To construct a prokaryotic promoter report system with wide applicability, a series of pFGH reporter vectors based on lacZ gene and pUC replicon were constructed from plasmid pFLX107 through the replacement of multiple cloning sites and sequence modifications. The plasmid with the lowest background activity was selected as the final report system with the lacZ gene deletion strain MC4100 as the host bacterium, following by testing with inducible promoter araBAD and the constitutive promoter rpsM. The background activity of pFGH06 was significantly lower than that of other plasmids of the same series, and even lower than that of reference plasmid pRCL at 28 °C (P<0.01). Further evaluation tests show that the plasmid pFGH06 could be used to clone and determine the activity of inducible promoter or constitutive promoter, and the complete recognition of the target promoter could be achieved through blue-white selection in the simulation test of promoter screening. Compared with the reported prokaryotic promoter report systems, pFGH06 has the advantages of smaller size, more multiple clone sites, adjustable background activity, high efficiency of promoter screening and recognition, thus with a wide application prospect.
Cloning, Molecular ; Escherichia coli/genetics* ; Genes, Reporter/genetics* ; Genetic Vectors/genetics* ; Lac Operon/genetics* ; Plasmids/genetics* ; beta-Galactosidase/genetics*

The expression of dentin sialophosphoprotein (DSPP) is the marker for cells differentiated into odontoblasts. This study attempted to analyze the DSPP promoter and build the reporter LacZ expression system driven by this promoter, which allows convenient and quick detection of odontoblast cells. First, we separated the human dental mesenchymal cells in which the expression of DSPP can be effectively induced by dexamethasone. Second, four 5' flanking regions of human DSPP gene (- 4 000-+54, -2 500-+54, -1 447-+54 and -1 027-+54) were analyzed, the results showed that the highest promoter activity lied in the -2 500-+54 region. The promoter activity is reduced when the 5' flanking region was extended from -2 500 to -4 000 bp upstream from the transcription start site; The promoter activity are also decreased when the 5' flanking regions were shorted from -2 500 to -1 447 bp and from -1 447 to -1 027 bp, indicating that potential suppresser elements are lied in the region between -4 000 and -2 500 bp and potential activator elements are lied in the region between -2 500 and -1 027 bp. Then we constructed the lentiviral report vector phDSPP-LacZ containing the -2 500-+ 54 promoter region in front of the LacZ gene. The expression of LacZ was detected using X-Gal staining in both human dental mesenchymal cells and immortalized human dental mesenchymal cells infected with phDSPP-LacZ. The phDSPP-LacZ lentiviral vector may provide a more convenient method to detect the expression of DSPP in human odontogenic differentiation, tooth development and tooth regeneration studies.
Cell Differentiation ; Extracellular Matrix Proteins ; genetics ; Genes, Reporter ; Humans ; Lac Operon ; Odontoblasts ; cytology ; Phosphoproteins ; genetics ; Promoter Regions, Genetic ; Sialoglycoproteins ; genetics

OBJECTIVETo explore a way of the gene therapy for acute spinal cord injury (ASCI) by vivo transfection of exogenous gene into spinal cord tissue.

METHODSTwenty-four rats of SD were divided into experiment group and control group (each group had 12 rats). After anaesthesia by abdominal cavity, lamina of thoracic vertebra of all rats were cut-open in prone position. Complex of plasmid and report gene-Lac Z, and plasmid without report gene-Lac Z were respectively injected into cavum subdural of SD rats of experiment group and control group by cation liposome (DOTAP) encapsulation. The rats were killed at the 2nd week after operation, spinal cord tissue of injected segments were detected by reverse transcription-polymerase chain raction (RT-PCR) and immunohistochemistry.

RESULTSIn experiment group, positive staining of beta-galactosidase can be clearly observed in neuron and glia cell of rat's spinal cord by immunohistochemistry detection. Lac Z mRNA in same area was also detected by RT-PCR. But, in control group, no above-mentioned positive results were found.

