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

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.

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.

Establishment of Stable High Expression Cell Line with Green Fluorescent Protein and Resistance Genes

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.

Influence of Insertion of the Last Sense Codon on Expression Efficiency of Green Fluorescent Protein Gene in Escherichia coli

We studied the relationship between insertion of the last sense codon (i.e., the codon preceding the stop codon) and the efficiency of gene expression. We inserted 64 kinds of last sense codon at the 5’ end of the stop codon of the green fluorescent protein (GFP) gene and introduced the modified GFP genes into Escherichia coli (E. coli). Measuring the fluorescence intensity of the GFP produced in E. coli showed that the last sense codon influenced GFP gene expression and when CCG was inserted as the last sense codon, fluorescence intensity of E. coli was increased to 2.09 fold. On the other hand, insertion of CUA caused decrease of fluorescence intensity to 0.33 fold. We hope that our findings, which may be applicable to gene engineering, will be useful for further studies of protein expression.

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

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.

Construction and expression of an optimized, novel human immunodeficiency virus type-1 lentiviral vector containing green fluorescent protein

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.

THE ENHANCED GREEN FLUORESCENT PROTEIN AS A MARKER FOR HUMAN TUMOR CELLS LABELLED BY RETROVIRAL TRANSDUCTION

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.

A Viral Expression Vector from Foxtail mosaic virus to Express Green Fluorescent Protein

[Objective]Foxtail mosaic virus(FoMV)infects gramineous and dicotyledonous plants.In this study,we sought to construct a viral vector based on FoMV to express exogenous proteins in plants.[Method]A recombinant viral expression vector was constructed by inserting the promotor of Potato virus X(PVX)and exogenous gene sequences into the 3’non-coding region of the FoMV coat protein gene.[Results]The plasmid pCB301-FoMV-CP-PVXprom-GFP expressed green fluorescent protein in inoculated Nicotiana benthamiana leaves.[Conclusion]A recombinant viral expression vector was constructed successfully.

Expression of green fluoscrescent protein gene in Sclerotinia sclerotiorum

Protoplasts of the pathogenic plant fungus,Sclerotinia sclerotiorum,were transformed using the pPGF plasmid,which contains green fluorescent protein gene,under the control of Aspergillus nidulans regulatory sequences. The pPGF plasmid was introduced by PEG/CaCl2 treatment. Positive transformants were harvested with hygromycin B (HYG) resistance as selective marker,and then were observed with green fluorescence phenomena in response to blue light,which suggested that GFP gene was cloned into genome DNA of S. sclerotiorum. The transformants were verified mitotically stable by Southern blotting analysis and passage culturing. This study is developed as an initial step for further research into infection mechanisms of S. sclerotiorum to plants and interactions with bio-control fungus.

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.

Fluorescence Tracking of Exogenous DNA in Genetic Transformation of the Chinese Oak Silkmoth Antheraea Pernyi via Sperm-Mediated Gene Transfer

Exogenous DNA expressing green fluorescent protein( GFP) and labeled with fluorescein isothiocyanate( FITC) was used to transform the Chinese oak silkmoth Antheraea pernyi( A. pernyi)via sperm-mediated gene transfer( SMGT). Sperms entry into the female reproductive system and eggs were observed using fluorescence microscopy. The ability of A. pernyi sperms to uptake exogenous DNA was confirmed,and transfer of the exogenous DNA was shown by GFP expression in the transgenic eggs. Our result suggested that SMGT could also be used to directly generate transgenic A. pernyi expressing functional genes of interest.

Application of Green Fluorescent Protein Gene (gfp) in the Symbiosis between Mesorhizobium Huakuii and Astragalus Sinicus

Green fluorescent protein (GFP) is a luminescent protein which was first discovered from A equoreavicto-ria[1]. The chromophore of wild type GFP consists of an imidazoione ring formed by cyclization of Ser65, dehydrated Tyr66 and Gly67. GFP can emit 510 nm green fluorescence (Emmax = 510 nm) by 395 nm

小鼠pLVX-Wnt3a-IRES-ZsGreen1慢病毒载体的构建及神经干细胞转染

目的构建共表达小鼠Wnt3a(mWnt3a)与绿色荧光蛋白(GFP)慢病毒载体,感染神经干细胞(NSCs),观察mWnt3a在NSCs中的表达。方法利用同源重组技术将mWnt3a基因插入慢病毒载体pLVX-IRES-ZsGreen1,构建pLVX-Wnt3a-IRES-Zs-Green1慢病毒重组质粒,通过瞬时转染法包装出病毒上清,感染NSCs,设为Wnt3a-NSCs组;同时设GFP感染NSCs组(GFP-NSCs组)和未感染NSCs组(NSCs组)作为对照。免疫荧光染色法对Wnt3a-NSCs组NSCs进行nestin鉴定;Real-Time PCR检测各组细胞mWnt3a mRNA的表达;Western blotting检测各组细胞mWnt3a、β-catenin蛋白的表达。结果经限制性内切酶检测、基因测序和绿色荧光观察证实成功构建了携带mWnt3a基因的重组慢病毒,且慢病毒滴度达3×108TU/mL。Wnt3a-NSCs组NSCs在荧光显微镜下证实有绿色荧光,且nestin表达阳性。Real-Time PCR和Western blotting结果显示感染后7 d,Wnt3a-NSCs组mWnt3a mRNA和蛋白以及β-catenin蛋白均明显高于GFP-NSCs组和NSCs组(P<0.01)。结论成功构建了表达mWnt3a基因的慢病毒载体,在体外培养条件下可以成功转染NSCs。

