Construction and in vitro analysis of recombinant adenovirus vector carrying red fluorescent protein reporter gene

AIM: To provide important tools for gene therapy and gene vaccine research by constructing an adenovirus vector containing red fluorescent protein ( RFP ) reporter gene with the approach of in vitro recombinant ligation. METHODS: The RFP gene fragment of pTurboRFP-N was digested and ligated into pShuttle transfer vector to construct recombinant vector pShuttle-TurboRFP-N. I- Ceu I/PI- Sce I were used to double digest recombinant vector pShuttle-TurboRFP-N and backbone of vector pH5'040.pkGFP-II. The target fragment was collected and ligated, and recombinant adenovirus vector AdH5'.040.CMV.RFP-N was obtained. After linearization, the vector was transfected into AD293 cells by liposome for virus packaging. The efficiency of virus packaging and RFP expression level in AD293 cells were examined using fluorescent microscope. In addition, the biological activity and titer of the virus were tested. Human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were infected with recombinant adenovirus vector AdH5'.040.CMV.RFP-N and control adenovirus vector AdH5.CMV.EGFP respectively. The infection efficiencies of the 2 vectors to different cell lines were compared by evaluating the expression levels of RFP and enhanced green fluorescent protein (EGFP). RESULTS: The recombinant adenovirus vector AdH5'.040.CMV.RFP-N was correctly constructed and confirmed by enzyme digestion. The virus was packaged by the vector in AD293 cells and had the ability to infect the target cells. The target gene in eukaryotic cells was also expressed. The number of recombinant adenoviruses and the titer of the virus after amplification and purification were 3.6×10¹⁵ vp/L and 1×10¹³ pfu/L,respectively. The infection efficiencies of recombinant adenovirus vector Ad5'.040.CMV.RFP-N to human lung cancer cell line A549 and breast cancer cell line MDA-MB-231 were higher than those in control adenovirus vector AdH5.CMV.EGFP (P<0.05). CONCLUSION: We have constructed recombinant virus vector carrying RFP reporter gene and provide an important tool for gene therapy and gene vaccine research. The reporter gene can be highly expressed in AD293 cells and has high infection efficiency to cancer cells. RFP is a good substitution and supplement to green fluorescent protein.     

Adenoviral-mediated high efficiency expression of enhanced green fluorescence protein gene in ex vivo expanded rat mesenchymal stem cells

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.     

Preparation of gfp-bcl-XL-contained recombinant adenovirus vector by the homologous recombination in bacteria

AIM: To prepare gfp-bcl-X_L-contained recombinant adenovirus(rAd-gfp-bcl-X_L).METHODS: Bcl-X_L gene was amplified from pEGFP-C₃-bcl-X_L, subcloned into shuttle plasmid and formed transfer plasmid of pAdTrack-CMV-bcl-X_L. Then pAdTrack-CMV-bcl-X_L 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-X_L and pAdEasy-1 into BJ5183. Recombinant adenovirus particle were produced and further amplified after the transfection of pAdEasy-1-gfp-bcl-X_L into 293 cells. PCR test indicated that the recombinant Ad contained bcl-X_L gene. The titer of the purified rAd-gfp-bcl-X_L was 6.5×10¹² PFU/L. CONCLUSIONS: The homologous recombination in bacteria is a convenient and high efficient method to prepare rAd-gfp-bcl-X_L. This affords a good gene transfer vector for the gene therapy in human’s diseases.     

Recombinant hFGF-10 adenovirus promotes proliferation of kerotinocytes

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.     

Construction of the vector for fusion protein gene driven by IGF-Ⅱ P3 promoter and its expression in hepatocellular carcinoma cells