CONCLUSIONEffective transfection of exogenous gene in vivo into spinal cord is a new hot spot for treatment of SCI. Thus certain nerve growth factor imput partly area of spinal cord injury can promote central nerve regrowth and avoid early secondary injury.

Acute Disease ; Animals ; Genetic Therapy ; Lac Operon ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; metabolism ; Spinal Cord Injuries ; therapy ; Transfection ; beta-Galactosidase ; analysis

To improve microbial lipid production, we inserted mTn-lacZ/leu2 into Trichosporon fermentans 2.1368-Leu(-) to obtain high lipid production mutants. By observing the LacZ chromogenic change, the positive reaction between Cerulenin (inhibitor of fatty acid synthase) and phosphate vanillin, a higher lipid-producing mutant 2.1368-Leu(-)-7 grown on corn-stalk hydrolysate was obtained. The lipid content of this mutant reached 38.30% (8.97% higher than that of the control) and the lipid production rate was 8.35% (20.63% higher than that of the control). The rate of sugar utilization was 77%, meaning that 100 g corn-stalk could be converted to 8.32 g lipid. The study provided an effective method for microbial lipid production by using cheap raw materials for biodiesel.
3-Isopropylmalate Dehydrogenase ; genetics ; Biofuels ; DNA Transposable Elements ; genetics ; Fermentation ; Lac Operon ; genetics ; Lipids ; biosynthesis ; Mutagenesis, Insertional ; Mutation ; Plant Stems ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; Trichosporon ; genetics ; metabolism ; Zea mays ; metabolism

OBJECTIVETo examine the influence of vascular endothelial growth factors (VEGF) in controlling the growth of an experimental osteosarcoma in mice by performing retrovirus-mediated sFlt-1 gene modification.

METHODSFrom March to October 2010 human osteosarcoma G-292 cells were in vitro infected with retroviral vectors encoding soluble Flt-1 or LacZ gene before transplanted into proximal tibiae of immune deficient SCID mice to establish experimental orthotopic osteosarcoma. Daily observation and biweekly microCT were performed to monitor tumor development and progression till sacrifice at 8 weeks after tumor cell inoculation for histological and molecular analyses.

RESULTSSuccessful transgene expression was confirmed in the culture media of sFlt-1 transduced G-292 cells using ELISA, and with positive X-gal staining of the LacZ transduced cells. Noteworthy tumors were grown in all mice on the tibiae receiving G-292 cell inoculation, with clear detection on microCT images starting 2 weeks after inoculation. Over the time period, tumors derived from sFlt-1 transduced G-292 cells were distinctively smaller in size compared to the ones from wide-type G-292 and G-292-LacZ cells. Histology showed typical osteosarcoma characteristics including severe cellular pleomorphism, bone erosions, and neo-vascularization. Real-time polymerase chain reaction indicated significantly higher sFlt-1 expression in sFlt-1 transduced groups than the wild-type G-292 or LacZ treated groups. Strong expression of oncogenes c-myc and c-fos were also obvious, along with the expression of VEGF in the primary tumor tissue.

CONCLUSIONRetrovirus-mediated sFLT-1 gene modification decelerates the osteosarcoma tumor growth in this murine model.

Animals ; Bone Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Female ; Genetic Vectors ; Humans ; Lac Operon ; Mice ; Mice, SCID ; Neovascularization, Pathologic ; metabolism ; pathology ; Osteosarcoma ; genetics ; metabolism ; pathology ; Retroviridae ; genetics ; Transgenes ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor Receptor-1 ; metabolism

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.
Antibodies, Neutralizing/immunology ; Cathepsin L/genetics/*metabolism ; *Cell Movement ; Cells, Cultured ; Comet Assay ; Dependovirus/genetics ; Endothelial Cells/cytology/*metabolism ; Fibroblast Growth Factor 2/genetics/immunology/*metabolism ; Gene Transfer Techniques ; Humans ; Immunoblotting ; JNK Mitogen-Activated Protein Kinases ; Lac Operon/genetics ; Mass Spectrometry ; Matrix Metalloproteinase 1/biosynthesis/genetics ; Muscle, Skeletal/*metabolism ; Neovascularization, Physiologic ; Plasminogen Activator Inhibitor 1/biosynthesis/genetics ; RNA, Messenger/biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

