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

To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.
Bacteriophage lambda ; genetics ; Chromosomes, Bacterial ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Knock-In Techniques ; methods ; Genes, Reporter ; genetics ; Lac Operon ; genetics ; Luciferases ; genetics ; Recombination, Genetic ; genetics

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

pBR322-Red is a newly constructed recombineering plasmid, which contains a part of the pBR322 vector, a series of regulatory elements of lambda-prophage and Red recombination genes. In the beginning, we studied the best working conditions of pBR322-Red, and then modified lac operon in E. coli W3110 chromosome using the plasmid as follow: Firstly, we knockout the lacI gene using Red-mediated recombineering with overlapping single stranded DNA oligonucleotides. Secondly, we substituded the lacA and lacY genes with lacZ, a report gene, by Red-mediated linearized double strands DNA homologous recombination. Finally, we detected the expression of lacZ on these loci for the first time. The results suggested that pBR322-Red system is suitable for modifying W3110 chromosome with various recombination strategies.
Bacteriophage lambda ; genetics ; Chromosomes, Bacterial ; genetics ; Escherichia coli ; genetics ; Gene Knock-In Techniques ; methods ; Gene Knockout Techniques ; methods ; Humans ; Lac Operon ; genetics ; Plasmids ; genetics ; Recombination, Genetic

OBJECTIVETo investigate the expression of neurotrophin-3 (NT-3) gene in Schwann cells of rat sciatic nerve introduced by an adenovirus vector in vivo.

METHODSA recombinant adenovirus vector for NT-3 (Ad-NT-3) was propagated in 293 packaging cells and titered with tissue culture infectious dose(50) (TCID(50)). Ad-NT-3 was injected directly into the rat sciatic nerve after transection and immediate repair. Immunohistochemical staining was employed to determine the expression of NT-3 in Schwann cells in rat sciatic nerve and the expressive intensity of the tissue slices of the sciatic nerve was measured with LEICA M550 image analysis system.

RESULTSOn the 2nd day after injection of Ad-NT-3, positive stain in the Schwann cells was apparent in the vicinity of anastomosis. NT-3 expression increased significantly on the 7th day (P<0.01) and then decreased 14-28 days after injection (P<0.01). There was no significant difference of NT-3 expression between the 14th and 28th day groups (P<0.05). Compared with the 2nd day group, the 14th and 28th day groups still maintained a relatively high level of NT-3 (P<0.01). Intact and repaired nerves, which were injected with adenovirus encoding LacZ genes (Ad-LacZ) or physiological saline served as controls, showed no NT-3-positive Schwann cells.

CONCLUSIONSAn adenovirus vector can be used to induce efficiently the expression of NT-3 gene in Schwann cells of rat peripheral nerves following nerve injury and repair, which suggests that neurotrophic factors can be introduced into Schwann cells with an adenovirus vector to promote peripheral nerve regeneration.

Adenoviridae ; genetics ; Animals ; Gene Expression ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Immunohistochemistry ; Lac Operon ; genetics ; Neurotrophin 3 ; genetics ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; metabolism ; Sciatic Nerve ; injuries

OBJECTIVETo explore the feasibility and safety of gene transfer into porcine myocardium via the pericardial cavity by a homemade easy device.

METHODSReplication-deficient recombinant adenoviral vector carrying LacZ report gene (Ad-LacZ) was constructed by the calcium phosphate precipitation method. Twelve healthy Chinese mini-swine were randomly divided into experimental group (n = 6) and control group (n = 6). Acute myocardial infarction (AMI) model was established by balloon occlusion of the distal part of D1 branch of left anterior descending (LAD) artery, at the same time the intrapericardial cavity injections were performed through the small incision of the abdominal wall below the xyphoid appendix using a homemade device. Then gene transfer was performed using a central venous catheter. The pericardium was pretreated with injection of a mixture of collagenase (1,200 U) and hyaluronidase (3,000 U) in both groups. Then 2.0 x 10(9) plaque formation unit (PFU) Ad-LacZ was injected into the pericardial cavity in experimental group, while 1 mL of normal saline was injected in the control group. The beta-galactosidase activity detection and X-gal staining of the ischemic myocardium were performed on the 3rd, 7th, and 28th day after injection.

RESULTSThe LAD artery was occluded completely and infarction and ischemia were detected by histological assessment In experimental group, the X-gal staining positive cells and beta-galactosidase activity quantification were detectable on the 3rd day after injection, increased markedly on the 7th day, and then declined on the 28th day. The transfer efficiencies indicated by the positive myocardial cells were 16.7%, 45.6% , 22.8% on the 3rd, 7th, 28th day, respectively. In control group, no positive cells and beta-galactosidase activity were observed.

CONCLUSIONAdenovirus can be transferred into ischemic myocardium and express target gene in the AMI model for four weeks with the homemade easy device via pericardial cavity pretreated by collagenase and hyaluronidase.

Adenoviridae ; genetics ; Animals ; Animals, Genetically Modified ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Vectors ; Heart ; Lac Operon ; Myocardium ; enzymology ; Pericardium ; physiology ; Punctures ; methods ; Swine ; Swine, Miniature ; beta-Galactosidase ; genetics

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

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 investigate the feasibility and features of 3 catheter-mediated approaches of gene transfer into heart, including direct myocardial injection (DMI), coronary artery perfusion (CAP), and intrapericardial cavity injection (ICI).

METHODSFifteen dogs were used, and 0.3 ml (1 x 10(9) pfu) of an adenovirus (Adex1SR LacZ) was injected into the heart by 3 methods. The dogs were killed 5 days following injection, and gene expressions in heart and liver were evaluated by histochemical analysis.

RESULTSThe results showed that (1) the CAP method was relatively less damaging and induced sparse LacZ expression in the myocardium, and the gene expression was also found in both vessels within the myocardium and liver; (2) gene transfer by DMI resulted in intense LacZ expression around the injection accompanied by a local inflammatory response; (3) LacZ expression elicited by ICI was detected in either the inner surface of the parietal pericardium or epicardial surface of the heart, and also in the myocardium underlying the visceral pericardium.

CONCLUSIONThree catheter-mediated methods of gene transfer into the heart may be used and a reasonable approach should be chosen according to purpose.

Adenoviridae ; genetics ; Animals ; Cardiac Catheterization ; Dogs ; Female ; Gene Expression ; Gene Transfer Techniques ; Genetic Vectors ; administration & dosage ; genetics ; Inflammation ; etiology ; Injections ; adverse effects ; methods ; Lac Operon ; genetics ; Liver ; metabolism ; Male ; Myocardium ; metabolism