AIM:To construct the shuttle plasmid vector for thymidine kinase(tk) and EGFP fusion protein gene driven by IGF-Ⅱ P3 promoter,and investigate the specific killing effect of the HSV-tk/GCV system on hepatocellular carcinoma(HCC) cells in vitro.METHODS:Recombinant shuttle plasmid vector was constructed by techniques of genetic recombination and screening,and identified by restriction digestion and sequencing analysis. Then the recombinant shuttle plasmid was transfected into HepG2 and HeLa cells by techniques of lipofectamine transfection and its expression was detected by fluorescence microscope and RT-PCR. Cell killing after ganciclovir(GCV) application was determined by MTT.RESULTS:Identification of pDC316-tkEGFP-P3 by enzyme digestion and sequencing analysis showed that the length,inserted location and direction of the target genes which were inserted into the recombinant were correct. It was found that enhanced green fluorescence protein could only be seen in HepG2 cells,but not in HeLa cells. The results of RT-PCR showed that only two bands could be seen in the samples of pDC316-tkEGFP-P3 transfected HepG2 cells. The MTT test showed the selective cytotoxicity of GCV to the transfected HepG2 cells.CONCLUSION:The shuttle plasmid vector carrying the tkEGFP fusion protein gene driven by IGF-Ⅱ P3 promoter has been constructed successfully and its specific expression in HepG2 cells provided a sound basis for targeted gene therapy for HCC.     

Construction and expression of recombinant adenovirus vector encoding human somatostatin receptor type 2

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.     

Conditionally replicating adenovirus activated by CXCR4 promoter in lung cancer

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.     

Construction and identification of recombinant adenovirus vector containing CTLA4Ig gene

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.     

Construction of eukaryotic expression vector with EGFP and hVEGF121 fusion protein and its expression in mesenchymal stem cells of rats

AIM: To construct a recombinant plasmid carrying enhanced green fluorescent protein and human vascular endothelial growth factor 121 gene and to detect its expression in rats mesenchymal stem cells (MSCs). METHODS: Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFP-C1. The recombinant plasmid pEGFP/hVEGF121 was identified with PCR, double enzyme digestion and DNA sequencing. Then this recombinant plasmid was transfected into rat's MSCs with lipofectamine. The expression of EGFP and VEGF121 protein were detected with fluorescence microscope and immunocytochemical staining, respectively. RESULTS: The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48h after transfection. CONCLUSIONS: The recombinant plasmid carrying enhanced green fluorescent protein and human vascular endothelial growth factor was successfully constructed and expressed positively in rat MSCs. It provides a good basis for further research on differentiation of MSC and VEGF gene therapy for ischemial cardiovascular disease.     

Morphological study of macrophages infected with constructed recombinant adenovirus carrying gp120 gene of Chinese HIV-1 strain

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 10^8.3 and 10^8.1 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.     

Construction of mouse pdx-1 gene eukaryotic expression vector and its expression in embryonic stem cells

AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5α was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5α was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.     

Construction of a recombinant adenovirus carrying IL- 12 -IRES-CK gene and its expression in hepatocyte

AIM: To construct a bicistronic recombinant adenovirus carrying creatine kinase (CK) and human interleukin 12 (hIL-12) gene, and then detect the expression of both genes in vitro and metabolic product of CK in vivo. METHODS: Two PCR products, CK and hIL- 12 genes linked by IRES, were inserted into adenoviral vector. Adenovirus particles carrying CK-IRES-IL- 12 gene was generated through homologous recombination, packaging and propagation. Rabbit hepatocytes were isolated and transfected with adenovirus particles. CK and IL-12 gene expression was confirmed by Western blotting and ELISA, respectively.RESULTS: The expression of CK and human IL-12 in hepatocyte were confirmed in vitro. Metabolic product of CK, phosphocreatine (PCr), could be detected in liver by non-invasive phosphorus-31 magnetic resonance spectroscopy (³¹P MRS) in vivo.CONCLUSION: In vivo detection of ectopic expression of CK gene in liver can be achieved by non-invasive ³¹P MRS through adenovirus transduction. The bicistronic recombinant adenovirus Ad5-hIL-12-IRES-CKb carrying CK and hIL- 12 provides a useful tool for the further study of using a reporter gene expression (CK) dynamically, non-invasively monitor a therapeutic gene expression (IL-12) in liver.     

Construction of recombinant adenoviruses expressing the helicobacter pylori CagA antigen

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.     