Nowadays, SUMO fusion system is important for recombinant protein production in Escherichia coli, yet a few aspects remain to be improved, including the efficacy for vector construction and protein solubility. In this study, we found the SUMO gene Smt3 (Sm) of Saccharomyces cerevisiae conferred an unexpected activity of constitutive prokaryotic promoter during its PCR cloning, and the gene coding regions of SUMOs in most species had a sigma70-dependent prokaryotic promoter embedded, through the prediction via the BPROM program developed by Softberry. By combining the characters of Sm promoter activity and the Stu I site (added at the 3'-terminal of Sm), and introducing a His-tag and a hyper-acidic solubility-enhancing tag, we further constructed a set of versatile vectors for gene cloning and expression on the basis of Sm'-LacZa fusion gene. Experimentally started from these vectors, several target genes were subcloned and expressed through blue-white screening and SDS-PAGE analysis. The results manifest a few of expectable advantages such as rapid vector construction, highly soluble protein expression and feasible co-expression of correlated proteins. Conclusively, our optimized SUMO fusion technology herein could confer a large potential in E. coli protein expression system, and the simultaneously established co-expression vector systems could also be very useful in studying the protein-protein interactions in vivo.
Amino Acid Sequence ; Base Sequence ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Lac Operon ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae Proteins ; biosynthesis ; genetics ; Small Ubiquitin-Related Modifier Proteins ; biosynthesis ; genetics ; Sumoylation

OBJECTIVETo evaluate the antitumor efficiency of IL-12 gene induced by RU486 regulatory system in a mouse model of orthotopically transplanted hepatoma.

METHODSThe orthotopic hepatoma model was prepared by inoculation of H22 hepatoma cells into the mouse liver. Murine interleukin-12 (IL-12) expressing plasmid pRS22 containing RU486 regulatory system was injected into mice by a hydrodynamic injection 3 days after H22 cells inoculation. Three days after hydrodynamic injection, the mice were induced with RU486 (250 µg/kg) consecutive intraperitoneal administration for 6 days. Blood samples were taken at 10 h after the first and third induction for the determination of IL-12, IFN-γ and NO. Five mice were sacrificed at 2 days after the treatment with RU486. The tumor size was measured. HE and immunohistochemical stainings were applied to evaluate the proliferative activity and angiogenesis in the tumors. The other 7 mice were kept to monitor their survival.

RESULTSIn mice receiving saline plus RU486, pRS-LacZ plus RU486, or pRS22 plus sesame oil, the liver tumors were big in size: (409.90 ± 137.03) mm(3), (271.80 ± 182.63) mm(3) and (251.00 ± 76.55) mm(3), respectively. Strong PCNA positive expression [(82.10 ± 4.62)%, (83.45 ± 2.34)% and (77.46 ± 2.99)%] and extensive microvessel density (74.58 ± 18.47, 63.60 ± 13.36 and 53.52 ± 11.74 per 400 × field), respectively, in these tumor tissues were observed after immunohistochemical staining. The survival period was shorter in these mice. In contrast, in mice treated with pRS22 plus RU486, the tumor was smaller in size. Extensive necrosis, weak PCNA proliferative activity (50.67 ± 8.09)%, and a marked paucity of microvessel density (25.38 ± 10.87) were seen. The survival of mice was obviously prolonged. Compared with the 3 control groups, a significant elevation of serum IL-12, IFN-γ and NO levels were detected in the mice treated with pRS22 plus RU486.

CONCLUSIONExpression of IL-12 gene can be effectively controlled by a RU486 regulatory system. The inducible IL-12 can delay the growth of orthotopically transplanted hepatoma and prolong the survival of mice.

