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 p...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.展开更多
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 rese...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 .展开更多
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 o...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-Ⅱwas induced by heat shock at 40°C.All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C.reinhardtii.展开更多
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 th...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.展开更多
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 inne...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.展开更多
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, C...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).展开更多
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...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展开更多
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 fluo...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.展开更多
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...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展开更多
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 obs...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.展开更多
BACKGROUND: Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein (EGFP) in in vitro cultured retinal gangli...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.展开更多
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...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.展开更多
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 ...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.展开更多
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 bi...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) .展开更多
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...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.展开更多
A constitutive expression vector pHY300-F1gfp was constructed to test the function of a promoter F1 subcloned from a rice epiphyte Bacillus brevis strain DX01. The DX01 cells harboring plasmid pHY300-F1gfp were detect...A constitutive expression vector pHY300-F1gfp was constructed to test the function of a promoter F1 subcloned from a rice epiphyte Bacillus brevis strain DX01. The DX01 cells harboring plasmid pHY300-F1gfp were detected to produce bright green fluorescence. Subsequently, the gfp-tagged B. brevis strain was released into the soil and its survival was investigated by PCR and the detection of green fluorescence. The spatial location of in situ gfp-tagged bacterial cells on the root surface of rice seedlings was visualized. All these results indicated that green fluorescent protein is an ideal molecular marker for detection of the activities of promoter F1, and it is also a reliable probe to monitor specific B. brevis bacteria in the environment.展开更多
We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constr...We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.展开更多
Objective: To investigate the invasion and metastasis of glioma in vivo by xenotransplanted tumor established by implanting C6 glioma cells transfected with green fluorescent protein (GFP) gene in vitro into the brain...Objective: To investigate the invasion and metastasis of glioma in vivo by xenotransplanted tumor established by implanting C6 glioma cells transfected with green fluorescent protein (GFP) gene in vitro into the brain of SD rats. Methods: C6 cells were transfected with a plasmid vector (pEGEP-N3) containing the GFP gene. Stable GFP-expressing clones were isolated and performed examination by flow cytometry and electron microscope. GFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later rats were killed and continuous brain sections respectively were examined by HE staining, immunohistochemistry method and fluorescence microscopy for detection of tumor cell invasion. Xenotransplanted tumor was primarily cultured to determine the storage of exotic GFP gene in vivo. Results: There were not obvious changes in cell cycle and ultrastructure for the cells transfected with GFP gene. C6 cells transfected with GFP gene maintained stable high-level GFP expression in the central nervous system during their growth in vivo. GFP fluorescence clearly demarcated the primary tumor margin and readily allowed for the detection of distant invasion on the single-cell level, which was evidently superior to HE and immunohistochemistry staining. There was not GFP gene loss of transfected cells in vivo. Conclusions: It is suggested that C6 cells transfected with GFP gene can be visualized by fluorescent microscopy after intracranial implantation. This model is an excellent experimental animal model in research on invasion of glioma.展开更多
The human immunodeficiency virus (HIV) lentiviral vector is an ideal vector for gene therapy. In the present study, the wild-type HIV-1 genome was segregated into four plasmids, and an optimized novel HIV-1 lentivir...The human immunodeficiency virus (HIV) lentiviral vector is an ideal vector for gene therapy. In the present study, the wild-type HIV-1 genome was segregated into four plasmids, and an optimized novel HIV-1 lentiviral vector containing green fluorescent protein and vesicular stomatitis virus G pseudo-capsule was constructed. The plasmids were pHR-CMV-EGFP, pCMVΔ8.9, pRSV-Rev, pCMV-VSV-G. The four plasmid system was co-transfected into 293T cells, and green fluorescent protein expression was observed. The present study obtained lentiviral particles by high-speed centrifugation, and the lentiviral particle titer was 4 × 108 TU/mL after centrifugation. Thus, an optimized novel HIV-1 lentiviral vector was successfully constructed.展开更多
Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tum...Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tumor cell lines were infected with LGSN amphotropic virus. Tumor cell lines that stably express EGFP were selected with G418. The integration and expression of EGFP gene were analyzed by polymerase chain reaction, and flow cytometry (FCM). Results: After gene transfection and ping-pong transduction, amphotropic producer line Am12/LGSN was generated with a stable green fluorescence signal readily detectable by FCM in up to 97% of examined cells. The viral titer in the supernatants was up to 8.2×105CFU/ml. After transduction and selection, G418-resistant leukemia K562, mammary carcinoma MCF-7, and bladder cancer 5637 cells were developed, in which the integration of both EGFP and neomycin resistance gene was confirmed by DNA amplification. In comparison with uninfected cells, FCM analysis revealed EGFP expression in up to 90% (range 85.5%–90.0%) of tumor cells containing LGSN provirus. Conclusion: The retroviral vector LGSN can effectively mark the human tumor cells with a stably EGFP expression which may be in studying tumor growth, metastasis and angiogenesis.展开更多
基金supported by grants from the National Natural Science Foundation of Hainan Province(30635)Foundation of Health Department of Hainan Province(2008-40)
文摘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.
基金Supported by the National Key Research and Development Program of China(No.2016YFC1402106)the Project of Innovation&Development of Marine Economy(HHCL201803)+1 种基金the National Natural Science Foundation of China(Nos.41406192,41376139)the Science and Technology Service Network Initiative of Chinese Academy of Sciences(No.KFJ-STS-ZDTP-023)
文摘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 .
基金Supported by the High Technology Research and Development Program of China (863 Program) (No. 2005AA601010-05)the Natural Science Foundation of Guangdong Province (No.5010492)the Technology Project of Shenzhen City
文摘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-Ⅱwas induced by heat shock at 40°C.All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C.reinhardtii.
