In the present study, packaging system composed of pAAV-CMV-GFP, pAAV-RC and pHelper were transfected into human embryonic kidney 293 cells (HEK293 cells) mediated by polyethyleneimine (PEI) to explore an optimal transfection condition. Different total plasmid DNA dosages (1, 2, 3, 4, 5, 6 μg) and different PEI/Plasmid ratios (1:1, 3:1, 5:1, 7:1) were tested with detection of green fluorescence protein (GFP) with ImagePro Plus6. 0 Software. Then transfection efficiency of the optimized transfection system was further observed for different time periods(12, 24, 36, 48, 60, 72 h). The results showed that total plasmid dosage of 4 μg/well with PEI/plasmid ratio of 3 : 1-5 : 1 was an efficient transfection condition. Transfection efficiency-time curve was an S-shaped curve. Transfection efficiency reached a plateau at 60 h after transfection. The optimized conditions for PEI-mediated transfection at the optimal time result in enhanced transfection efficiency of triple plasmid into HEK293 cells.
Green Fluorescent Proteins ; HEK293 Cells ; Humans ; Plasmids ; Polyethyleneimine ; Transfection ; methods

OBJECTIVETo construct strains containing green fluorescent protein (GFP) to study gene regulation in Saccharomyces albicans cells during the infection process.

METHODSpACT1-GFP was constructed, and Saccharomyces albicans CAI4 was transformed. The expression of GFP in yeast and hyphal compartments was observed with microscopy.

RESULTS99% of Saccharomyces albicans cells containing pACT1-GFP fusion displayed significant fluorescence levels both in the yeast and hyphal compartments. The fluorescence intensity in two compartments had no obvious difference.

CONCLUSIONpACT1-GFP can be expressed stably in the yeast cells.

Candida albicans ; Green Fluorescent Proteins ; Humans ; Plasmids ; Saccharomyces ; Saccharomyces cerevisiae

OBJECTIVE: To construct a eukaryotic expression vector of human tissue factor pathway inhibitor-2 (TFPI-2) and to investigate the effect of TFPI-2 gene on the growth of acute monocytic leukemia cell line (SHI-1). METHODS: The cDNA of TFPI-2 was obtained by genetic chemical synthesis, the TFPI-2 gene and the linear vector fragment were ligated and inserted into the multiple cloning site of PEGFP-N1 vector, and the eukaryotic expression vector PEGFP-N1-TFPI-2 was transfected SHI-1 cells, then the obtained SHI-1 cells was observed by fluorescence microscopy; MTT assay was used to detect the effect of TFPI-2 gene on the relative growth rate of SHI-1 cells at the different time-point; RT-PCR was used to detect TFPI-2 mRNA expression levels in the cells of each group before and after TFPI-2 transfection; TFPI-2 protein expression was detected by Western blot. The cells which successfully transfected with PEGFP-N1-TFPI-2 vector were named as SHI-1-TFPI-2 (experimental group), and the cells transfected with the empty vector pEGFP-N1 and the untransfected cells were named as SHI-1-V and SHI-1-P and used as the control group. RESULTS: The human TFPI-2 gene eukaryotic expression vector PEGFP-N1-TFPI-2 was successfully constructed, then the transfected into SHI-1 cells, observed by fluorescence microscopy 24 hours later, as a result, the PEGFP-N1-TFPI-2 was successfully transferred into SHI-1 cells, and the number of fluorescent cells increased after 48 h and 72 h. RT-PCR showed that the gray scale ratio of TFPI-2 gene to β- actin in the experimental group was higher than that in the control group. The gray scale ratio was 0.51±0.04 in SHI-1-V group, 0.52±0.03 in SHI-1-P group, 0.87±0.08 in SHI-1-TFPI-2 group, and the difference between SHI-1-TFPI-2 and SHI-1-V, SHI-1-P group was statistically significant (P＜0.05). CONCLUSION The expression of TFPI-2 gene in PEGFP-N1-TFPI-2 can inhibit the growth of SHI-1 cells, which provides a research direction for gene therapy of leukemia in the future.
Eukaryota ; Genetic Vectors ; Glycoproteins ; metabolism ; Green Fluorescent Proteins ; Humans ; Transfection