Animals ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; therapy ; Cell Line, Tumor ; Female ; Genetic Therapy ; Humans ; Interferon-gamma ; blood ; Interleukin-12 ; genetics ; metabolism ; Lac Operon ; Liver Neoplasms ; genetics ; metabolism ; pathology ; therapy ; Mice ; Mice, Inbred BALB C ; Mifepristone ; pharmacology ; Neoplasm Transplantation ; Neovascularization, Pathologic ; pathology ; Nitric Oxide ; blood ; Plasmids ; genetics ; Proliferating Cell Nuclear Antigen ; metabolism ; Random Allocation

To prepare a kind of effective non-viral transduction vector, which can deliver exogenous gene into the brain, this vector can be injected through vein system and has the ability to penetrate blood brain barrier. Several groups of materials proportion, type of oil phase, water-oil ratio, phosphatides-cholesterol ratio, temperature of steaming, ultrasonic temperature and time were compared for optimization. Well-constructed immunoliposomes encapsuling LacZ gene were infused into rats through tail vein. 48 h after injection, expression product beta-galactosidase of LacZ gene was detected by histochemistry staining to convince the validity of immunoliposomes as non-viral vectors. The best proportion of synthesis immunoliposomes is as following: phosphatides-cholesterol ratio is 1:1, lipids/drug is 100:1, the type of oil phrase is dichloromethane, oil-water ratio is 4:1, temperature of steaming is 30 degrees C, ultrasonic temperature and time is 10 degrees C and 5 min. At last, 10% trehalose was added as a stabilizer. The entrapment rate is 87.24% and antibody coupling rate is 69%. When immunoliposomes were infused into rats, the expression of LacZ gene could be observed in the brain and periphery organs. Through the best proportion of materials, gene delivering immunoliposomes had been synthesized successfully. This non-viral vector can deliver exogenous gene penetrating blood brain barrier and express in the brain, and will be well-used in the field of gene therapy of cerebral diseases.
Animals ; Antibodies, Monoclonal ; administration & dosage ; pharmacokinetics ; Blood-Brain Barrier ; Brain ; blood supply ; immunology ; metabolism ; Drug Delivery Systems ; methods ; Genetic Vectors ; Lac Operon ; genetics ; Liposomes ; administration & dosage ; immunology ; pharmacokinetics ; Male ; Particle Size ; Plasmids ; Polyethylene Glycols ; administration & dosage ; pharmacokinetics ; Rats ; Receptors, Transferrin ; immunology ; Tissue Distribution ; beta-Galactosidase ; genetics ; metabolism

The genus Rhodococcus is of considerable interest in recent years, stemming from their diverse applications in biodegradation, bioremediation, biotransformation and biosurfactant. Using Nocardia/Rhodococcus-Escherichia coli shuttle plasmid pNV18.1 as the backbone vector, we tested the driven efficiency of promoters Ptac and PlacZ of E. coli and Pami-1/Pami-2 of R. ruber in host R. rhodochrous ATCC 33278 by overexpression of nitrile hydratase. Results showed that the specific activity of nitrile hydratase per dry cell weight in engineered Rhodococcus strains driven by Ptac, Pami-1, Pami-2 and PlacZ was 7.5, 6.3, 5.3 and 1.8 times of that in the wild, respectively. It indicated that these promoters could be well recognized by RNA polymerase of Rhodococcus. We further expressed the beta-galactosidase reporter gene (lacZ) in R. ruber driven by promoter PlacZ. Results indicated that lacZ was an appropriate reporter gene for genetic or metabolic engineering research of Rhodococcus.
Escherichia coli ; genetics ; Gene Expression Regulation, Bacterial ; Genes, Reporter ; genetics ; Lac Operon ; genetics ; Promoter Regions, Genetic ; genetics ; Rhodococcus ; enzymology ; genetics ; beta-Galactosidase ; genetics