重组慢病毒转染兔骨髓间充质干细胞与脱钙骨基质构建转基因组织工程材料

背景:缺损组织的修复重建受限于自体或异体可替代移植材料的来源问题而导致临床应用受限,转基因干细胞和组织工程材料研究开辟了新的治疗思路。目的:探究增强型绿色荧光蛋白重组慢病毒转染兔骨髓间充质干细胞在体外的生物学特性以及与脱钙骨基质体外构建转基因组织工程材料的生物矿化特性。方法:细胞贴壁及密度离心法获得兔骨髓间充质干细胞,增强型绿色荧光蛋白重组慢病毒以感染复数为100转染第5代兔骨髓间充质干细胞,体外观察转染细胞与未转染细胞的增殖能力、细胞表型、细胞周期以及成骨诱导后碱性磷酸酶、Runx2、骨钙素表达的差异;增强型绿色荧光蛋白重组慢病毒转染骨髓间充质干细胞与脱钙骨基质在体外构建转基因组织工程材料,对其进行扫描电镜观察及元素能谱分析。结果与结论:增强型绿色荧光蛋白重组慢病毒成功转染骨髓间充质干细胞后,在转染24,48 h细胞增殖较未转染细胞缓慢(P <0.05);在转染72 h后,细胞表型未发生变异,细胞周期、细胞增殖能力以及成骨诱导后碱性磷酸酶、Runx2、骨钙素表达量与未转染细胞无明显差异(P> 0.05);增强型绿色荧光蛋白标记的骨髓间充质干细胞在脱钙骨基质支架上有较好的生物相容性,根据荧光表达强度推测目的基因在2周左右发挥最大生物学功能,且出现了钙磷矿化物沉积,体现出优越的生物矿化特性。

Microbubble-enhanced ultrasound exposure improves gene transfer in vascular endothelial cells

AIM： To explore the effects of ultrasound exposure combined with microbubble contrast agent （SonoVue） on the permeability of the cellular membrane and on the expression of plasrnid DNA encoding enhanced green fluorescent protein （pEGFP） transfer into human umbilical vein endothelial cells （HUVECs）. METHODS： HUVECs with fluorescein isothiocyanatedextran （FD500） and HUVECs with pEGFP were exposed to continuous wave （1.9 MHz, 80.0 mW/cm^2） for 5 min, with or without a SonoVue. The percentage of FD500 taken by the HUVECs and the transient expression rate of pEGFP in the HUVECs were examined by fluorescence microscopy and flow cytornetry, respectively. RESULTS： The percentage of FDS00-positive HUVECs in the group of ultrasound exposure combined with SonoVue was significantly higher than that of the group of ultrasound exposure alone （24.0%± 5.5% vs 66.6% ± 4.1%, P 〈 0.001）. Compared with the group of ultrasound exposure alone, the transfection expression rate of pEGFP in HUVECs was markedly increased with the addition of SonoVue （16.1% ± 1.9% vs 1.5% ± 0.2%, P 〈 0.001）. No statistical significant difference was observed in the HUVECs survival rates between the ultrasound group with and without the addition of SonoVue （94.1% ± 2.3% vs 91.1% ± 4.1% ）. CONCLUSION： The cell membrane permeability of HUVECs and the transfection efficiency of pEGFP into HUVECs exposed to ultrasound are significantly increased after addition of an ultrasound contrast agent without obvious damage to the survival of HUVECs. This non- invasive gene transfer method may be a useful tool for clinical gene therapy of hepatic tumors.

Construction of Eukaryotic Expression Vector Containing B7-1/GFP Gene and Its Expression in Osteosarcoma Cell Line

Objective： To construct eukaryotic expression vector containing B7-1/GFP geneand study its expression in osteosarcoma cell line LM8. Methods： By using gene cloning technique, eukaxyotic expression vector pEGFP-C1 was used to construct the murine B7-1 recombinant plasmid （pEGFP-C1/B7）. Recombinant plasmid was transfected into LM8 cells with liposome and was confirmed by restriction endonuclease digestion and DNA sequencing. The expression of the fusion protein was detected using fluorescence microscope and Western blot analysis. Results： The recombinant eukaryotic expression plasmid pEGFP-C1/B7 was successfully constructed, which was confirmed by DNA sequencing, RT-PGR and restriction enzymes analysis. The green fluorescent protein could be detected in the transfected LM8 with fluorescence microscope. The expected B7-1 and green fluorescent protein （GFP） fusion protein was detected by RT-PCR and Western blot. Conclusion： The eukaryotic expression vector containing B7-1/GFP gene was constructed successfully, and it could be expressed in LM8 after transfection.

