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Transfection of bone marrow mesenchymal stem cells using green fluorescence protein labeled hVEGF165 recombinant plasmid mediated by liposome 被引量:5
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作者 Tao Wang Tian-An Liao Shao-Bo Zhong 《Asian Pacific Journal of Tropical Medicine》 SCIE CAS 2013年第9期739-742,共4页
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
关键词 Vascular endothelial growth factor green fluorescent protein Bone MARROW MESENCHYMAL stem cells PLASMID
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Multilineage Differentiation of Dental Pulp Stem Cells from Green Fluorescent Protein Transgenic Mice 被引量:5
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作者 Brian E.Grottkau P.Prasad Purudappa 《International Journal of Oral Science》 SCIE CAS CSCD 2010年第1期21-27,共7页
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
关键词 dental pulp stem cells multilineage differentiation green fluorescent protein
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In vivo transfection of enhanced green fluorescent protein in rat retinal ganglion cells mediated by ultrasound-induced microbubbles 被引量:3
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作者 Hong Su Su Liu +3 位作者 Zhigang Wang Wenyue Xie Bing Jiang Haibo Xiong 《Neural Regeneration Research》 SCIE CAS CSCD 2009年第6期413-417,共5页
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
关键词 ultrasound contrast agent MICROBUBBLE retinal ganglion cells in vivo gene therapy enhanced green fluorescent protein
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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? 被引量:3
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作者 Yansong Wang Gang Lü 《Neural Regeneration Research》 SCIE CAS CSCD 2010年第17期1303-1307,共5页
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
关键词 neural stem cells brain-derived neurotrophic factor TRANSPLANTATION green fluorescent protein spinal cord injury neural regeneration
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Gene transfer into primary cultures of fetal neural stem cells by a recombinant adenovirus carrying the gene for green fluorescent protein 被引量:6
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作者 Yong FU Shen-qing WANG +3 位作者 Ying-peng LIU Guo-peng WANG Jian-ting WANG Shu-sheng GONG 《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》 SCIE CAS CSCD 2008年第4期299-305,共7页
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
关键词 Recombinant adenovirus vector Viral infection Fetal neural stem cells green fluorescent protein
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Transient expression of the enhanced green fluorescent protein(egfp) gene in Sargassum horneri 被引量:1
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作者 PANG Yunlong LI Yan +2 位作者 LIU Zhengyi CUI Yulin QIN Song 《Journal of Oceanology and Limnology》 SCIE CAS CSCD 2019年第2期651-656,共6页
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
关键词 green FLUORESCENT protein (GFP) particle BOMBARDMENT SARGASSUM horneri TRANSGENESIS
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Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii 被引量:3
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作者 吴锦霞 胡章立 +2 位作者 王潮岗 黎双飞 雷安平 《Chinese Journal of Oceanology and Limnology》 SCIE CAS CSCD 2008年第3期242-247,共6页
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 con... 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. 展开更多
关键词 expression efficiency green fluorescent protein (GFP) HSP70A-RBCS2 RBCS2 transformation efficiency
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Constructing retroviral vector carrying green fluorescent protein(GFP)and investigating the expression of GFP in primary rat myoblast 被引量:1
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作者 Shuling Rong Yongxin Lu +4 位作者 Yuhua Liao Xiaolin Wang Xiaoqing Li Jiahua Zhang Yanli He 《Journal of Nanjing Medical University》 2006年第4期197-200,共4页
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
关键词 green fluorescent protein TRANSFECTION MYOBLAST
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The Influence of Heat Treatment on Interaction of Green Tea Flavonoids with Milk Proteins 被引量:1
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作者 Zerrin Yüksel Onür Elif Avci 《Journal of Agricultural Science and Technology(B)》 2018年第8期530-541,共12页
Several heat treatment norms are applied in the dairy industry during the manufacture of various products. The knowledge on the influence of temperature on the proteins-flavonoid binding is critical for optimization o... Several heat treatment norms are applied in the dairy industry during the manufacture of various products. The knowledge on the influence of temperature on the proteins-flavonoid binding is critical for optimization of process conditions. The aim of this study was to establish the effect of heat treatment on the interaction between milk proteins and flavonoids and also determine when to apply heat treatment. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis revealed that free flavonoids decreased in the presence of milk proteins and a binding ratio (%) was calculated based on this decrease. Three different heat treatment norms were selected (80, 85 and 90℃×10 min). Analyses were carried out in both sodium caseinate and skimmed milk samples to observe the possible effect of milk serum proteins on the interaction. The binding ratio between flavonoids and proteins was found to be higher in the samples where green tea extract (GTE) was added before heat treatment than in the samples where GTE was added after heat treatment in both the skimmed milk and sodium caseinate systems. Results of protein surface hydrophobicity (PSH) index and protein partition also demonstrated that heat treatment must be applied after the addition of GTE to improve the amount of GTE binding to milk proteins. 展开更多
关键词 FLAVONOIDS MILK proteinS green TEA heat treatment RP-HPLC.
