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1.
AIM:To construct recombinant adenovirus vector containing brain derived neurotrophic factor, (BDNF) gene using bacterial homogenous recombination, and investigate the expression in expanded rat mesenchymal stem cells (rMSC) in vitro.METHODS:BDNF gene and proBDNF gene were subcloned into adenovirus shuttle plasmid pAdTrack-CMV containing enhanced green fluorescent protein gene (EGFP) expression cassette, forming shuttle vector of pAdTrack-BDNF, and pAdTrack-proBDNF, and co-transformed into BJ5183 bacterial cells with adenovirus backbone vector pAdEasy-1 using chemical transformation. After the recombinant adenovirus vector was obtained, the identified recombinant adenovirus plasmid DNA was digested with Pac I and transfected to 293 cells to package recombinant adenovirus particles. rMSC were infected by recombinant adenovirus and EGFP expression was detected using fluorescent microscope. Infection efficiency was assessed by flow cytometrics. Western blotting identified expression of Ad -proBDNF and Ad-BDNF in rMSC. rMSC infected with Ad -proBDNF and Ad-BDNF were induced to differentiate into neuron-like cells. rMSC infected with Ad -proBDNF and Ad-BDNF were injected into nude mice and assessd in vivo.RESULTS:We successfully constructed the recombinant adenovirus Ad -proBDNF and Ad-BDNF that expressed in expanded rMSC in vitro.CONCLUSION:Recombinant adenovirus high-effectively mediates Ad -proBDNF and Ad-BDNF expression in expanded rMSC in vitro and in vivo.  相似文献   

2.
AIM:To investigate the feasibility and infection efficiency of MSCs with replication-deficient adenovirus containing delivered gene, and whether enhanced green fluorescence protein (EGFP) gene track the change during rMSCs differentiating neuron-like cells. METHODS: Rat marrow mesenchymal stem cells (rMSCs) were expanded in low density in vitro. Under the control of CMV promoter, pAd-EGFP-Vector was constructed by homologous recombination in E.coil BJ 5183, and the recombinant virus was produced in HEK 293 packaging cell line. rMSCs infected with Ad-EGFP were observed and analyzed with fluorescence microscope. Infection efficiency was assessed by microscopical scoring and flow cytometrics. After withdrawing serum and exposure to β-mercaptoethanol medium, rMSCs infected with Ad-EGFP was induced to differentiate into neuron-like cells. As a control, the plasmid of pTrack-EGFP also was transfected into rMSCs to evaluate transfection efficiency. RESULTS:The results showed that Adenovirus vector (AdVec) delivered EGFP gene with high efficiency to marrow mesenchymal stem cells. Gene expression analysis showed that 36%±2 % of rMSCs infected with recombinant adenovirus expressed the transgene of EGFP at high levels. However, the transfection of plasmid pTrack-EGFP using routine method of lipofectamin mixed with plasmid DNA (pTrack-EGFP) was not easily successful and the transfection efficiency was much lower. rMSCs infected with Ad-EGFP in different passage could differentiate into typical morphology alike neural cells after withdrawing serum and exposure to β-mercaptoethanol medium. Immuno-staining with neuron-specific enolase (NSE), a neuronal marker, was strong positive, which suggested that rMSCs infected with Ad-EGFP had the potential to differentiate into neurons or neuron-like cells. CONCLUSION:The AdVec system can deliver target gene into MSCs and EGFP gene carried by AdVec can track the change during rMSCs differentiating into neuron-like cells.  相似文献   

