Green fluorescent protein (GFP) gene was inserted into the pseudogene (psi) region of genome of rabies virus rHep-Flury strain, and a recombinant rabies virus carrying GFP, designated as HEP-GFP, was rescued by reverse genetics system. It was demonstrated that green fluorescent protein could be expressed in the chimeric virus after 5 passages in BHK-21 cell line. The research indicated that the pseudogene (psi) region in the genome of rHEP-Flury strain, as an independent functional unit in the process of virus assembly, could independently carry and express exogenous genes.
Animals ; Cell Line ; Cricetinae ; Green Fluorescent Proteins ; genetics ; Rabies virus ; genetics ; Recombinant Proteins ; genetics

Recently, we have reported a new gfp gene isolated from Aequorea macrodactyla. The protein purified from expressed E. coli exhibited an excitation peak at 476 nm and an emission peak at 496 nm. However, the drawback of only maturing to fluorescence at low temperature limited its applications. In this paper, we further describe twelve mutants of GFPxm. Seven mutants produced enhanced fluorescence when expressed in E. coli at higher temperature (37 degrees C). After six hours of induction at 25 degrees C, 32 degrees C and 37 degrees C respectively, the relative fluorescent intensities of GFPxm16, GFPxm18 and GFPxm19 were higher than that of EGFP, moreover GFPxm16 and GFPxm163 could preserve high fluorescent intensity even expressed at 42 degrees C. Four mutants of the seven could reach high expression level in three kind of mammalian cells. Another 6 mutants had red-shift of excitation-emission maxima, and longest excitation-emission maxima were 514nm and 525nm. Another three mutants had two excitation peaks, and one mutant had only one UV-excitation peak. The most exciting result is the mutant of OFPxm with orange color. The mutant has an excitation peak at 509 nm and an emission peak at 523nm. 523nm is yellowish green but the protein is orange observed by eyes. The mutant could reach high expression level and matured at higher temperature but the fluorescent intensity was comparatively low because of low quantum yield.
Animals ; Electrophoresis, Polyacrylamide Gel ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Luminescent Proteins ; genetics ; metabolism ; Mutation ; Temperature

In genetic modification of plants, once the transformants are obtained, selection markers are no longer required in mature plants. At present, the Cre/lox site-specific recombination system is most widely used to eliminate the selectable marker genes from the transgenic plants. In this study, attempt was made to favour the selection of marker-free plants in the re-transformation method. Green fluorescent protein (GFP) can be directly visualized in living cells, tissues or organisms under UV illumination. This advantage of GFP is exploited in the development of a practical approach in which GFP is used as a visual marker to monitor the removal of the selectable marker gene from transgenic plants. For that purpose, the pGNG binary vector was constructed, in which the GFP gene (gfp) was linked to the expression cassette Nos P-nptII-NosT and the two units were cloned between two directly-orientated lox sites. The CaMV 35S promoter was placed before the first lox site and used to drive GFP expression. The beta-glucuronidase gene (gus) of Escherichia coli was cloned behind the second lox site without a promoter, thus would not be expressed in this position. Tobacco plants were first transformed with pGNG and selected on kanamycin (Kan)-containing media. Regenerated transgenic shoots were readily singled out by GFP fluorescence. The GFP-expressing plants were then re-transformed with pCambia1300-Cre containing hygromycin phosphotransferase gene (hpt) as a selectable marker gene. The Cre-mediated recombination resulted in the elimination of lox-flanked genes, herein gfp and nptII, from the plant genome and brought the GUS gene next to the 35S promoter. Our data demonstrated that transgenic plants free of nptII were easily selected by monitoring the loss of green fluorescence, and at the same time, GUS (here as a target protein) was expressed in the nptII-free plants. Finally, hpt and cre were removed from the progenies of the nptII-free plants by gene segregation.
Genetic Markers ; Green Fluorescent Proteins ; genetics ; Plants, Genetically Modified ; genetics ; Plasmids ; Recombination, Genetic ; Tobacco ; genetics

OBJECTIVETo construct a lentiviral expression vector of has-miR-338-3p and verify its target gene.

