Objective To construct the responsive plasmid PTRE-HIF-1αof Tet-on gene expression system and examine its expression. Methods RT-nested PCR was performed on the total RNA extracted from hypoxia HepG2 cells to obtain the cDNA of HIF-1α, which was inserted into the responsive plasmid PTRE2hyg. DNA sequencing was performed after the recombinant of responsive plasmid PTRE-HIF-1α was identified by endonuclease digestion. This recombinant vector was transfected into HepG2Tet-on cells by means of liposome and its expression was examined by RT-PCR and Western blot under the control of deoxycycline. Results The amplified products were confirmed as the cDNA of HIF-1α by DNA sequencing. The responsive plasmid PTRE-HIF-1α verified by edonuclease digestion, was capable of expression in HepG2Tet-on cells and could be controlled by deoxycycline. Conclusion The responsive plasmid PTRE-HIF-1α of Tet-on expression system is constructed successfully, and it can express under the regulation of deoxycycline in the HepG2Tet-on cells.
【Abstract】Objective To provide experimental evidence for gene therapy of thrombophilia diseases by constructing eukaryotic expression plasmid of human thrombomodulin(hTM) gene and transfecting the plasmid into COS7 cell and human umbilical vein endothelial cells(HUVECs). Methods The coding fragment of hTM gene was amplified by PCR. Both hTM gene and pcDNA3.1(+)/neo empty vector were digested with HindⅢ and EcoRⅠ. Two digested fragments were combined into pcDNA3.1/hTM with T4DNA ligase. After identification, the pcDNA3.1/hTM was transfected into COS7 cell and HUVECs using cation liposome. The expression of hTM mRNA and protein on the COS7 cell and HUVECs was detected by RTPCR and immunohistochemistry respectively. Results The hTM recombinant plasmid was confirmed by double endonuclease digesting and sequencing. It was transfected into COS7 cell and HUVECs successfully with liposome.Conclusion The pcDNA3.1/hTM plasmid can be successfully constructed and highlevel hTM can be expressed in eukaryotic cells. All of this provides us experimental evidence for gene therapy and further study of TM anticoagulant mechanism.
OBJECTIVE: To construct a plasmid which has a reporter gene for exploring the role of human telomerase reverse transcriptase(hTRT) in in-vitro cell cultivation. METHODS: hTRT was cut by restricted enzyme from plasmid pGRN145 and inserted to plasmid pEGFP-C1 (enhanced green fluorescent protein). RESULTS: Restricted enzyme analysis and DNA sequencing showed that the sequence of the pEGFP -hTRT transgenic plasmid was correct. CONCLUSION: The recombinant vector pEGFP-hTRT has been successfully constructed, and it can be used as a transgenic plasmid in generating immortalized cell lines.
This study aims to construct the plasmid of human Runx1 and observe its possible effects on Runx1 gene expression in human pulmonary adenocarcinoma cells (A549). The shRNA sequence targeting human Runx1 was designed and synthesized, then inserted into pSuper plasmid by DNA recombination technology. The recombinant plasmid was confirmed by bacterial colonies PCR, enzyme digestion analysis and DNA sequencing. A549 cells were transfected by Runx1 shRNA plasmid. The inhibition efficiency of pSuper-Runx1-shRNA plasmid on Runx1 at mRNA level and protein level were measured with real-time PCR and Western blot. The results of real-time PCR and Western blot indicated that the mRNA and protein levels of Runx1 in A549 cells were inhibited by the pSuper-Runx1-shRNA expression plasmid, and the inhibition rate were 33% and 50%, respectively.
Objective To detect the difference of periostin expression in small cell lung cancer (SCLC) cell, and explore its effect on chemoresistance of SCLC patients. Methods The expression of periostin in mRNA and protein was detected by RT-PCR and Western blot analysis in SCLC H69 and multidrug resistant strain H69AR. The expression of periostin was up-regulated by recombinant plasmid-periostin in H69 cell. The survival rate in the transfected group was different from that of the negative control group and uninterrupted group. Results The expression of periostin mRNA and protein in the sensitive strain H69 was lower than that of the multidrug resistant strain H69AR (P<0.05). The recombinant periostin-plasmid was transfected into H69 cells and at the same concentration of chemotherapeutic drugs (cisplatin, etoposide) the survival rate increased significantly (P<0.05). The positive expression rate of periostin in SCLC tissues was 67.44%, and the sensitivity of the chemotherapy group was lower than that of the drug resistant group (P<0.05). Conclusion The expression of periostin in SCLC cell H69 is significantly lower than that of the multidrug resistant strain H69AR and overexpression of periostin increases resistance of the sensitive strain H69 and hence periostin may be involved in SCLC chemoresistance.
Objective To investigate the effects of recombinant adenovirus-mediated co-transfection of carcinoembryonic antigen (CEA) gene and erythropoietin (EPO) gene on promoting hematopoietic stem cells directly producing erythrocyte vaccine against colon cancer. Methods The expression adenovirus vectors carrying CEA and EPO or green fluorescent protein (GFP) gene were constructed respectively, and recombinant adenovirus carrying CEA, EPO or GFP were packaged and produced respectively. The bone marrow-derived mesenchymal stem cells (MSCs) of mice were isolated and cultured in vitro by anti-CD117 magnetic bead separation, and were transfected with CEA (CEA group), EPO (EPO group) or GFP (blank vector group), co-transfected with CEA and EPO (CEA-EPO group). The expressionsof CEA and EPO gene and its protein after transfection in supernatant fluid of culture were detected by realtime-PCR and Western blot method in each group. We had checked and obtained the vaccine with co-transfection of CEA gene and EPO gene by cell red line marker antibody CD71 and GPA, then we carried on experiments with the vaccine in vitro and in vivo. There were 4 groups in our trail: blank vector group, CEA group, EPO group, and CEA-EPO group. Results We had successfully gathered the hematopoietic stem cells, flow cytometry analysis result showed that there were significant differences before and after purification for positive selected samples (P<0.05). The expressions of double genes (CEA-EPO gene) and protein showed CEA-EPO gene were successfully transfected into the hematopoietic stem cells. We had confirmed erythrocyte vaccine with co-transfection of CEA and EPO gene by antibody CD71 and GPA with flow cytometry. The monocytes cytotoxicity on colon cancer cell line CT26 showed that lysis of target cells of CEA-EPO group were higher than those of other 3 groups when in proportion of 40∶1 (P<0.05). In the experimentation of neoplasma format, the volume of tumor and mortality were smaller or lower, but survival time was longer of CEA-EPO group in2 weeks after treatment (P<0.05). Conclusions The erythrocyte vaccine with co-transfection of CEA gene and EPO gene has efficient anti-tumor effects on colon cancer. Not only can promote hematopoietic stem cell directly producing erythrocyte vaccine, but also can produce tumor antigen vaccine against colon cancer.