OBJECTIVETo investigate the roles of Domain I and Domain II of hepatitis C virus (HCV) 5' untranslated region (UTR) in the translation initiation activity of HCV 5'UTR in different host cell lines.

METHODSThe eukaryotic expression plasmid pCMVNCRLuc (pCN1), in which full-length HCV 5'UTR regulates firefly luciferase expression, was modified by deleting Domain I and the downstream single-stranded sequence (43 bp in total) from the UTR (pCNl-d2), Domain I with the downstream single-stranded sequence and Domain II (118 bp in total) from the UTR (pCNl-d3), or the total UTR (pCNl-d5). The modified plasmids were transfected via liposome into different cell lines with pRL-TK plasmid co-transfected as the normalization control. At 36 h after the transfection, the total cellular RNA was harvested for semi-quantitative RT-PCR, and the relative expression activities of luciferase were assayed with a dual luciferase reporter gene assay system. The translation initiation activities of the truncated HCV 5'UTRs in different translation systems were analyzed.

RESULTSDeletion of Domain I and the downstream single-stranded sequence caused no significant changes of the translational activity of HCV 5'UTR in Hela or C6 cells, but decreased the translational activity by 46% in L-02 cells and increased the translational activity by 46% in 293T cells. Deletion of both Domain I and Domain II resulted in decreased translational activity of HCV 5'UTR by 51% in HeLa cells, but increased the translational activity by 40% in L-02 cells, 60% in C6 cells and 135% in 293T cells.

CONCLUSIONSDomain I and Domain II of HCV 5'UTR perform cell type-specific roles in HCV IRES-driven translation initiation.

5' Untranslated Regions ; Genes, Reporter ; HeLa Cells ; Hepacivirus ; genetics ; Humans ; Luciferases ; Plasmids ; Protein Biosynthesis ; genetics ; RNA, Viral ; genetics ; Transfection

Pyrosequencing is a tool based on bioluminescence reaction for real-time analyzing DNA sequences. The sensitivity of pyrosequencing mainly depends on luciferase in reaction mixture. However, the instability of pyrosequencing reagents caused by fragile wild Photinus pyralis luciferase (PpL) in conventional pyrosequencing usually leads to unsatisfied results, which limits the application of pyrosequencing. In order to improve the stability of pyrosequencing reagents, the coding sequences of mutant thermostable Luciola lateralis luciferase (rt-LlL) was synthesized, and inserted into the plasmid of pET28a(+) to express the thermostable rt-LlL with a 6 x His-tag in the N terminal. The purified rt-LlL with the molecular mass of 60 kDa was obtained by Ni-affinity chromatography. The specific activity of rt-LlL was determined as 4.29 x 10(10) RLU/mg. Moreover, the thermostability of rt-LlL was investigated, and the results showed that rt-LlL had activity at 50 degrees C, and remained 90% of activity after incubated at 40 degrees C for 25 min. Finally, rt-LlL was used to substitute commercial Photinus pyralis luciferase in conventional pyrosequencing reagent to get thermostable pyrosequencing reagent. Comparing with conventional pyrosequencing reagent, the thermostable pyrosequencing reagent is more stable, and it's activity would not lose when incubated at 37 degrees C for 1 h. This study laid foundation of establishing reliable and stable pyrosequencing system which would be applied in Point-of-Care Testing.
Animals ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Fireflies ; enzymology ; Luciferases ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Sequence Analysis, DNA ; methods

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.
Anemia/blood/*therapy ; Animals ; Base Sequence ; Blood Urea Nitrogen ; Cell Hypoxia ; Creatinine/blood ; Erythropoietin/biosynthesis/*genetics/secretion ; Gene Expression Regulation ; Genes, Reporter ; Genetic Therapy ; HeLa Cells ; Humans ; Injections, Intramuscular ; Kidney/pathology ; Luciferases, Firefly/biosynthesis/genetics ; Molecular Sequence Data ; Plasmids/*genetics ; Promoter Regions, Genetic ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/biosynthesis/genetics/secretion ; Response Elements ; Transcriptional Activation ; Uremia/blood/*therapy

OBJECTIVETo establish a bioluminescent MDA-MB-231 cell line which can stably express luciferase and green fluorescent protein to allow bioluminescent imaging in nude mouse models bearing human triple-negative breast cancer xenografts.