METHODSThe pre-miR-338-3p was synthesized and inserted into pLV-THM, and the recombinant plasmid pLV-THM-miR-338-3p was confirmed by restriction endonuclease analysis and DNA sequencing. 293T cells were co-transfected with the lentiviral vector pLV-THM-miR-338-3p, psPAX2 and pMD2.G, and the supernatant containing the lentivirus particles was harvested to determine the virus titer and used to infect SW-620 cells. Flow cytometry was employed for sorting the GFP-positive cells. The expression of miR-338-3p was determined using real-time RT-PCR and the expression of SMO protein was detected with Western blotting in the infected SW-620 cells. The invasiveness of the infected SW-620 cells was assessed using Transwell assay.

RESULTSRestriction enzyme digestion and DNA sequencing demonstrated successful construction of the lentiviral vector pLV-THM-miR-338-3p. SW-620 cells infected with pLV-THM-miR-338-3p showed a significantly increased expression of miR-338-3p, and the overexpression of miR-338-3p suppressed the expression of SMO protein and the invasiveness of the cells.

CONCLUSIONThe successful construction of the lentiviral vector pLV-THM-miR-338-3p and the establishment of a SW-620 cell line with miR-338-3p overexpression provide the basis for further study of the molecular function of miR-338-3p in colorectal carcinoma. MiR-338-3p can suppress SMO gene expression to inhibit the invasiveness of colorectal carcinoma cells.

Cell Line, Tumor ; Gene Expression ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Lentivirus ; genetics ; MicroRNAs ; genetics

The main aim of this study was to transform the enhanced green fluorescent protein gene (egfp) into biocontrol fungus Paecilomyces lilacinus strain 9410. We constructed the expression vector pUPNGT of the fusion gene nptII-egfp using pcDNA3.1(-) as a helper plasmid. The egfp gene was then transformed into P. lilacinus strain 9410 via Agrobacterium tumefaciens-mediated transformation. PCR and Southern blotting analysis showed that the egfp gene was integrated into the genomes of the tested transformants and the integration manner was single-copy. The transformants could generate green fluorescence when they were excited by 488 nm blue laser. These results indicated that the egfp gene had been successfully transformed into P. lilacinus 9410 and expressed in the tested transformants. Our work may provide a new approach to assess environmental safety and practical biocontrol efficacy ofP. lilacinus under different conditions.
Agrobacterium tumefaciens ; genetics ; Green Fluorescent Proteins ; genetics ; Paecilomyces ; genetics ; metabolism ; Polymerase Chain Reaction ; methods ; Transformation, Genetic

The transfection efficiency of oligonucleotide and plasmid to the HL-60 cell line with lipofectaminePLUS was compared through observing the transfection rate and the expression duration of exogenous gene in the target cells. The results showed that the transfection rate of oligonucleotide to the HL-60 was about 90% - 95% and it had no obvious attenuation within 84 h. However, the plasmid transfection rate was only 5% -25% and it was decreased significantly within 60 h. It was suggested that the transfection of oligonucleotide with liposomes was better than that of plasmid.
Green Fluorescent Proteins/genetics ; HL-60 Cells ; Liposomes ; Oligonucleotides/*genetics ; Plasmids/*genetics ; Transfection

AIMTo study polyethylenimine (PEI)-mediated in vivo gene transfection into testis cells and preliminary functional research of spermatogenic cell-specific gene NYD-SP12 using this method.

METHODSPEI/DNA complexes were introduced into the seminiferous tubules of mouse testes using intratesticular injection. Transfection efficiency and speciality were analyzed on the third day of transfection with fluorescent microscopy and hematoxylin staining. The long-lasting expression of the GFP-NYD-SP12 fusion protein and its subcellular localization in spermatogenic cells at different stages were analyzed with fluorescent microscopy and propidium iodide staining.

RESULTSWith the mediation of PEI, the GFP-NYD-SP12 fusion gene was efficiently transferred and expressed in the germ cells (especially in primary spermatocytes). Transfection into Sertoli cells was not observed. The subcellular localization of the GFP-NYD-SP2 fusion protein showed dynamic shifts in spermatogenic cells at different stages during spermatogenesis.

