. [Method] Primers specific for OsWRKY78 gene were designed according to the OsWRKY78 full length sequence in Genbank. The gene was cloned by RT-PCR method. The gene was then recombined into a plasmid expression vector carrying green fluorescent protein (GFP) gene, pBinGFP. The recombinant was confirmed by PCR and enzyme digestion. The recombinant plasmid pBinGFP-OsWRKY was transformed into Arabidopsis through Agrobacterium tumefaciens strain GV3101 and transgenic plants were obtained. [Result] Measured by fluorescence microscopy, the expression of OsWRKY78 and GFP fusion protein in root tip cells was localized in the nucleus. [Conclusion] This study laid the foundation for further investigating the function of OsWRKY78 gene and its role in related signal transduction and provided theoretical basis for exploring the relation between OsWRKY78 gene and brown planthoppers.

F-Actin Visualization in Generative and Sperm Cells of Living Pollen of Rice Using a GFP-Mouse Talin Fusion Protein

Green fluorescent protein (GFP) fused to the F-actin binding domain of mouse talin labels the actin cytoskeleton in the living generative and sperm cells of a third generation transgenic rice (Oryza sativa L.) plant, A005-G-T-1-2. Observations were made on pollen at four major developmental stages, viz. I. uni-nucleate microspore stage; II. early bi-cellular pollen stage; III. late bi-cellular pollen stage; and IV. tri-cellular pollen stage. At each of these developmental stages vegetative nucleus, generative nucleus/ cell, and sperm cells were seen undergoing continuous and coordinated motion and migration. These movements seemed to be influenced by associated microfilament networks existing in the pollen. Based on these observations we propose that it is the interaction between the microfilament networks (usually one existing in the central cytoplasm and another in the cortex) that controls the dynamic movement of the vegetative nucleus, generative nucleus/cell and sperm cells. Furthermore, we have also observed that there is an array of microfilaments (oriented mostly parallel to the long axis of the cell) existing in the generative and sperm cells. As far as we are aware, this is the first report showing the existence of microfilaments in living generative and sperm cells of rice pollen. The implication and significance of the existence of microfilaments in generative and sperm cells in rendering self-propelled motion of these cells in relation to their passage and movement in the pollen tube and embryo sac for fertilization were discussed.

Actin Visualization in Living Immature Pollen of Rice Using a GFP-Mouse Talin Fusion Protein

Green fluorescent protein (GFP) fused to the F_actin binding domain of mouse talin labels the actin cytoskeleton in the immature pollen of stable transformed rice (Oryza sativa L.) plants. Actin microfilaments could be visualized only in the late_developmental stage of the immature pollen. During this developmental stage, microfilaments, initially composed of very short fibrils, develop into a very complex and novel network that sometimes totally and sometimes partially encloses the vegetative nucleus and the spherical shaped generative cell in the central cytoplasm of the immature pollen. The behavior of the actin microfilamentous structure throughout the late_developmental stage of the immature pollen is extremely dynamic, and the likelihood of this structure in generating forces for vegetative nucleus and generative cell movement in the immature pollen has been discussed. No actin filaments were visualized in the spherical generative cells.

Soluble Expression and Rapid Quantification of GFP-hepA Fusion Protein in Recombinant Escherichia coli

To establish a rapid quantification method for heparinase I during its production in recombinant Escherichia coli, a translational fusion vector was constructed by fusing the N terminus of heparinase I to the C terminus of a green fluorescent protein mutant （GFPmutl）. As a result, not only was the functional recombinant expression of heparinase I in E. coli accomplished, but also a linear correlation was obtained between the GFP fluorescence intensity and heparinase I activity, allowing enzyme activity to be quantified rapidly during the fermentation.

Multilineage Differentiation of Dental Pulp Stem Cells from Green Fluorescent Protein Transgenic Mice

Aim The aim of this study was to confirm the multilineage differentiation ability of dental pulp stem cells （DPSCs） from green fluorescent protein （GFP） transgenic mice. The expression of GFP in DPSCs was also observed during differentiation. Methodology DPSCs were harvested from the dental pulp tissue of transgenic nude mice, and then transferred to osteogenic, adipogenic, and chondrogenic media. The morphological characterization of induced cells was observed by microscopy and histological staining. The expression of marker genes was measured by RT-PCR. Results The endogenous GFP and multilineage potential of transgenic DPSCs had no influence on each other. Moreover, the results of fluorescence microscopic imaging suggest that there was no significant decline of GFP expression during DPSCs differentiation. Conclusion As the population of GFP labeled DPSCs can be easily identified, this will be a promising method for tracking DPSCs in vivo.

In vivo transfection of enhanced green fluorescent protein in rat retinal ganglion cells mediated by ultrasound-induced microbubbles