Construction of recombinant plasmid pEGFP-HSP70 and its expression in rat neural stem cells

AIM：To explore the method of constructing recombinant plasmid pEGFP-HSP70 and its expression in neural stem cells. METHODS：Total RNA was acquired from the fetal liver tissue of SD rat. cDNA complete sequence of heat-shock protein 70 (HSP70) gene was amplified by RT-PCR, and cloned into an eukaryotic expression vector containing the enhanced green fluorescent protein (EGFP) reporter gene, pEGFP-C2. Sequencing analysis was performed to confirm the recombinant plasmid pEGFP-HSP70. The technique of nucleofector transfection was used to transfect the recombinant plasmid pEGFP-HSP70 into neural stem cells. RESULTS：HSP70 cDNA sequence was correctly cloned into the eukaryotic expression vector pEGFP-C2. The recombinant plasmid pEGFP-HSP70 was constructed successfully. Compared with control group, the fluorescence intensities in pEGFP-C2 group and pEGFP-HSP70 group were significantly increased. The fluorescence intensity in pEGFP-HSP70 group after 24 h of transfection was significantly decreased compared with other time points of 7 d, 14 d and 21 d. The expression level of HSP70 significantly increased 1 d, 7 d and 14 d after transfection compared with control group. CONCLUSION：The neural stem cells can be directly used as gene action target cells. The HSP70 expression level in the stem cells is closely related to the time after transfection.     

Construction of lentiviral vector carrying the Ang-1 gene and its expression in the rMSCs

AIM: To construct lentiviral vector carrying the angiopoietin-1 (Ang-1) gene,and make it express Ang-1 in the rat mesenchymal stem cells (rMSCs).METHODS: The cDNA encoding the CDS of Ang-1 gene was obtained from the placenta of the adult Fisher 344 rats with RT-PCR.After digestion with restrication endonuclease,the Ang-1 gene was recombined to construct the transfer plasmid PNL-Ang1-IRES2-EGFP.The three-plasmid system of lentiviral vector was consisted of PNL-Ang1-IRES2-EGFP,the packaging plasmid HELPER,and the envelope plasmid VSVG,which were co-transfected to 293T cells mediated by lipofectamin2000 to produce lentiviral particles.The rMSCs were infected by obtained lentiviral particles.The insertion of Ang-1 gene was detected by PCR,the mRNA expression of Ang-1 in rMSCs was detected with RT-PCR,the protein expression of Ang-1 was observed with immunocytochemistry and Western blotting methods.RESULTS: The result of sequencing showed that the cloned Ang-1 gene was consistent with the sequence reported in GenBank.After digestion with restrication endonuclease,the 1 512 bp fragment of Ang-1 gene and the 10.5 kb vector fragment of PNL-IRES2-EGFP were observed with gel electrophoresis.The insertion of Ang-1 gene in viral genome was confirmed.The EGFP expression was observed with the fluorescent microscope.In infected rMSCs,the mRNA and protein expressions of Ang-1 were confirmed.CONCLUSION: Lentiviral vector carrying Ang-1 gene has been successfully constructed.The infected rMSCs are able to express the Ang-1 mRNA and Ang-1 abundantly.This will facilitate the following exploratory development of Ang-1 gene-modified rMSCs.     

Construction of recombinant plasmid pEGFP-NGB and its expression in cultured neuroglial cells

AIM: To establish recombination plasmid pEGFP-NGB and to investigate the expression of pEGFP-NGB in culture neuroglia cells. METHODS: The NGB cds was isolated by using RT-PCR method with total RNA extracted from fetal Kunming mouse brain, then the NGB cds was cloned into the eukaryotic expression vector pEGFP-C1 of EGFP reported green fluorescence protein. The expression vector of recombinant plasmid pEGFP-NGB was successfully constructed. GeneJamer transfection reagent was used to transfer recombinant plasmid pEGFP-NGB into culture neuroglial cells. The mRNA and protein expression of pEGFP-NGB in culture neuroglial cells were investigated. RESULTS: The positive clone sequencing was consistent with the sequence of Genbank. The NGB mRNA and protein expression of pEGFP-NGB in culture neuroglial cells were detected at high levels. The high expression of green fluorescence protein was observed by fluorescence microscope in culture neuroglial cells. CONCLUSION: The expression vector of recombinant plasmid pEGFP-NGB was successfully constructed and green fluorescence protein was expressed in cultured neuroglial cells.