CONCLUSIONPEI can efficiently mediate gene transfer into spermatocytes. Thus, it might be useful for the functional research of spermatogenic-cell specific genes such as the NYD-SP12 gene. In our study, the NYD-SP12 protein was visualized and was involved in the formation of acrosome during spermatogenesis. Our research will continue into the detailed function of NYD-SP12 in spermatocytes.

Animals ; Green Fluorescent Proteins ; Homeodomain Proteins ; genetics ; physiology ; Humans ; Male ; Mice ; Polyethyleneimine ; Spermatogenesis ; physiology ; Transfection ; methods

OBJECTIVETo compare three different methods in transfection of plasmid pGPU6/GFP/Neo into chicken embryo fibroblast.

METHODSDifferent doses (1.25 μl,2 μl,2.5 μl) of Lipofec-tamin2000, Gbfectene-Elite and HilyMax were used to transfect 1 μg plasmid pGPU6/GFP/Neo. The transfection efficiency was observed by the fluorescence and the cell viabilities were measured.

RESULTSThe transfection efficiency of HilyMax was significant higher than that of Lipofectamin2000 and Gbfectene-Elite (86.85%±2.32% compared with 48.33%±3.24% and compared with 37.35%±5.41%; F=18.882, P<0.05). The transfection efficiency of 2.5 μl HilyMax was highest(90.53%±1.15%). The cell viability of Lipofectamin2000 was significantly lower than that of HilyMax and Gbfectene-Elite(65.76%±5.78% compared with 89.54%±0.86% and compared with 82.45%±3.56%;F=90.676, P<0.05).

CONCLUSIONHilyMax has the highest transfection efficiency and the lowest cell toxicity in transfection of plasmid pGPU6/GFP/Neo into chicken embryo fibroblast, with the optimal ratio of 2.5 μl HilyMax:1 μg plasmid.

Animals ; Cell Line ; Chick Embryo ; Fibroblasts ; cytology ; Green Fluorescent Proteins ; genetics ; Plasmids ; genetics ; Transfection ; methods

Cell Line ; Embryonic Stem Cells ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Lentivirus ; genetics ; Transfection

BACKGROUND AND OBJECTIVEThe p53 as a transcription factor in cell stress was activated to regulate cell cycle and programmed cell death to inhibit tumor growth. Usually, p53 is kept in non-activated state through various mechanisms, including the action of p53 C-terminal negative regulatory sequences. The purpose of the study is to prepare the two types p53 recombinant adenoviruses that carry full-length p53 as well as deletion of negative regulatory sequences at p53 C-terminus and to detect exogenous GFP expression in human lung cancer cell infected-virus by FCM scatter plot.

METHODSUsing pAdEasy-Track vector system the p53 recombinant plasmids was constructed and the homologous recombinants in E. coli was produced. The three kinds of recombinant adenovirus in L293 cells was generated, sequencing proved. Exogenous GFP expression in human lung cancer 801D cells infected-virus was detected by FCM scatter plot.

RESULTSp53 recombinant adenoviruses named Ad-p53(wtp), Ad-p53(del) and Ad-(empty carrier) were produced. Results of sequences indicate that the Ad-p53(del) was deletion of 111 bases before stop codon TGA and of 3 untranslated region at p53, the Ad-p53(wtp) no loss of any p53 base, the Ad-(empty carrier) no p53 sequence. FCM scatter plot indicate the percentage of 801D cells expressed GFP with three kinds of viral infection was almost same and was increased with the virus density. 801D contains ratio of cells with different fluorescence intensity.

CONCLUSIONThe preparation of recombinant adenovirus, Ad-p53(del), pA-p53(wtp) and Ad-(empty carrier). The cells expressed-GFP can be quantitatively detected by FCM scatter plot. It was provide that the reliability of the virus system and accurate method for selecting viruses density to infecting cells.

Adenoviridae ; genetics ; Animals ; Flow Cytometry ; methods ; Genes, p53 ; Green Fluorescent Proteins ; genetics ; Humans ; Mice ; Recombination, Genetic