BACKGROUNDSteroid-induced osteonecrosis of the femoral head (ONFH) is a common clinical disease, with a high disability rate. At present, efficient prevention and treatment of steroid-induced ONFH is still lacking. The peroxisome proliferator-activated receptor-γ (PPARγ) is recognized as an important pathogenic gene for the development of steroid-induced ONFH. RNA interference (RNAi) is a tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target proteins. Therefore, down-regulation of PPARγ expression by RNAi may prevent the incidence of steroid-induced ONFH.

METHODSAccording to the principles of siRNA design, three duplex siRNA sequences (971 - 989, 1253 - 1271 and 1367 - 1385) derived from the PPARγ gene (NM_001082148) were synthesized. These duplexes were annealed, purified and ligated into 1.0-cytomegalovirus (CMV) shuttle vector. The shuttle vector was transfected into HEK293 cells. The HEK293 generated recombinant adenovirus vector carrying PPARγ siRNA sequences was purified and the titer of recombinant adenovirus was determined.

RESULTSAfter the annealing of single-strand DNA oligo encoding short hairpin RNA (shRNA) sequences, products were identified by gel electrophoresis. These products were ligated into the 1.0-CMV shuttle vector and the recombinant shuttle vectors 1.0-CMV-971, 1.0-CMV-1253 and 1.0-CMV-1367 were constructed. These sequences of these recombinant vectors were confirmed. We then successfully constructed the recombinant adenovirus vector carrying siRNA targeting PPARγ. After purification, the virus titer was higher than 10(10) plaque forming unit (PFU)/ml.

CONCLUSIONIn this study, three recombinant adenovirus shuttle vectors carrying siRNA targeting PPARγ, including shuttle vectors 1.0-CMV-971, 1.0-CMV-1253 and 1.0-CMV-1367, were successfully constructed and high titers of recombinant adenovirus were obtained.

Adenoviridae ; genetics ; Genetic Vectors ; genetics ; PPAR gamma ; genetics ; RNA, Small Interfering ; genetics

In this study, the recombinant adenovirus (Ad) vector containing dual reporter gene [i.e. human transferrin receptor gene (TFRC) and firefly luciferase reporter gene] was constructed to provide a novel experimental tool for magnetic resonance (MR) and bioluminescence dual-modality molecular imaging. The cDNA of TFRC was amplified by polymerase chain reaction (PCR) and cloned into the multiple cloning site of pShuttle-CMV-CMV-Luciferase vector. After identification by Sfi I digestion and sequencing, pShuttle-TFRC-Luciferase vector and the adenoviral backbone vector (pAdeno) were subjected to homologous recombination. The correct recombinant plasmid was then transfected into 293 packaging cells to produce adenoviral particles and confirmed by PCR. After infection of human colorectal cancer LOVO cells with Ad-TFRC-Luciferase, the expressions of transferrin receptor (TfR) and luciferase protein were detected respectively by Western blotting and bioluminescence imaging in vitro. The results showed that TFRC gene was successfully inserted into the adenoviral shuttle vector carrying luciferase gene. DNA sequence analysis indicated that the TFRC gene sequence in the shuttle plasmid was exactly the same as that reported in GenBank. The recombinant plasmid was identified correct by restriction digestion. Ad-TFRC-Luciferase recombinant adenovirus was constructed successfully, and the virus titer was 1.6×10(10) pfu/mL. Forty-eight h after dual reporter gene transfection, the expressions of TfR and luciferase protein were increased significantly (P<0.01). It was concluded that the recombinant adenovirus vector with dual reporter gene was successfully established, which may be used for in vivo tracing target cells in multimodality imaging.
Adenoviridae ; genetics ; Genes, Reporter ; genetics ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Molecular Imaging ; methods

