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.

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.

Rapid Purification of Enhanced Green Fluorescent Protein from Escherichia coli

As an excellent reporter molecule, enhanced green fluorescent protein (eGFP) was widely used for gene expression and regulation and was generally expressed in Escherichia coli strain. A rapid procedure consisting of ammonium sulfate precipitation, size exclusion chromatography, and anion exchange chromatography was devel- oped for the purification of eGFP. Based on the proposed procedure, recombinant eGFP with an electrophoretic pu- rity was achieved in combination with an overall yield of 66% and a purification factor of 17.9. The fluorescent spectrometry of purified eGFP and lysate from E. coli strain expressing eGFP exhibited the same wavelength of ex- citation and emission maxima, indicating that the purification procedure did not influence the construct and fluo- rescent characteristics of desired protein. The procedure mentioned was easy to scale up for the purification of large quantities of eGFP.

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

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

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

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.

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.

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.

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.

Labeling embryonic stem cells with enhanced green fluorescent protein on the hypoxanthineguanine phosphoribosyl transferase locus

To label embryonic stem (ES) cells with enhanced green fluorescent protein (EGF P) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for t he first time to provide a convenient and efficient way for cell tracking and ma nipulation in the studies of transplantation and stem cell therapy Methods Homologous fragments were obtained by polymerase chain reaction (PCR), from whic h the gene targeting vector pHPRT EGFP was constructed The linearized vector was introduced into ES cells by electroporation The G418 r6TG r cell clones were obtained after selection with G418 and 6TG media The integration patterns of these resistant cell clones were identified with Southern blotting Results EGFP expressing ES cells on the locus of HPRT were successfu lly generated They have normal properties, such as karyotype, viability and di fferentiation ability The green fluorescence of EGFP expressing cells was main tained in propagation of the ES cells for more than 30 passages and in different iated cells Cultured in suspension, the 'green' ES cells aggregated and forme d embryoid bodies, retaining the green fluorescence at varying developmental sta ges The 'green' embryoid bodies could expand and differentiate into various t ypes of cells, exhibiting ubiquitous green fluorescence Conclusions This generation of 'green' targeted ES cells is described in an efficient proto col for obtaining the homologous fragments by PCR Introducing the marker gene in the genome of ES cells, we should be able to manipulate them in vitro and use them as vehicles in cell replacement therapy as well as for other biomedical a nd research purposes

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.

Production of Transgenic Korean Native Cattle Expressing Enhanced Green Fluorescent Protein Using a FIV-Based Lentiviral Vector Injected into MII Oocytes

The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins. Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer, but these approaches have been largely ineffective; however, a third approach, lentivirus-mediated transgenesis, has successfully produced transgenic livestock. In this study, we generated transgenic （TG） Korean native cattle using perivitelline space injection of viral vectors, which expressed enhanced green fluorescent protein （EGFP） systemically. Two different types of lentiviral vectors derived from feline immunodeficiency virus （FIV） and human immunodeficiency virus （HIV） carrying EGFP were injected into the perivitelline space of MII oocytes. EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group （47.5% ± 2.2% v.s. 22.9% 4± 2.9%）. Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers. Ten heifers were successfully impregnated and delivered 10 healthy calves. All of these calves expressed EGFP as detected by in vivo imaging, PCR and Southern blotting. In addition, we established an EGFP-expressing cell line from TG calves, which was followed by nuclear transfer （NT）. Recloned 8-cell embryos also expressed EGFP, and there were no differences in the rates of fusion, cleavage and development between cells derived from TG and non-TG calves, which were subsequently used for NT. These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle. Moreover, our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells.

The labeling of C57BL/6j derived embryonic stem cells with enhanced green fluorescent protein

Objective To labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application.Methods The EGFP gene was controlled by the hybrid CA promoter/enhancer (CMV enhancer/ chicken beta-actin promoter/ beta-actin intron) to construct the vector of the transgene, pCA-EGFP. The vector was transfected into MESPU35 by electroporation.Results We generated EGFP expressing ES cells demonstrating normal properties. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages as well as in differentiated cells. Cultured in suspension, the 'green' ES cells aggregated, and formed embryoid bodies maintaining the green fluorescence at varying developmental stages. The 'green' embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence. Conclusions The hybrid CA promoter/enhancer used to control the EGFP expressing ES cells, resulted in more intense and ubiquitous activity. The EGFP transfected cells yield bright green fluorescence, which can be visualized in real time and in situ. In addition, the ES cells, MESPU35, are derived from C57BL/6j mice, which are the most widely used in oncology, physiology and genetics. Compared to 129 substrains, C57BL/6j mice avoid a number of potential problems apparent in the other strains.

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.

Fusion of EGFP and porcine α 1,3GT genes decrease GFP expression

Objective:To investigate the effect of fusion proteins expressed by the fused gene of porcineα1,3 galactosyltransierase(α1,3 GT) and enhanced green fluorescent protein(EGFP) on the green fluorescence intensity of EGFP.Methods:The fragment containingα1.3GT was firstly recovered after the pcDNA3.1-α1.3GT recombinant vector were digested with BamHl and EcoRI,and then,the resultant fragment was ligated to the pEGFP-N1 vector which was also digested with the same enzymes.The new recombinant eukaryotic expression pEGFP/a 1,3GT vector was obtained and sequenced.The pEGFP/α1,3GT was used to transfect human lung carcinoma cells A549 and HEKC 293FT,and the expression of EGFP was quantitatively analyzed by fluorescent microscope and flow cytometry.Results:The positive percentage of A549 was 80.5%,and that of 293 FT was 86.5%48 hours after the two cell lines both were transfected by pEGFP-N1.The positive percentage of A549 was 75.8%,and that of 293 FT was 81.2%48 hours after the two cell lines were transfected by pEGFP/α1.3GT.The mean fluorescence intensities of A549 transfected with pEGFP-N1 and pEGFP/α1.3GT were 1.21 and 0.956,respectively when compared with that of A549 without transfection.Meanwhile,the those of the 293FT that were transfected with pEGFP-N1 and pEGFP/αl,3GT were 7.66 and 1.00.respectively when compared with that of 293FT cells without transfection.Conclusions:These results suggested that the expression of EGFP gene fused with porcineα1,3GT gene was partly inhibited.