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Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells 被引量:13
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作者 Qin FANG Eng Khuan Seng +1 位作者 Wen DAI Lan-lan ZHANG 《中国病毒学》 CSCD 2007年第5期397-404,共8页
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
关键词 草鱼呼肠孤病毒 VP6蛋白 增强绿色荧光蛋白 杆状病毒表达系统 共表达
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Bacillus brevis DX01 Labeling by Green Fluorescent Protein Gene and Its Colonization in Rice Seedling Roots
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作者 陈云鹏 沈大棱 《Journal of Shanghai Jiaotong university(Science)》 EI 2005年第S1期26-31,共6页
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
关键词 green FLUORESCENT protein BACILLUS brevis COLONIZATION
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Expression of the Capsid Precursor Protein gene of Foot-and-mouth Disease Virus and Green Fluorescent Protein Gene in BHK-21 Cells Mediated by Retroviral Vector
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作者 LI Jiong LIU Yan-hong +4 位作者 AN Fang-lan LIU Jun-lin LIU Xiang-tao SHANG You-jun YIN Hong 《畜牧兽医学报》 CAS CSCD 北大核心 2010年第S1期70-75,共6页
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
关键词 retroviral vector FMDV capsid precursor protein gene green fluorescent protein gene BHK-21 cell
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SIGNIFICANCE OF THE EXPRESSION OF GREEN FLUORESCENT PROTEIN ON DETECTION OF GLIOMA INVASION IN VIVO
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作者 李侠 章翔 +3 位作者 吴景文 高大宽 刘先珍 梁景文 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 2001年第1期39-43,共5页
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
关键词 GLIOMA green fluorescent protein INVASION Flow cytometry
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Construction and expression of an optimized, novel human immunodeficiency virus type-1 lentiviral vector containing green fluorescent protein
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作者 Xia Li Xueling Ma +6 位作者 Lijing Zhao Hang Gao Hongjuan Wang Li Du1 Juan Wang Nan Li Kangding Liu 《Neural Regeneration Research》 SCIE CAS CSCD 2011年第7期542-545,共4页
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
关键词 gene expression gene therapy human immunodeficiency virus 1 green fluorescent protein LENTIVIRUS neural regeneration
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THE ENHANCED GREEN FLUORESCENT PROTEIN AS A MARKER FOR HUMAN TUMOR CELLS LABELLED BY RETROVIRAL TRANSDUCTION
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作者 傅建新 王玮 +3 位作者 白霞 卢大儒 阮长耿 陈子兴 《Chinese Journal of Cancer Research》 SCIE CAS CSCD 2002年第2期126-130,共5页
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
关键词 green fluorescent protein Gene transfer Retroviral vector Cultured tumor cells
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Studies on the Sructure, Mechanism of Green Fluorescent Protein and Its Application
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作者 Liu Zu-qiang Hu Min Qi Yi-peng 《Wuhan University Journal of Natural Sciences》 CAS 2000年第2期252-252,共1页
The green fluorescent protein (GFP) from jellyfishAequorea victoria has unique superiority as a kind of biological label. GFP has been widely used in all fields of biology based on the recent studies on its structure,... The green fluorescent protein (GFP) from jellyfishAequorea victoria has unique superiority as a kind of biological label. GFP has been widely used in all fields of biology based on the recent studies on its structure, characteristic and mechanism of bioluninescence. 展开更多
关键词 green fluorescent protein biological label APPLICATION
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Establishment of Stable High Expression Cell Line with Green Fluorescent Protein and Resistance Genes
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作者 张胜桃 刘文励 +2 位作者 何培根 龚非力 杨东亮 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2006年第3期298-300,共3页
In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected... In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium. 展开更多
关键词 green fluorescent protein resistance gene stable transfection
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Mapping subgenomic promoter of coat protein gene of Cucumber green mottle mosaic virus
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作者 LIU Mei LIU Li-ming +2 位作者 WU Hui-jie KANG Bao-shan GU Qin-sheng 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2020年第1期153-163,共11页
Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMM... Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMMV)-based virus vectors have been constructed, SGP of the coat protein(CP) has not yet mapped. To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site(TSS) as previous study identified by random amplification of c DNA ends(RACE). Secondly, the region from nucleotides –110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein(MP) and CP. To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction. The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CP TSS. For the downstream of CP TSS, we analyzed the expression of enhanced green fluorescent protein(EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS. To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis. Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced. To our best knowledge, this is the first mapping of CGMMV SGP. 展开更多
关键词 Cucumber green mottle mosaic virus coat protein subgenomic promoter
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Expression of Green Fluorescent Protein Gene with Baculovirus Vectorin Insect Cells
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作者 Hu Jianhong Zhu Fanxiu +1 位作者 Qi Yipeng Huang Yongxiu 《Wuhan University Journal of Natural Sciences》 CAS 1997年第1期117-121,共5页
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
关键词 green fluorescent protein(GFP) BACULOVIRUS transfer vector insect cells polyhedrin gene neomycin resistance gene
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Jellyfish Green Fluorescent Protein(GFP) as a Reporter for Fusarium gramminearum Development on Wheat
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作者 QI Jun-xian LIU Tai-guo +4 位作者 XU Ying CHEN Huai-gu GAO Li LIU Bo CHEN Wan-quan 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2014年第10期2177-2183,共7页
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
关键词 Fusarium head blight (FHB) green fluorescent protein (GFP) resistance evaluation
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