3.
AIM: To construct a conditionally replicating adenovirus vector activated by CXCR4 promoter and to evaluate its ability of lysing the lung cancer cells specifically. METHODS: Human CXCR4-E1A gene amplified by PCR was cloned into the shuttle plasmid pDC316-GFP to construct the recombinant shuttle plasmid pDC316-CXCR4-GFP. The recombinat shuttle plasmid and adenovirus genomic plasmid pBHG-lox-E1, 3Cre were transfected into 293 cells to construct the recombinant adenovirus CRAd-CXCR4-GFP. PCR was used to detect the target gene fragments, and the viral titer was determined. A549 cells with the highest mRNA expression of CXCR4 were screened out from 5 kinds of lung cancer cell lines by real-time PCR. CXCR4 promoter activity and adenovirus replication numbers were detected in A549 cells after transfection of CRAd-CXCR4-GFP and Ad-NULL. CRAd-CXCR4-GFP and Ad-NULL were transfected into A549 cells and 16HBE cells, the apoptotic rates were detected by flow cytometry and the viability was analyzed by CCK-8 assay. RESULTS: The recombinant plasmid pDC316-CXCR4-GFP was constructed successfully. Green fluorescence was observed in 293 cells under fluorescent microscope after co-transfection of pDC316-CXCR4-GFP and pBHG-lox-E1, 3Cre at 11 d. Green fluorescence was observed in 293 cells after infection of amplified 3rd generational adenovirus. PCR showed that the purpose gene was successfully integrated in recombinant adenovirus genome. The virus in the supernatant reached a titer of 1×1013 PFU/L. The mRNA expression of E1A and E4 in the A549 cells after transfection of CRAd-CXCR4-GFP was markedly increased compared with Ad-NULL group. Compared with Ad-NULL group and empty control group, the apoptotic rate and the viability of A549 cells in CRAd-CXCR4-GFP group had no significant difference in the first 4 d, the apoptotic rate increased significantly at 5 d, and the cell viability declined significantly at 5 d, but the apoptotic rate and the viability of 16HBE cells in each group had no significant difference within 5 days. CONCLUSION: The conditionally replicating adenovirus vector CRAd-CXCR4-GFP has been successfully constructed, which has the ability of lysing lung cancer cells specifically.  相似文献   

4.
AIM: To study the reversal effects of multidrug resistance by transfecting tumor necrosis factor α (TNF-α) cDNA and multidrug resistant 1 (MDR1) gene antisense RNA into multidrug resistant breast cancer cell line MCF-7/ADR. METHODS: The recombinant vector of enhanced green fluorescent protein (EGFP) with MDR1 antisense RNA and recombinant vector of red fluorescent protein (DsRed2) with TNF-α cDNA were constructed by RT-PCR and DNA recombinant techniques. The recombinant vectors were transfected into multidrug resistant breast cancer cell line MCF-7/ADR. The cell growth curves, cell apoptosis rates, MDR1 gene expression at mRNA and P-gp levels, and the sensitivity to ADR were determined before and after the transfection. RESULTS: After the transfection, cells showed lower growth rate, higher apoptosis rate, lower MDR1 expression at mRNA and P-gp levels, and the sensitivity to ADR increased significantly. CONCLUSION: Transfection of TNF-α cDNA and MDR1 antisense RNA into multidrug resistant breast cancer cells may have good effects on reversal of multidrug resistance.  相似文献   

5.
AIM: To construct recombinant adenovirus vector carrying the gene of human somatostatin receptor type 2 （SSTR2） for gene therapy of pancreatic carcinoma.METHODS: SSTR2 cDNA was inserted into adenovirus shuttle plasmid pDC316, named pDC316-SSTR2.pDC316-SSTR2 was cotransfected with rescue plasmid pBHGlox (delta) E1, 3Cre into 293 cells by liposome reagent.Ad-SSTR2 was generated by site-specific recombination and confirmed by PCR.Ad-SSTR2 was propagated in 293 cells and purified.The titer of viral stock was determined by end-point dilution assay.Western blotting was used to determine the expression of SSTR2 protein after human pancreatic carcinoma cell capan-2 was infected with recombinant adenovirus.RESULTS: pDC316-SSTR2 was successfully constructed.Recombinant adenovirus Ad-SSTR2 was acquired by pDC316-SSTR2 and pBHGlox (delta) E1, 3Cre cotransfected into 293 cells.Ad-SSTR2 was characterized by PCR.The virus titer was 6.0×1012 pfu/L.SSTR2 protein was detected after adenovirus infected capan-2 48 h with Western blotting.CONCLUSION: The recombinant adenovirus vector encoding human SSTR2 is successfully constructed and correctly expressed in pancreatic carcinoma cells.This investigation provides the basis for study of gene therapy of pancreatic carcinoma.  相似文献   

