No abstract available.
Ovarian Neoplasms* ; Transfection*

OBJECTIVETo construct a eukaryotic vector which could express bone morphogenetic protein-7 (bmp-7) in MC3T3-E1.

METHODSBone morphogenetic protein-7 gene was obtained by RT-PCR from human embryo kidney. And after sequencing and electrophoresis the obtained aim DNA fragment was inserted into eukaryotic expression plasmid pcDNA3.1 (+) by using restricted endonuclease and ligase. The DNA sequence of the newly-constructed plamids was proved right by the gene technic company. And then the new plasmids containing right sequence aim gene were transfected into MC3T3-E1 cells by Lipofectamine 2000. 72 h after transfecting, RT-PCR was performed to show the transfected cells containing the aim gene, and the whole protein of the transfected cells were gathered and used as samples in the next Western blot to test the expression of bmp-7 gene.

RESULTSDNA sequencing indicated the sequence of the obtained bmp-7 was identical to the reported ones in GeneBank. The electrophoretic map of the products of RT-PCR and restriction enzyme digestion played another evidence that the newly-constructed plasmids were bmp-7/pcDNA3.1(+). The results of Western blot showed that the transfected cells could express BMP-7.

CONCLUSIONThe construction of a eukaryotic vector which could express BMP-7 in MC3T3-E1 was successful.

OBJECTIVETo observe the therapeutic effect of CDglyTK gene mediated by synthetic radiation-inducible promoter in the treatment of Tca8113 cells.

METHODSCDglyTK gene in pCEA-CDglyTK was subcloned into pcDNA3.1 (+) to construct plasmid pcDNA3.1 (+)-CDglyTK, and then the synthetic radiation-inducible promoter in pMD18 -T -E was inserted into pcDNA3.1 (+) -CDglyTK to construct plasmid pcDNA3.1 (+ )/E -CDglyTK. The recombinant plasmid was transfected into Tca8113 cells by lipofectamine, and then exposed to 3 Gy irradiation. Cytotoxicity was evaluated by MTT. The expression of CDglyTK gene was detected by RT-PCR. The apoptosis and proliferation were examined by flow cytomtery.

RESULTSThe plasmid pcDNA3.1 (+)/E-CDglyTK was constructed successfully. The comparative survival rate of Tca8113 cells was markedly decreased by induction irradiation. Up-regulation of CDglyTK expression was found in Tca8113 cells exposed to irradiation. The apoptosis index (AI) of Tca8113 cells exposed to irradiation was higher than that of Tca8113 cells without irradiation, the other way round, the proliferation index (PI) of Tca8113 cells exposed to irradiation was lower than that of Tca8113 cells without irradiation.

CONCLUSIONThe synthetic radiation-inducible promoter can be served as a molecular switch to improve the expression of CDglyTK gene in Tca8113 cells, and low dose induction radiation can significantly improve the therapeutic efficiency.

TGF-p receptor mutation is now considered as one of the carcinogenic process of many tumors. To evaluate whether there is an abnormality in TGF-p type II receptor in osteosarcoma cell lines, we performed Northern analysis, cross-linking assay, luciferase activity and TGF-p growth inhibition assay in four osteosarcoma cell lines: G292, U202, HOS and SaOS. We also transfected the tumor cells with normal TGF-p type II receptor sequence to find if there is a possibility of gene therapy in osteosarcoma. In Northern analysis, Type II receptor expressions were decreased at SaOS, U202 and HOS cell lines. In cross-linking assay, all four cell lines didnt show type II receptor at their cell surface. The growth of these tumor cells were not suppressed by TGF-p. From these findings, we concluded that the normal production of TGF-p type II receptor was impaired in osteosarcoma. The transfection of these tumor cells with normal type II receptor sequence restored growth inhibition by TGF-p. This means even though TGF-p type II receptor is abnormal in osteosarcoma, we can restore its function by transfection of normal sequence. We think that the TGF-p type Il receptor gene therapy can be one of the treatment method for osteosarcoma in the future.
Cell Line* ; Genetic Therapy ; Luciferases ; Osteosarcoma* ; Transfection

To construct a eukaryotic expression plasmid containing the luciferase reporter gene (Fluc) to quickly detect apoptosis. Four amino acids, Asp-Glu-Val-Asp (DEVD), the recognize motif of Caspase-3, were introduced into the middle of the Fluc-C and N fragment. Meanwhile, four amino acids, Asp-Glu-Val-Gly (DEVG), were selected as a negative control. Subsequently, the recombinant gene was cloned into the N and C terminal end of the split intein, and named as pFluc-DEVD and pFluc-DEVG. Then the plasmids were transfected into cells and renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. Then the apoptosis level was detected by the double luciferase reporter gene and the Western blotting analysis. The results showed that when apoptosis occurred, the content of firefly luciferase expressed in the pFluc-DEVD plasmid transfected group was about 3 times higher than pFluc-DEVG plasmid transfected group. Furthermore, Western blotting detection indicated that the Fluc level was significantly increased in pFluc-DEVD transfected group when pre-treated by apoptosis stimulants. The activation degree of Caspase-3 was closely related to the expression of Fluc, and had a significant statistical difference. These results confirmed that firefly luciferase protein expressed by pFluc-DEVD plasmid can be cleaved by the intracellular Caspase-3 enzyme, and this plasmid can accurately reflect the cell apoptosis level, which provides a useful method for quantitative detection of apoptosis.
Apoptosis ; Genes, Reporter ; Luciferases, Firefly ; Transfection