BACKGROUND： Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein （EGFP） in in vitro cultured retinal ganglial cells （RGCs）. OBJECTIVE： To investigate the feasibility of ultrasound-mediated microbubble destruction for EGFP transfection in rat RGCs, and to compare efficiency and cell damage with traditional transfection methods. DESIGN, TIME AND SETTING： In vivo, gene engineering experiment. The study was performed at the Central Laboratory, Institute of Ultrasonic Imaging, Chongqing Medical University from March to July 2008. MATERIALS： Eukaryotic expression vector plasmid EGFP and microbubbles were prepared by the Institute of Ultrasonic Imaging, Chongqing Medical University. The microbubbles were produced at a concentration of 8.7 × 10^11/L, with a 2-4 μm diameter, and 10-hour half-life in vitro. METHODS： A total of 50 Sprague Dawley rats were randomly assigned to four groups. Normal controls （n = 5） were infused with 5 μL normal saline to the vitreous cavity; the naked plasmid group （n = 15） was infused with 5 pL EGFP plasmid to the vitreous cavity; in the plasmid with ultrasound group （n = 15）, the eyes were irradiated with low-energy ultrasound wave （0.5 W/cm^2） for a total of 60 seconds （irradiated for 5 seconds, at 10-second intervals） immediately following infusion of EGFP plasmids to the vitreous cavities. In the microbubble-ultrasound group （n = 15）, the eyes were irradiated with the same power of ultrasonic wave immediately following infusion of microbubbles containing EGFP plasmids to the vitreous cavities. MAIN OUTCOME MEASURES： After 7 days, retinal preparations and EGFP expression in RGCs were observed by fluorescence microscopy. RGC quantification in the retinal ganglion cell layer was performed. In addition, EGFP mRNA expression was semi-quantitatively determined by RT-PCR. RESULTS： The transfection efficiency of EGFP to RGCs by microbubbles with ultrasound was significantly greater than the other groups, and no obvious damage was detected in the RGCs. CONCLUSION： Under irradiation of low-frequency ultrasound waves, ultrasound-mediated microbubble destruction was effective and resulted in safe transfection of the EGFP gene to the RGCs.

Repair of spinal cord injury by neural stem cells transfected with brain-derived neurotrophic factor-green fluorescent protein in rats A double effect of stem cells and growth factors?

Brain-derived neurotrophic factor（BDNF）can significantly promote nerve regeneration and repair.High expression of the BDNF-green fluorescent protein（GFP）gene persists for a long time after transfection into neural stem cells.Nevertheless,little is known about the biological characteristics of BDNF-GFP modified nerve stem cells in vivo and their ability to induce BDNF expression or repair spinal cord injury.In the present study,we transplanted BDNF-GFP transgenic neural stem cells into a hemisection model of rats.Rats with BDNF-GFP stem cells exhibited significantly increased BDNF expression and better locomotor function compared with stem cells alone.Cellular therapy with BDNF-GFP transgenic stem cells can improve outcomes better than stem cells alone and may have therapeutic potential for spinal cord injury.

Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein

Objective: To evaluate the transduction efficiency of a recombinant adenovirus carrying the gene for green fluorescent protein (Ad-GFP) into the primary cultures of fetal neural stem cells (NSCs) by the expression of GFP. Methods: The Ad-GFP was constructed by homologous recombination in bacteria with the AdEasy system; NSCs were isolated from rat fetal hippocampus and cultured as neurosphere suspensions. After infection with the recombinant Ad-GFP, NSCs were examined with a fluorescent microscopy and a flow cytometry for their expression of GFP. Results: After the viral infection, flow cytometry analysis revealed that the percentage of GFP-positive cells was as high as 97.05%. The infected NSCs sustained the GFP expression for above 4 weeks. After differentiated into astrocytes or neurons, they continued to express GFP efficiently. Conclusion: We have success- fully constructed a viral vector Ad-GFP that can efficiently infect the primary NSCs. The reporter gene was showed fully and sustained expression in the infected cells as well as their differentiated progenies.

Expression and Purification of Recombinant MP-GFP Protein in Escherichia coli

Guided pesticide is an unique compound resulted from the conjugation with carrier （amino acid, protein, sugars, etc） and the active pesticide ingredient. One of the attributes of the guided pesticide is its potential to accumulate at the site of the damaged points caused by pest or at the site of entry to the target pests, such as via inhalation, cuticular penetration, and oral digestion. Movement protein （MP） is a kind of protein coded by plant virus. A genetic fusion between green fluorescent protein （GFP） and movement protein resulted in the expression of a fluorescent fusion MP-GFP protein, which was fully biologically active in mediating the cell-to-cell spread of virus. In order to obtain a suitable carrier for a pesticide, fluorescent carrier MP-GFP was constructed. It was found that the recombinant MP-GFP protein was the inclusion body. The results indicated that optimized cultural condition for expression of recombinant MP-GFP protein was incubation at 37°C for 2 h and induction with 0.2 mmol L-1 IPTG （isopropyl-b-dthiogalactopyranoside） at 25°C for 4 h. MP-GFP protein was purified by using Ni-NTA resin. The expressed recombinant MP-GFP protein had both the fluorescence character of report GFP gene and moving character of movement protein. It could provide a guided carrier for studying the guided pesticide. It could also provide convenience for studying the delivery and distribution of the guided pesticide ingredients in the plant.

Using of green fluorescent reporter gene (GFP) to monitor the fate of Fusarium moniliforme mycoparasitized by Trichoderma viride

Fusarium moniliforme Sheld.is a rice pathogenic fungus and causes the disease called Bakanae,which has increasingly damaged rice production in the recent years. Trichoderma spp. has been one of the most widely used biological control agent of plant disease. By geneticaly labelling F. moniliforme with the GFP reporter gene, we have studied the antagonistic action of Trichoderma viride against this pathogenic fungus. The binary GFP reporter vector pCHF3-35S∷GFP was constructed, which carries the gfp gene driven by the CaMv35S promoter. The vector was transformed into F. moniliforme via Agrobacterium.The mycoparasitism of T.viride against F.moniliforme was tested by dual culture and examined with fluorescence microscope. The result of the dual culture showed that the T.viride maintained a strong competitive ability against F. moniliforme , by growing on the top of the pathogen colony. Fluorescence microscope observation indicated that attacked hyphae of F. moniliform were distorted, swollen or broken. This indicate an enzymatic by T.viride to degrade the host cell walls and used the cell contents as a source of nutrients (Fig 1) .

Expression of Green Fluorescent Protein Gene with Baculovirus Vectorin Insect Cells

The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.