METHODSThe lentivirus carrying luc2, eGFP and neo fusion genes were packaged in 293T cells via calcium phosphate co-precipitation. Human triple-negative breast cancer cell line MDA-MB-231 was infected by the lentivirus, and the positive cell clones were tested for eGFP and luc2 expressions by fluorescence microscopy and Xenogen IVIS200 bioluminescent imaging system, respectively. MTT assay, transwell invasion assay and wound healing assay were performed to evaluate the changes in the proliferation, invasion and migration abilities of the infected cells. The cells were then orthotopically implanted into the right second mammary fat pat of female BALB/c nude mice. The tumor growth was monitored by the in vivo imaging system every week, and the tumor tissues were harvested to evaluate the in vivo stability and tumorigenicity of the modified cells using cryosection and HE staining.

RESULTSThe lentivirus-infected MDA-MB-231cells could stably express luc2 and eGFP, and the luciferase activity reached 9689 phontons/s/per cell. No significant changes occurred in the biological activities of the lentivirus-infected MDA-MB-231 cells. We successfully established the nude mouse model bearing orthotopically implanted human triple-negative breast cancer cells.

CONCLUSIONThe modified MDA-MB-231 cell line can be detected sensitively at the primary implantation site and distant metastasis site in nude mice, which provides a convenient and sensitive platform for the research of metastatic mechanism and new antitumor drugs of human triple-negative breast cancer. The combination of eGFP and luc2 is superior to single reporter gene.

Animals ; Brain Neoplasms ; secondary ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Female ; Genes, Reporter ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Luciferases ; biosynthesis ; genetics ; Luminescent Measurements ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism

We developed a method for monitoring of miRNA activity in live cells by a secreted luciferase gene based plasmid sensor named as Gsensor. Firstly, we constructed pAAV2neo-Gluc-MCS-polyA as "empty Gsensor", which contained multiple cloning sites (MCS) for miRNA target inserted. To detect miR142-3p activity, miR142-3p Gsensor and miR142-3p Gsensor-3 were constructed by inserting one or three complementary miR142-3p targets into pAAV2neo-Gluc-MCS-ployA. Subsequently, miR142-3p Gsensor and miR142-3p Gsensor-3 were respectively transfected into U937 cells and Gluc activity was assayed in the supernatant 48 h post transfection. Results showed that both of them effectively indicated miR142-3p activity of inhibiting Gluc expression compared with empty Gsensor. Simultaneously, miR142-3p Gsensor also demonstrated the inhibition of miR142-3p activity by Anti-miR142 when they were cotransfected into U937 cells. This implied one copy of miRNA target in Gsensor was sensitive enough for investigation of miRNA activity. We further analyzed factors affecting Gsensor function including time and dose, and found that miR142-3p activity sensed by miR142-3p Gsensor rose within 48 h post transfection and approached stable thereafter. Transfected dose varying among 0.001-0.05 pg/cell had little effect on its function. Using miR142-3p Gsensor, we further detected miR142-3p activity in HEK293, U937, K562, SP2/0 and P815 cells. Results suggested that miR142-3p activity was high in U937, K562, SP2/0 and P815 cells and almost negative in HEK293. miR142-3p activity was positively correlated with its relative copies in HEK293, U937 and K562 detected by QRT-PCR. In conclusion, Gsensor proved to be an effective tool for monitoring of miRNA activity in live cells, and provide a new method for monitoring miRNA activity in vitro.
Genes, Reporter ; genetics ; Genetic Vectors ; HEK293 Cells ; Humans ; K562 Cells ; Luciferases ; biosynthesis ; genetics ; MicroRNAs ; metabolism ; Transfection ; U937 Cells