This study was aimed to construct a recombinant adenovirus vector for antisense klf4 gene through AdEasy system. Human klf4 cDNA was reversely inserted into the multiple cloning sites (MCS)of the pShuttle-CMV by using the backbone plasmid AdEasy-l, the antisense klf4 gene was constructed through homologous recombination in E.coli BJ5183, then the adenoviruses were packaged and amplified in the HEK 293 ce1ls. The adenovirus vector for antisense klf4 gene confirmed by PCR, restriction analysis and DNA sequencing. After being transfected with the adenoviruses at 200 MOI for 48 hours, total RNA and protein were extracted from human umbilical vein endothelial cells (HUVEC). Klf4 mRNA and KLF4 protein expression levels were evaluated by Real-time PCR and Western-blot. The results showed that the recombinant adenovirus vector for antisense klf4 gene was successfully constructed, recombinant adenovirus could suppress the expression of klf4 mRNA and KLF4 protein in HUVECs. It is concluded that the adenovirus vector for antisense klf4 gene has been constructed successfully, which provides the material basis for further studying the biologic function and potential application of klf4.
Adenoviridae ; genetics ; Antisense Elements (Genetics) ; Genetic Vectors ; Humans ; Kruppel-Like Transcription Factors ; genetics ; Plasmids ; Transfection

Adenoviridae ; classification ; genetics ; pathogenicity ; Adenoviridae Infections ; epidemiology ; prevention & control ; virology ; Age Distribution ; Child, Preschool ; Humans ; Infant ; Infant, Newborn

The purpose of this study was to construct a recombinant conditionally replicating adenovirus (CRAd) expressing programmed cell death 5 (pdcd5). Pdcd5 gene was inserted in the E3 region of SG600-a CRAd in which the key genes for virus replication E1a and E1b were controlled under the human telomerase reverse transcriptase promoter (hTERTp) and the hypoxia response element (HRE) respectively, and with a deletion of 24 nucleotides within CR2 region of E1a. The insertion and orientation of all recombined plasmids were confirmed by restriction enzyme digestion and polymerase chain reaction (PCR). The infection efficiencies of a recombined virus carrying enhanced green fluorescent protein (EGFP) in leukemic cell lines were observed by using fluorescence microscope. The relative pdcd5 expression levels of K562 after being infected with SG611-pdcd5 were detected by real-time quantitative PCR. The results showed that the construction of SG611-pdcd5 was completed and confirmed. Pdcd5, hTERTp, HRE, skeleton and fiber11 of recombinant adenovirus SG611-pdcd5 were successfully amplified. The infection efficiencies of SG611-EGFP were all above 70% in both leukemic K562 and MEG-01 cell lines. SG611-pdcd5 expressed pdcd5 with high efficiency in leukemic cells as compared with Ad-pdcd5 or SG611 (p < 0.001). The expression level of pdcd5 increased gradually along with the increase of MOI. It is concluded that the triple-regulated adenovirus of SG611-pdcd5 containing the pro-apopro-tic gene pdcd5 has been successfully established with high pdcd5 expression level in leukemic cells, indicating that the recombinant adenovirus, SG611-pdcd5, promises further development of targeted tumor gene therapy.
Adenoviridae ; genetics ; Apoptosis Regulatory Proteins ; genetics ; Genetic Therapy ; methods ; Neoplasm Proteins ; genetics ; Oncolytic Viruses ; genetics ; Promoter Regions, Genetic ; Telomerase ; genetics

OBJECTIVETo construct adenoviral vectors expressing mature miRNA-30a and miRNA-30e.

METHODSThe target mmu-miR-30a and mmu-miR-30e genes amplified from mouse genome were digested and linked to the shuttle plasmid pSES-HUS, which was then transformed into competent AdEaseier cells for recombination. The confirmed recombinant plasmids were transfected into Hek-293 cells for production of the adenoviruses pAd-mmu-miR-30a and pAd-mmu-miR-30e. The obtained adenoviruses were used to infect Mefs cells, and the cellular expressions of mmu-miR-30a and mmu-miR-30e were detected using fluorescence quantitative PCR.

RESULTSmmu-miR-30a (357 bp) and mmu-miR-30e (324 bp) containing the restriction sites were amplified and linked to the shuttle plasmid pSES-HUS, which was successfully recombined with AdEasy1. After packaging in Hek-293 cells, the adenoviral vectors were obtained, which caused an increase of mmu-miR-30a expression by 26.46∓7.46 folds and mmu-miR-30e expression by 2.76∓0.25 folds in transfected Mefs cells.