6.
AIM:To construct the recombinant adenoviral vector containing human fibroblast growth factor 10 (hFGF-10) gene, and to study the effect of the recombinant adenovirus on the proliferation of kerotinocytes. METHODS:HFGF-10 gene was amplified by PCR and ligated with shuttle vector pAdTrack-CMV to get the recombinant plasmid pAdTrack-CMV-hFGF-10, which was linearized with PmeI and transferred into Escherichia coli BJ5183 containing the adenoviral bone plasmid pAdEasy-1 for homologous recombination to obtain the recombinant adenoviral plasmid pAdEasy-hFGF-10. The recombinant adenoviral plasmid was then transfected into HEK-293 cell line to package and amplify the recombinant adenovirus. The expression of hFGF-10 in HaCat cells infected with the recombinant adenovirus was detected by Western blotting. The influence of the recombinant adenovirus on the proliferation of kerotinocytes was checked by MTT. RESULTS:The recombinant adenovirus containing hFGF-10 gene was successfully constructed, which effectively infected HaCat cells. The result of Western blotting showed that a protein in culture media of the infected HaCat cells reacted with hFGF-10 antibody. The recombinant adenovirus stimulated the proliferation of kerotinocytes. CONCLUSION:HaCat cells infected with the recombinant adenovirus expresses and secrets hFGF-10 protein, which promotes the proliferation of HaCat cells.  相似文献   

7.
AIM:To construct a recombinant adenovirus expression vector containing CTLA4Ig gene.METHODS:The CTLA4Ig gene derived from the plasmid PCDNA3.0/CTLA4Ig by using polymerase chain reaction (PCR) was inserted into the backward position of cytomegalovirus (CMV) immediate early promoter of the shuttle plasmid (pAdTrack-CMV). After being identified by endonuclease, PCR and sequencing, the recombinant shuttle plasmid pAdTrack-CTLA4Ig was co-transformed into E.coli. BJ5183 cells with the adeoviral backbone plasmid pAdEasyl-1 to obtain the homologous recombination. The adenovirus was generated in 293 cells. A series methods such as PCR and fluorescence microscope was employed to identify the generated recombinant adenovirus.RESULTS:Recombinant CTLA4Ig adenoviruses were constructed and the titer of virus was generally up to 1.65×1012 phaque forming units per liter (PFU/L).CONCLUSION:Success in constructing recombinant pAdTrack-CTLA4Ig will be the base of the further research on its expression in the mammalian cells, and be potenially used in the prevention of transplant rejection and autoimmunity diseases.  相似文献   

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11.
AIM:To optimize the IκBα mutant (IκBαM) gene derived from human placenta tissue by deleting N-terminal phosphorylation sites of serine 32/36,and to construct and identify its replication-deficient recombinant adenovirus (AdIκBαM).METHODS:The IκBαM gene (203-1 003 bp) was acquired by positional cloning,followed by subcloning it into pShuttle and pGEM-T vectors for further PCR,double digestion,DNA sequencing and homology analysis.Subsequently,the expression unit of pShuttle-IκBαM containing CMV promoter,IκBαM cDNA and poly A signals was inserted into Ad5 vector,after which the resultant recombinant adenovirus AdIκBαM was packaged in 293 cells by cotransfection with lipofectamine.Western blotting analysis and electrophoretic mobility shift assay were utilized to detect the AdIκBαM-mediated expression of IκBαM gene in 293 cells and its suppressive effect on phorbol myristate acetate (PMA)-induced nuclear factor κB (NF-κB) activation in ECV304 cells,respectively.RESULTS:The relevant nucleotide and amino acid sequence of IκBαM gene was consistent with that of GenBank (accession number M69043).The titer of the prepared AdIκBαM was 4.0×1012 pfu/L.Moreover,the IκBαM gene was expressed in 293 cells,and potently inhibited the PMA-induced NF-κB activation in ECV304 cells in a dose-dependent manner.CONCLUSION:AdIκBαM is a nonvel vector for both efficient transfer and expression of IκBαM gene as well as specific inhibition of NF-κB activity,providing a promising future for gene therapy of asthma.  相似文献   

