Most studies related to determining the expression profile of genes and specific promoters used histochemical localization of the reporter gene, gusA. While the histochemical method for visualizing gusA expression suffers from several limitations in the determination of gene expression and location, especially in the tissues with high background acitivty. To solve this problem, a transient expession vector pBI221-RFP/GFP, was constructed using GFP and RFP as double fluorescent marker genes. This vector used CaMV 35S promoter to drive GFP and determine the transforming efficiency. It analyzed expression profile of the target gene and promoter through the RFP activities of the tranformed tissues. Through the specific promoter AGPL1 from watermelon and E8 promoter from tomato, it is resistible to use this vector to study the expression patterns of promoters. Results indicated that the pBI221-RFP/GFP is a very efficient transient expression vector that can be verify the functions of the genes and promoters.
Citrullus ; genetics ; Gene Expression Regulation, Plant ; Genes, Plant ; Genes, Reporter ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; metabolism ; Luminescent Agents ; metabolism ; Luminescent Proteins ; genetics ; metabolism ; Lycopersicon esculentum ; genetics ; Promoter Regions, Genetic

Adenosine Triphosphatases ; chemistry ; Antineoplastic Agents ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Drug Screening Assays, Antitumor ; Female ; Humans ; Liver Neoplasms ; pathology ; Luminescence ; Luminescent Measurements ; Male ; Middle Aged ; Tumor Cells, Cultured

Immunoassays greatly contribute to veterinary drug residue analysis. However, there are few reports on detecting neomycin residues by immunoassay. Here, a rapid and sensitive chemiluminescent enzyme immunoassay (CLIEA) was successfully developed for neomycin residue analysis. CLIEA demonstrated good cross-reactivity for neomycin, and the IC50 value was 2.4 ng/mL in buffer. The average recovery range was 88.5%-105.4% for spiked samples (10, 50, and 100 μg/kg), and the coefficient of variation was in the range of 7.5%-14.5%. The limit of detection of CLEIA was 9.4 μg/kg, and this method was compared with the liquid chromatography-tandem mass spectrometry method using naturally contaminated samples, producing a correlation coefficient of >0.95. We demonstrate a reliable CLIEA for the rapid screening of neomycin in milk.
Animals ; Anti-Bacterial Agents ; metabolism ; Drug Residues ; metabolism ; Food Contamination ; analysis ; Immunoenzyme Techniques ; veterinary ; Limit of Detection ; Luminescent Measurements ; veterinary ; Milk ; chemistry ; Neomycin ; metabolism

AIMTo establish a new and simple flow injection method for the rapid determination of acetylspiramycin (ASPM).

METHODSASPM was determined by chemiluminescence (CL) method combined with flow injection (FI) technology, which was based on the inhibitive effect of ASPM on the chemiluminescence reaction of the luminol-K3Fe (CN)6 system.

RESULTSThe decrease of chemiluminescence intensity was proportional to the logarithm of ASPM concentration (0.1-100) microgram.L-1, the detection limit was 40 ng.L-1 (3 sigma). The whole process, including sampling and washing, could be completed in 0.5 min with a RSD less than 3.0% (n = 5).

CONCLUSIONThe FI-CL method is of both high sensitivity and good selectivity giving a throughput of 120 h-1. The proposed method was applied successfully to the determination of ASPM in pharmaceutical preparations and human urine without any pre-treatment. It was found that the ASPM concentration reached its maximum after being orally administrated for two hours.

Anti-Bacterial Agents ; analysis ; blood ; urine ; Flow Injection Analysis ; Humans ; Luminescent Measurements ; Luminol ; chemistry ; Male ; Microchemistry ; Spiramycin ; analogs & derivatives ; analysis ; blood ; urine

Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.
Amebicides/*pharmacology ; Animals ; Flow Cytometry ; Gentamicins/*pharmacology ; Green Fluorescent Proteins/*chemistry ; Host-Parasite Interactions ; Leishmania mexicana/drug effects/genetics/*growth & development ; Leishmaniasis, Cutaneous/*parasitology ; Luminescent Agents/*chemistry ; Macrophages, Peritoneal/parasitology ; Mice ; Mice, Inbred BALB C ; Organisms, Genetically Modified ; Spectrometry, Fluorescence

OBJECTIVETo label the primary articular chondrocytes overexpressing human insulin-like growth factor (hIGF 1) with green fluorescent protein (GFP) for repair of articular cartilage defects in rabbits.

METHODSGFP cDNA was inserted into pcDNA3.1 hIGF 1 to label the expression vector. The recombinant vector, pcGI, a mammalian expression vector with multiple cloning sites under two respective cytomegalovirus promoters/enhancers, was transfected into the primary articular chondrocytes with the help of lipofectamine. After the positive cell clones were selected by G418, G418-resistant chondrocytes were cultured in medium for 4 weeks. The stable expression of hIGF 1 in the articular chondrocytes was determined by in situ hybridization and immunocytochemical analysis and the GFP was confirmed under a fluorescence microscope. Methyl thiazolyl tetrazolium (MTT) and flow cytometer methods were employed to determine the effect of transfection on proliferation of chondrocytes. Gray value was used to analyze quantitatively the expression of type II collagen.

RESULTSThe expression of hIGF 1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hIGF 1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks. After the transfection of IGF 1, the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of type II collagen.

CONCLUSIONSThe hIGF 1 eukaryotic expression vector containing GFP marker gene has been successfully constructed. GFP, which can be visualized in real time and in situ, is stably expressed in articular chondrocytes overexpressing hIGF 1. The labeled articular chondrocytes overexpressing hIGF 1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.