CONCLUSIONWe have successfully constructed the adenoviral vectors expressing the mature miRNAs.

Adenoviridae ; genetics ; Animals ; Base Sequence ; Genetic Vectors ; HEK293 Cells ; Humans ; Mice ; MicroRNAs ; genetics ; Plasmids

Adenoviridae ; genetics ; Cell Line ; Genetic Vectors ; Humans ; Nitric Oxide Synthase Type III ; genetics ; Virus Replication

OBJECTIVETo construct a replication-defective adenovirus containing TK gene and investigate the killing effects of TK gene against human liver cancer cells SMMC-7721.

METHODSThe recombinant adenovirus ADV-TK was constructed using homologous recombination in the cells. SMMC-7721 cells transfected with recombined adenovirus were exposed to GCV, and the cell viability was measured by MTT assays.

RESULTSThe recombinant adenovirus containing TK gene was successfully constructed. Transfection by the recombinant adenovirus ADV-TK and GCV exposure significantly suppressed the growth of SMMC-7721 cells.

CONCLUSIONA replication-defective adenovirus containing TK gene has been successfully constructed, and in combination with GCV, the recombinant adenovirus produces significant killing effect against SMMC-7721 cells in vitro.

Adenoviridae ; genetics ; Cell Line, Tumor ; Genetic Therapy ; Genetic Vectors ; Humans ; Liver Neoplasms ; therapy ; Thymidine Kinase ; genetics

Conditionally replication adenovirus M4, which was constructed in our lab, was proved to have good clinical application prospect for its good anti-tumor and anti-metastasis effect. However, clinically applying M4 faces many problems. One of the most important is the safety of M4. In this study, we investigated the safety of M4 by comparing with Adv-TK, which was proved to be safe in I-III phase clinical trials. M4 and Adv-TK were injected into mice via the tail vein separately, and the mice were sacrificed at the indicated time. Blood was collected for biochemical tests, the liver was harvested for hematoxylin and eosin (H&E) staining and viral quantification, and splenic lymphocytes were separated for adenovirus specific cellular immune response. Our results showed that M4 had no obvious effect on mouse general symptoms. A transient reversible infiltration of inflammatory cells in collect abbacy was only observed in M4 group, and a transient slight increase in Cr level was detected both after M4 and Adv-TK injection. The adenovirus specific cellular immune response induced by M4 was similar to that by Adv-TK, and the distribution and metabolism of M4 in the mouse liver were also similar to those of Adv-TK. It was concluded that conditionally replication adenovirus M4 had the same safety as Adv-TK. The study provides safety basis for the coming clinical trials of M4.
Adenoviridae ; genetics ; Animals ; Cell Line ; HEK293 Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Virus Replication ; genetics

OBJECTIVETo construct the adenovirus vector containing recombinant human catalase (CAT) and to express the recombinant gene in vitro.

METHODSTotal RNA was extracted from human leukocytes and full-length human CAT cDNA was obtained with RT-PCR method. The CAT gene was cloned into pcDNA3.1(+) vector and pcDNA3.1(+)CAT was constructed. The positive clones were confirmed by the restriction enzyme digestion and gene sequencing. The CAT gene was cloned into the entry vector pENTR1A, and pENTR1A-CAT vector was constructed. By LR reaction pENTR1A-CAT and pAd/CMV/V5-DEST was recombined in vitro, and the recombinant adenovirus pAd/CMV/V5-DEST-CAT was obtained. The positive pAd/CMV/V5-DEST-CAT was confirmed by sequencing and transfected into 293A cells with Pac I linearization and Lipofectamine 2 000, and the recombinant virus particles were packaged and amplified in the cells. The expression of CAT protein and CAT enzyme activities of the recombinant virus were determined by Western blot and 240 nm UV absorption methods.

RESULTHigh expression of recombinant adenovirus was obtained and the expressed human catalase had high enzyme activity.

CONCLUSIONAd/CMV/V5-DEST-CAT vector containing human catalase gene has been constructed successfully; and the expressed enzyme in 293A cells has high activity.

Adenoviridae ; genetics ; Catalase ; genetics ; metabolism ; Cell Line ; Genetic Vectors ; Humans ; Transfection