12.
AIM: To construct a replication-defective recombinant adenovirus, which expresses the CagA gene. METHODS AND RESULTS: The CagA gene was amplified by PCR. This heterogeneous gene was cloned into shuttle vector pAdTrack-CMV. The recombinant adenovirus DNA was obtained by the homologous recombination between the shuttle vector and adenovirus DNA in E.coli 5183. After linearization， the recombinant adenovirus DNA was transfected into 293 cells and the recombinant adenovirus was obtained. Through this technique, the replication- defective recombinant adenovirus AdEasyCagA was constructed. CONCLUSIONS: The replication-defective recombinant adenoviruses AdEasyCagA was constructed successfully. The work will make a good foundation for studying the effects of the replication-defective recombinant adenovirus on Th1/Th2 balance in asthma and be useful for finding a new pathway to prevent and cure asthma.  相似文献   

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14.
AIM:To construct the lentiviral vectors with green fluorescent protein(GFP) and luciferase(Luc) reporter genes driven by human myosine light chain 2v gene promoter(pMLC2v) and to investigate their expression in human cardiomyocyte(HCM) cell line and human lung cancer cell line A549. METHODS:Human pMLC2v-specific lentiviral vectors with GFP(pMLC2v-GFP) or Luc(pMLC2v-Luc) were constructed and transfected into HCM and A549 cell lines. The expression characteristics of the reporter genes were observed by confocal fluorescent microscopy and bioluminescence detection. Common(nonspecific) promoter-driven GFP(GFPC) or red fluorescent protein(RFPC) lentiviral vectors were used as controls. RESULTS:Both cell lines expressed GFP and RFP 3 days after transfected with the nonspecific vectors. HCM specifically expressed GFP and Luc 3 weeks after transfected with the pMLC2v-GFP or pMLC2v-Luc vectors. However, A549 cells didn't show the similar expression pattern. CONCLUSION:The pMLC2v-GFP and pMLC2v-Luc lentiviral vectors are specific for newly proliferative cardiomyocytes, indicating that they can be used as reliable tools for tracking the differentiation of stem cells into cardiomyocytes in vivo.  相似文献   

15.
AIM: To prepare gfp-bcl-XL-contained recombinant adenovirus(rAd-gfp-bcl-XL).METHODS: Bcl-XL gene was amplified from pEGFP-C3-bcl-XL, subcloned into shuttle plasmid and formed transfer plasmid of pAdTrack-CMV-bcl-XL. Then pAdTrack-CMV-bcl-XL was linealinzed with PmeI and co-transformed into BJ5183 bacteria with adenovirus genomic plasmid of pAdEasy-1. The identified recombinant adenovirus plasmid was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles. The target gene was detected by PCR.RESULTS: There were about 35% positive recombinant bacterial clones after the co-transformation of pAdTrack-CMV-bcl-XL and pAdEasy-1 into BJ5183. Recombinant adenovirus particle were produced and further amplified after the transfection of pAdEasy-1-gfp-bcl-XL into 293 cells. PCR test indicated that the recombinant Ad contained bcl-XL gene. The titer of the purified rAd-gfp-bcl-XL was 6.5×1012 PFU/L. CONCLUSIONS: The homologous recombination in bacteria is a convenient and high efficient method to prepare rAd-gfp-bcl-XL. This affords a good gene transfer vector for the gene therapy in human’s diseases.  相似文献   