OBJECTIVE: To explore the effects of the rat FVIII light chain (rLC) on the activity of human FVIII heavy chain hHC745 and hHC1690. METHODS: hHC745, hHC1690, human FVIII light chain (hLC) and rLC were cloned into adeno-associated virus serotype 8 (AAV8) expression vectors with CB promoter (ubiquitous expression), respectively, and transfected into 293T cells using a dual-chain strategy of co-expression of HC and LC. The cultured cell supernatant was collected at 48 hours after transfection. The plasmids (pAAV8-CB-hHC745, pAAV8-CB-hHC1690, pAAV8-CB-hLC and pAAV8-CB-rLC) were hydrodynamically injected into hemophilia A (HA) mice via lateral tail vein. Forty-eight hours after injection, the peripheral blood of the mice was collected through retroorbital venous plexus and the plasma was obtained by centrifugation. The activity of FVIII was detected by activated partial thromboplastin time (aPTT) assay, and the antigen expression level of FVIII was determined by enzyme-linked immunosorbent assay (ELISA). The specific activity of FVIII was calculated based on the activity and the antigen expression level. RESULTS: In 293T cells, the coagulation activity of FVIII was significantly enhanced when hHC745 and hHC1690 combined with rLC compared with them combined with hLC. The FVIII activity of hHC745+rLC was about 4.6 times higher than that of hHC745+hLC, while hHC1690+rLC was about 2.9 times higher than that of hHC1690+hLC. The data from ELISA showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.1 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 2.8 times compared with hHC1690+hLC. In HA mice administrated with hydrodynamic injection, the FVIII activity of hHC745+rLC and hHC1960+rLC was significantly higher than that of hHC745+hLC and hHC1690+hLC at comparable expression level. The FVIII activity of hHC745+rLC was significantly higher than that of hHC745+hLC, increasing to about 5.1 times, while, that of hHC1690+rLC increasing to about 2.1 times than hHC1690+hLC. ELISA results also showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.2 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 1.8 times compared with hHC1690+hLC. In addition, the activity of hHC1690 combined with rLC was significantly higher than that of hHC745 combined with rLC. CONCLUSION rLC can significantly enhance the coagulation activity of FVIII when co-expressed with hHC of different length including hHC745 and hHC1690.
Rats ; Humans ; Mice ; Animals ; Dependovirus/genetics* ; Transfection

OBJECTIVETo study the influence of C-terminal domain of ADAMTS13 on its cleaving activity.

METHODSThe full-length wild-type (WT) and C-terminal domain truncated type (TT, TSP8 + CUB domains were deleted) of human ADAMTS13 recombinant protein were transfected into and permanent expressed on Hela cells. Western blot and R-CBA were used to directly detect the activities of the two recombinant proteins under the static and stressed condition respectively. ELISA was used to compare the binding abilities of the two proteins by coating with vWF.

RESULTSThe recombinant proteins were identified by Western blot with anti-his-tag or anti-ADAMTS13 antibodies. With pretreatment of 1.5 M urea, the enzyme activity of TT was significantly higher than that of WT, and so did in binding ability with vWF While, only WT could cleave vWF under high stress.

CONCLUSIONThe distal carboxyl-terminal TSP8 together with CUB domains of ADAMTS13 may affect the enzyme activity by regulating the binding of ADAMTS13 to vWF in different conditions, and they are very important for the enzyme activity under high stress force condition.

In order to study the potential of Venus, lentiviral vector, applied to acute myeloid leukemia, the recombinant vector Venus-C3aR was transfected into 293T packing cells by DNA-calcium phosphate coprecipitation. All virus stocks were collected and transfected into HL-60, the GFP expression in HL-60 cells was measured by flow cytometry. The expression level of C3aR1 in transfected HL-60 cells was identified by RT-PCR and flow cytometry. The lentiviral toxicity on HL-60 was measured by using CCK-8 method and the ability of cell differentiation was observed. The results indicated that the transfection efficacy of lentiviral vector on HL-60 cells was more than 95%, which meets the needs for further study. C3aR1 expression on HL-60 cells increased after being transfected with recombinant lentiviral vector. Before and after transfection, the proliferation and differentiation of cells were not changed much. It is concluded that the lentiviral vector showed a high efficacy to transfect AML cells and can be integrated in genome of HL-60 cells to realize the stable expression of interest gene. Meanwhile, lentiviral vector can not affect HL-60 cell ability to proliferate and differentiate.
Genetic Vectors ; HL-60 Cells ; Humans ; Lentivirus ; genetics ; Transfection

Gene therapy has been considered as one of the optimal treatments. Although, at the beginning of this century, a series of unexpected side effects brought gene therapy into depression, the improved lentiviral vectors, which characterised by high efficiency transfection, stable expression in target cells and good biosafety, have been applied in clinical trials in recent years and acquired a certain clinical improvements. Nowadays gene therapy becomes an eye-catching field. This review discusses the gene therapy how blocked by lentiviral vectors, the high efficiency and biosafety of lentiviral vectors, the improvement of lentiviral vector preparation and so on.
Animals ; Genetic Therapy ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Transfection

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