Animals ; Cartilage, Articular ; metabolism ; Cells, Cultured ; Chondrocytes ; metabolism ; Flow Cytometry ; Genetic Markers ; Genetic Therapy ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Luminescent Agents ; RNA, Messenger ; analysis ; Rabbits

In vivo tumor imaging technique method based on bioluminescence principle was established to evaluate the anti-tumor effect of paclitaxel mixed micelle (PMM). MDA-MB-231 tumor cells with luciferase reporter vectors were firstly implanted into nude mice, and subsequently the luciferase substrate was regularly injected during intraperitoneal administration of PMM. Then the tumor size, growth and the intensity of light signals were monitored with in vivo imaging technique. The method of luciferase tumor in vivo imaging could be real-time, reliable and exact in labeling and reflecting the growth of tumors, and the observed results were consistent with that by conventional method, so it would be a feasible approach to study anti-tumor effect of drugs. The anti-tumor effect of paclitaxel mixed micelle was observed by this method, and the results showed that this formulation could inhibit growth of tumor, and the anti-tumor rate of it was about 85%.
Animals ; Antineoplastic Agents, Phytogenic ; administration & dosage ; pharmacology ; therapeutic use ; Breast Neoplasms ; drug therapy ; pathology ; Cell Line, Tumor ; Female ; Humans ; Luminescent Measurements ; Male ; Melanoma, Experimental ; drug therapy ; pathology ; Mice ; Mice, Inbred C57BL ; Mice, Nude ; Micelles ; Neoplasm Transplantation ; Paclitaxel ; administration & dosage ; pharmacology ; therapeutic use ; Particle Size ; Tumor Burden ; drug effects

OBJECTIVETo determine the correlation between results of ATP bioluminescence tumor chemosensitivity assay (ATP-TCA) of human ovarian cancer specimens in vitro and clinical chemo-therapeutic responses of patients.

METHODSThirty-four freshly taken ovarian cancer specimens (28 cases) and ascites (6 cases) and 9 chemotherapeutic drugs were tested in vitro for cancer chemosensitivity by ATP-TCA.

RESULTSAmong the 34 ovarian cancer cases, the efficacy of ATP-TCA is 94.0%, the sensitivity, the specificity, the positive and negative predicting values, and an overall predicting value in vitro and vivo were 90.0%, 91.7%, 94.7%, 84.6% and 90.6%, respectively.

CONCLUSIONThe results of ATP-TCA assay are correlated well with clinical treatment responses. The assay may be an important and useful method for individual-based chemotherapy of cancers.

Adenosine Triphosphate ; metabolism ; Adult ; Aged ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Carcinoma ; drug therapy ; pathology ; Cisplatin ; pharmacology ; Cyclophosphamide ; pharmacology ; Drug Resistance, Neoplasm ; Drug Screening Assays, Antitumor ; methods ; Female ; Humans ; Luminescent Measurements ; Middle Aged ; Ovarian Neoplasms ; drug therapy ; pathology ; Paclitaxel ; pharmacology ; Predictive Value of Tests ; Sensitivity and Specificity

OBJECTIVETo label human insulin-like growth factor-I (hIGF-I) eukaryotic expression vector with green fluorescent protein (GFP) for the repair of articular cartilage defects.

METHODSGFP cDNA was inserted into pcDNA(3.1)-hIGF-1 to construct the co-expression vector with two multiple cloning sites mammalian expression vector under two cytomegalovirus promoters/enhancers respectively. Recombinant pcGI was transfected into NIH 3T3 cells with the help of lipofectamine.

RESULTSEnzyme digestion and agarose gel electrophoresis analysis revealed that pcGI vector contained correct GFP and hIGF-I cDNA. Expression of hIGF-1 and GFP was confirmed in transfected NIH 3T3 cells by immunocytochemical analysis and fluorescence microscopy.

CONCLUSIONShIGF-I eukaryotic expression vector has been successfully labeled with GFP.

Cartilage, Articular ; injuries ; Cells, Cultured ; Eukaryotic Cells ; cytology ; physiology ; Fibroblasts ; cytology ; physiology ; Gene Expression Regulation ; Genetic Therapy ; methods ; Genetic Vectors ; Green Fluorescent Proteins ; pharmacology ; Humans ; Immunohistochemistry ; Insulin-Like Growth Factor I ; genetics ; therapeutic use ; Luminescent Agents ; pharmacology ; Sensitivity and Specificity ; Transfection ; methods ; Wounds and Injuries ; diagnosis ; therapy

The HUMACTBP2 locus was investigated to collect population genetic data in the Korean population and to evaluate the applicability for the forensic field. An Automatic fluorescent-based sequencer (377 automatic DNA sequencer, ABI) was used to detect amplified fragments of the HUMACTBP2 locus electrophoresed on 4% denaturing polyacrylamide sequencing gels. ACTBP2 allelic ladder consisting of different sizes of 18 alleles was constructed and employed as an internal size standard in combination with a GS-350 size standard for precision of allele-band sizing. By utilizing different fluorescent dyes, both the allelic ladders and samples were able to be analyzed in the same lane by 99% orecision of allele-band sizing. Among the Korean population (n=224), 26 alleles in the range of 239-313 bp are determined. allele No. 6 is found 45 times (0.100) which is mostly frequent, and the rest of allele is distributed with their relative frequency of 0.002-0.100. The comparison between observed and expected numbers of homozygous and heterozygous individuals confirms that ACTBP2 locus is in the state of Hardy-Weinberg equilibrium among the Korean population. The heterozygosity is 0.9389+/-0.0034(93.89%), and the power of discrimination(PD) and power of exclusion(PEX) are calculated to be 0.991(99.1%)and 0.890(89.0%), respectively, showing the high informativeness for individual identification. Thus, these results mean that the HUMACTBP2 locus can effectively be used for the forensic application.
Alleles ; DNA ; Fluorescent Dyes ; Gels ; Genetic Variation*