16.
AIM: To construct a recombinant adenovirus carrying gp120 gene of Chinese HIV-1 strain,which can infect mouse bone marrow-derived macrophages (BMM). METHODS: Co-transfection of shuttle and backbone plasmids of AdMax system into 293Ad5+ cells was performed, followed by viral packaging, propagation and purification. These viruses were subject to Karber TCID50 titration. The expression of gp120 protein in 293Ad5+ cells was determined by ELISA. The viral titration was validated by a multiplicity of infection (MOI) test with BMM. RESULTS: The titers of the outcome viruses, including AdMax-HIV-1 gp120 (Ad-gp120) and its vector control Ad-GFP, were 108.3 and 108.1 TCID50/mL, respectively. Both recombinant adenoviruses infected BMM with similar capacity of 293Ad5+ cell infection, which validated the TCID50 titration.The gp120 protein was positive in 293Ad5+ cell lysates. BMM activation was observed morphologically after Ad-gp120 infection as compared with Ad-GFP-infected cells. CONCLUSION: Functional adenovirus containing HIV-1 gp120 of prevalent strains in China was successfully constructed. Infection of Ad-gp120 causes BMM activation.  相似文献   

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19.
AIM: To construct pNTAP-PRAK eukaryotic expression plasmid and to establish a stable HEK293 cell line expressing tandam affinity purification (TAP)-tagged PRAK. METHODS: Human PRAK coding region was subcloned into pNTAP vector to construct a recombinant plasmid called pNTAP-PRAK, then DH5α E.coli was transformed with the recombinant plasmid. After identified by PCR, digestion with restriction endonuclease and sequencing, the correct recombinant expression plasmid was transfected with PolyFect liposome transfection reagent to HEK293 cells. The cell line with stable expression of exogenous TAP tagged-PRAK gene was established by screening of antibiotic G418. The expression and localization of the fusion protein TAP tagged-PRAK were detected by Western blotting and immunofluorescence assay. RESULTS: All the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pNTAP-PRAK was constructed correctly. The result of Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection followed by G418 screening. The result of immunofluorescence assay showed that the expression product TAP tagged-PRAK distributed mainly in the nucleus. CONCLUSION: The eukaryotic expression vector pNTAP-PRAK was successfully constructed and the cell line stably expressing TAP tagged-PRAK was established. TAP tag didnt influence the localization of exogenous PRAK.  相似文献   

20.
AIM: To construct a lentiviral vector for stable delivery of the ER-α36gene and to detect its effect on SGC7901 cell growth. METHODS: The efficient RNAi targeting sequences identified for the ER-α36gene were screened. The Oligo DNA was synthesized with target sequences and annealed to form double-stranded DNA. Then it was digested by XhoI and EcoR I and connected with GV307 vector to produce LV-ER-α36-RNAi lentiviral vector. PCR was used to screen the positive clones and sequence. The LV-ER-α36-RNAi, pHelper 1.0 and pHelper 2.0 plasmids were co-transfected into 293T cells for producing lentiviral vector and infecting SGC7901 cell line. Fluorescence microscopy, real-time PCR and Western blotting were used to detect the transfection efficiency and gene silencing effect. 17β-estrodial at concentration of 1×10-10 mol/L was used to stimulate the recombinant cell line, and the action on the growth of gastric cancer cells and the expression of Src, ERK1/2 and cyclin D1 were determined. RESULTS: DNA sequencing analysis confirmed the identity of recombinant shRNA expression vectors. Immunofluorescence assay demonstrated that transfection efficiency was above 80%. Transfection of LV-ER-α36-RNAi significantly knocked down the expression of ER-α36 at mRNA and protein levels with tetracycline (TeT) simulating as revealed by real-time PCR and Western blotting. Compared with control group, the growth of the recombinant cell line declined and the expression of Src, ERK1/2 and cyclin D1 and the activation of Src decreased (P<0.05).CONCLUSION: Lentiviral vectors that silenceER-α36expression are constructed successfully and can be used to study the role of ER-α36 in gastric cancer. The ER-α36is related with many kinds of cancer cell growth, including gastric cancer cells.  相似文献   

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