Transformation of Upland Cotton (Gossypium hirsutum L.) with gfp Gene as a Visual Marker

The green-fluorescent protein （gfp） gene was evaluated as a screening marker during cotton （Gossypium hirsutum L.） transforming and plant regeneration. High expression of GFP （green-fluorescent protein） was observed in transgenic cells as early as 42 h after co-culture with Agrobacterium. Most of the stable transformation events were detected in the cells of primary vascular tissue. GFP transient expression could be detected on all the explants after co-culturing for 4 d, however, the highest GFP stable expression was recorded when the explants were co-cultured for 3 d. We believe the transient and stable expression of a foreign gene in genetic transformation were two relative but different events, because high transient expression did not surely lead to high stable transformation. Under the same conditions of in vitro culture, transgenic and non-transgenic calli exhibited different morphological characters on different stages of development. High concentration of plant growth regulators （PGRs） was efficient for somatic embryogenesis of the transgenic calli, which means that the transgenic calli need relatively higher dose of hormone for further growth and somatic embryogenesis than non-transgenic ones. Strong GFP-expression was observed in leaf, stem, petioles, floral tissues, and seedlings of T~ progeny. Segregation ratios of eight transgenic lines were scored for expression of GFP in the T~ progeny that providing further evidence of stable transformation. These results proved that GFP is a powerful reporter gene for protocol optimization, selection, and monitioring in whole transformation events.

Retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells

Objective To develop retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2 in mesenchymal stem cells.Methods Mesenchymal stem cells from New Zealand white rabbits were transduced with retroviral pLEGFP-BMP_2 vector by the optimized retroviral transduction protocol.Fluorescent microscopy's examination was to evaluate the results of the transduction,flow cytometer's analysis was to evaluate the transduction efficiency and the Fluorescence-activated cell sorting method was to sort the transduced cells.Bioactivity test from C_2C_12K_4 cells was to show the expression and bio-activity of the fusion gene.Results Fluorescent microscopy showed the success of the transduction.By flow cytometer's analysis,the mean efficiency of the transduction with EGFP was(42.8±6.1)% SD.Transduced cells were sorted efficiently by the fluorescence-activated cell sorting method and after sorting,almost of those showed the expression of BMP_2.Fluorescently and strongly bioactivity test for C_2C_12K_4 cells demonstrated that fluorescent materials were located the surface of cells and the activity of luciferase increased compared with the control.Analysis of long-term expression showed there was no difference between 2 week-time point and 3 month-time point of culture post-sorting.Conclusion Mesenchymal stem cells can be transduced efficiently by retrovirus-mediated transfer of the fusion gene encoding EGFP-BMP_2,the highly pure transduced cells are obtained by the fluorescence-activated cell sorting technique,the expressed chimeric protein embraced the double bioactivity of EGFP and BMP_2,and moreover,the expression had not attenuated over time.

Gene-modified leucoconcentrate for personalized ex vivo gene therapy in a mini pig model of moderate spinal cord injury

We previously demonstrated that gene-modified umbilical cord blood mononuclear cells overexpressing a combination of recombinant neurotrophic factors are a promising therapeutic approach for cell-mediated gene therapy for neurodegenerative diseases,neurotrauma,and stroke.In this study,using a mini pig model of spinal cord injury,we proposed for the first time the use of gene-modified leucoconcentrate prepared from peripheral blood in the plastic blood bag for personalized ex vivo gene therapy.Leucoconcentrate obtained from mini pig peripheral blood was transduced with a chimeric adenoviral vector(Ad5/35 F)that carried an enhanced green fluorescent protein(EGFP)reporter gene in the plastic blood bag.The day after blood donation,the mini pigs were subjected to moderate SCI and four hours post-surgery they were intravenously autoinfused with gene-modified leucoconcentrate.A week after gene-modified leucoconcentrate therapy,fluorescent microscopy revealed EGFP-expressing leucocytes in spinal cord at the site of contusion injury.In the spleen the groups of EGFP-positive cells located in the lymphoid follicles were observed.In vitro flow cytometry and fluorescent microscopy studies of the gene-modified leucoconcentrate samples also confirmed the production of EGFP by leucocytes.Thus,the efficacy of leucocytes transduction in the plastic blood bag and their migratory potential suggest their use for temporary production of recombinant biologically active molecules to correct certain pathological conditions.This paper presents a proof-of-concept of simple,safe and effective approach for personalized ex vivo gene therapy based on gene-modified leucoconcentrate autoinfusion.The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee(approval No.5)on May 27,2014.