文摘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.
基金National Natural Science Foundation of China (Grant Nos 30470074,30671615)Innovation Project of the Chinese Academy of Sciences (KSCX2-YW-N-021).
文摘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.
基金supported in part by the Special Fund for Agro-scientific Research in the Public Interest(201303016)the China Agriculture Research System(CARS-03)from the Ministry of Agriculture of China+3 种基金by the Project of International Scientific and Technical Cooperation(2013DFG31930)the National Key Technologies Research and Development Program of China(2012BAD19B04)the Breeding and Cultivation of Novel GM Varieties(2013ZX08002001)863 Program(2012AA101501)from the Ministry of Science and Technology of China
文摘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).
文摘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
基金supported by the National Natural Science Foundation of China (31500237)
文摘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.
文摘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
基金funded by The Peabody Foundation Inc.,the Anthony and Constance Franchi Fund for Pediatric Orthopaedics at the Mass General Hospital for Children, and the National Natural Science Foundation of China (30801304)Foundation for the Author of National Excellent Doctoral Dissertation of PR China (FANEDD 200977)
文摘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.
基金the National Natural Science Foundation of China,No.30430230
文摘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.
基金the Natural Science Foundation of Liaoning Province, No. 20052096
文摘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.
基金Project (No. 30672308) supported by the National Natural ScienceFoundation of China
文摘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.
文摘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) .
文摘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.
基金Item supported by the McKnight founda-tion and science and technology commission of Shanghai (No.KBH1322093)
文摘A constitutive expression vector pHY300-F1gfp was constructed to test the function of a promoter F1 subcloned from a rice epiphyte Bacillus brevis strain DX01. The DX01 cells harboring plasmid pHY300-F1gfp were detected to produce bright green fluorescence. Subsequently, the gfp-tagged B. brevis strain was released into the soil and its survival was investigated by PCR and the detection of green fluorescence. The spatial location of in situ gfp-tagged bacterial cells on the root surface of rice seedlings was visualized. All these results indicated that green fluorescent protein is an ideal molecular marker for detection of the activities of promoter F1, and it is also a reliable probe to monitor specific B. brevis bacteria in the environment.
基金supported by the National Programs for High Technology Research and Development of China (2006AA10A204)the Gansu Key Technologies R&D Program(ZGS-052-A41-0006-03)the Programs for Director Fund of Lanzhou Veterinary Research Institute
文摘We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.
基金This work was supported by a grant from the National Natural science foundation of China (No. 39970752).
文摘Objective: To investigate the invasion and metastasis of glioma in vivo by xenotransplanted tumor established by implanting C6 glioma cells transfected with green fluorescent protein (GFP) gene in vitro into the brain of SD rats. Methods: C6 cells were transfected with a plasmid vector (pEGEP-N3) containing the GFP gene. Stable GFP-expressing clones were isolated and performed examination by flow cytometry and electron microscope. GFP-expressing cells were stereotactically injected into the brain parenchyma of SD rats to establish xenotransplanted tumor. Four weeks later rats were killed and continuous brain sections respectively were examined by HE staining, immunohistochemistry method and fluorescence microscopy for detection of tumor cell invasion. Xenotransplanted tumor was primarily cultured to determine the storage of exotic GFP gene in vivo. Results: There were not obvious changes in cell cycle and ultrastructure for the cells transfected with GFP gene. C6 cells transfected with GFP gene maintained stable high-level GFP expression in the central nervous system during their growth in vivo. GFP fluorescence clearly demarcated the primary tumor margin and readily allowed for the detection of distant invasion on the single-cell level, which was evidently superior to HE and immunohistochemistry staining. There was not GFP gene loss of transfected cells in vivo. Conclusions: It is suggested that C6 cells transfected with GFP gene can be visualized by fluorescent microscopy after intracranial implantation. This model is an excellent experimental animal model in research on invasion of glioma.
基金the National Natural Science Foundation of China, No. 30770755
文摘The human immunodeficiency virus (HIV) lentiviral vector is an ideal vector for gene therapy. In the present study, the wild-type HIV-1 genome was segregated into four plasmids, and an optimized novel HIV-1 lentiviral vector containing green fluorescent protein and vesicular stomatitis virus G pseudo-capsule was constructed. The plasmids were pHR-CMV-EGFP, pCMVΔ8.9, pRSV-Rev, pCMV-VSV-G. The four plasmid system was co-transfected into 293T cells, and green fluorescent protein expression was observed. The present study obtained lentiviral particles by high-speed centrifugation, and the lentiviral particle titer was 4 × 108 TU/mL after centrifugation. Thus, an optimized novel HIV-1 lentiviral vector was successfully constructed.
文摘Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tumor cell lines were infected with LGSN amphotropic virus. Tumor cell lines that stably express EGFP were selected with G418. The integration and expression of EGFP gene were analyzed by polymerase chain reaction, and flow cytometry (FCM). Results: After gene transfection and ping-pong transduction, amphotropic producer line Am12/LGSN was generated with a stable green fluorescence signal readily detectable by FCM in up to 97% of examined cells. The viral titer in the supernatants was up to 8.2×105CFU/ml. After transduction and selection, G418-resistant leukemia K562, mammary carcinoma MCF-7, and bladder cancer 5637 cells were developed, in which the integration of both EGFP and neomycin resistance gene was confirmed by DNA amplification. In comparison with uninfected cells, FCM analysis revealed EGFP expression in up to 90% (range 85.5%–90.0%) of tumor cells containing LGSN provirus. Conclusion: The retroviral vector LGSN can effectively mark the human tumor cells with a stably EGFP expression which may be in studying tumor growth, metastasis and angiogenesis.