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

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

Self-assembling amphipathic peptides (SAPs) have alternating hydrophilic and hydrophobic residues and can affect the thermal stabilities and catalytic properties of the fused enzymes. In this study, a novel multifunctional tag, S1vw (HNANARARHNANARARHNANARARHNARARAR) was developed to modify fused enzymes. After fusing S1vw at the enzymes/proteins N-terminus through a PT-linker, the crude enzymatic activities of polygalacturonate lyase and lipoxygenase were enhanced 3.1- and 1.89-fold, respectively, compared to the wild-type proteins. The relative fluorescence intensity of the green fluorescent protein was enhanced 16.22-fold. All the three S1vw fusions could be purified by nickel column with high purities and acceptable recovery rates. Moreover, S1vw also induced the thermostabilities enhancement of the fusions, with polygalacturonate lyase and lipoxygenase fusions exhibiting 2.16- and 3.2-fold increase compared with the corresponding wild-type, respectively. In addition, S1vw could enhance the production yield of green fluorescent protein in Escherichia coli and Bacillus subtilis while the production of GFP and its S1vw fusion changed slightly in Pichia pastoris. These results indicated that S1vw could be used as a multifunctional tag to benefit the production, thermal stability and purification of the fusion protein in prokaryotic expression system.
Escherichia coli ; Green Fluorescent Proteins ; Hydrophobic and Hydrophilic Interactions ; Peptides ; Pichia ; Recombinant Fusion Proteins ; metabolism

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

OBJECTIVETo investigate and evaluate the appropriate virus titer and transfection efficiency of recombinant adenovirus p53 into the oral dysplastic epithelial cells (POE-9n) and provide reference for oral precancerosis research.

METHODSThe transfection sensitivity of adenovirus into oral dysplastic epithelial cells was evaluated by the recombinant adenovirus p53 containing green fluorescent protein (rAd-GFP). Different titre rAd -p53 was transfected into oral dysplastic epithelial cells to evaluate the effects of rAd-p53 on cell proliferation inhibition by MIT assay. The expression of exogenous p53 gene in POE-9n cells was detected by immunocytochemistry.

RESULTSMore than 95% POE-9n cells were transfected by rAd-GFP with MOI from 100 to 500 and there was no statistical difference between different MOI values (r=-0.124, P>0.05). It was found that rAd-p53 had significant inhibition effects on POE-9n cell proliferation with MOI from 100 to 500, and there were no significant differences at 96 h and 120 h after the transfection on cell proliferation inhibition (P>0.05). P53 protein was well expressed in rAd-p53 transfected POE-9n cells.

CONCLUSIONExogenous p53 can be successfully transfected into POE-9n cells by rAd-p53 and the virus titer of MOI 100 was high enough to ensure efficient transfection.

Adenoviridae ; Cell Proliferation ; Epithelial Cells ; Genes, p53 ; Genetic Vectors ; Green Fluorescent Proteins ; Humans ; Transfection

To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide (AS-ODNs) by insonated gas-filled lipid microbubbles, SF6-filled microbubbles were prepared by sonication-lyophilization method. An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles. Three levels of mixing speed, different durations of mixing and various delay time before ultrasound were examined, separately. Transfection efficiency was detected by fluorescence microscopy. Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared. From the results, there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells. AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration, but there is a negative relationship with delay time before ultrasound. The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r x min(-1) for 30-60 s with less than 60 s delay before ultrasound. For a successful transfection, long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids, because these vectors depend on endocytosis and membrane fusion to realize transfection. Unlike liposomes and cationic lipid-polymer hybrids, gas-filled lipid microbubbles depend on sonorporation effect to realize transfection. Therefore, the incubation of gene and microbubbles before mixing cells may not be necessary. Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.
Cell Line, Tumor ; Green Fluorescent Proteins ; Humans ; Microbubbles ; Oligodeoxyribonucleotides, Antisense ; genetics ; Sulfur Hexafluoride ; Transfection ; methods ; Ultrasonics