We chose Gaussia luciferase (Gluc), a secreted luciferase gene as reporter to real-time detect and dynamically monitor hydrodynamic injection gene expression. First, we constructed an expression vector pAAV2neo-Gluc. Then Huh7 and HepG2 cells were transfected with pAAV2neo-Gluc and the activity of Gluc in the supernatant and cell lysates were assayed. Results showed that the Gluc activity in the supernatant was about 100 higher than that in cell lysates, indicating the expressed Gluc existing mainly as a secreted form as reported. Live bioluminescence imaging of mice hydrodynamic injected pAAV2neo-Gluc showed whole body distribution, while the pAAV2neo-Fluc primarily located in the liver. Then we injected different doses of pAAV2neo-Gluc into mice by tail-vein hydrodynamic injection, took minor amount of blood from mice tails at different time points and measured the luciferase activity to investigate dynamic changes of Gluc expression and secretion in vivo. The results suggested that the time courses of Gluc expression were highly consistent among each dose groups. The luciferase activity in blood could be detected as early as 2 h after injection, reached the peak at about 10 h and gradually decreased from then on. The expression level of Glue was positively correlated with the dose of injected plasmid DNA. To further detect the assay sensitivity of the ex vivo Gluc measurement method, we investigated three additional groups of mice injected with lower doses of 0.001 microg, 0.01 microg and 0.1 microg pAAV2neo-Gluc respectively. Results revealed that activity of Gluc in blood could be detected even at dose as low as 0.001 microg DNA, suggesting the assay sensitivity was extremely high. In conclusion, a real-time ex vivo detection method of dynamically monitoring of gene expression in vivo by hydrodynamic injection can be a valuable means for the study of gene expression regulation in vivo.
Animals ; Gene Expression Regulation ; Genes, Reporter ; genetics ; Genetic Therapy ; Hep G2 Cells ; Humans ; Hydrodynamics ; Injections, Intravenous ; Liver ; metabolism ; Luciferases ; administration & dosage ; biosynthesis ; genetics ; pharmacokinetics ; Male ; Mice ; Mice, Inbred BALB C ; Transfection

We established xenograft mouse models for studying human lung cancer by using an in vivo imaging system. We first transfected pGL4.17 (luc2/neo) plasmid into human non-small lung cancer A549 cells and screened cell lines stably expressing a luciferase reporter gene with G418. Then we analyzed the correlation of luciferase activity and cells number by in vitro bioluminescence. Furthermore, we compared cell growth characteristics by cell counting. We selected suitable clones and inoculated subcutaneously into nude mice or intravenously into SCID mice to construct lung cancer xenograft models. Using an in vivo imaging system, we monitored the growth and metastasis of the tumors. Finally, we verified the extents of tumorigenesis and metastasis by tissue sections with Hematoxylin and Eosin (HE) staining. In our study, we successfully established the xenograft mouse models for in vivo imaging with luciferase expressed lung cancer cells. These models provided convenient, sensitive, intuitive and stable tools for studying the mechanisms of lung cancer progression and development of anticancer drug.
Animals ; Carcinoma, Non-Small-Cell Lung ; pathology ; Cell Line, Tumor ; Disease Models, Animal ; Humans ; Luciferases ; biosynthesis ; genetics ; Lung Neoplasms ; pathology ; Mice ; Mice, Nude ; Mice, SCID ; Neoplasm Metastasis ; pathology ; Neoplasm Transplantation

Estrogen Receptor (ERalpha) is a member of superfamily of ligand-activated transcription factors which play critical roles in many biological processes. To screen novel modulators of ERalpha for drug development and biological function research, we developed a mammalian one-hybrid-based high-throughput screening model for ERalpha modulator. We cloned the ERalpha LBD gene from the total mRNA of fat tissue by RT-PCR and fused it with the GAL4 DNA binding domain of pBIND-GAL4 plasmid to construct a chimara expression plasmid pBIND-GAL4-Eralpha(LBD). The L02 cells was cotransfected with pBIND-GAL4-ERalpha(LBD) and a GAL4-responsive luciferase reporter plasmid pGL3-GAL4, and following treatment with test compounds for 24 h, the activities of luciferase were detected to evaluate the transactivities of ERalpha modulators. After manner optimizations of transfection conditions, Estradiol, an agonist control, induced the expression of luciferase in a dose-dependent with EC50 of 0.17 micromol/L, the maximum folds of induction was about 28.1. Tamoxifen, an antagonist control, efficiently suppressed the estradiol-mediated luciferase induction with EC50 of 0.10 micromol/L. Using this screening model, we discovered four ERalpha agonists from 2000 natural and synthetic compounds.
3T3-L1 Cells ; Animals ; Chimera ; metabolism ; DNA-Binding Proteins ; biosynthesis ; genetics ; Estrogen Receptor Modulators ; chemistry ; isolation & purification ; Estrogen Receptor alpha ; agonists ; Genes, Reporter ; genetics ; Genistein ; chemistry ; isolation & purification ; HeLa Cells ; Humans ; Luciferases ; genetics ; metabolism ; Mice ; Models, Chemical ; Saccharomyces cerevisiae Proteins ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; Transfection

OBJECTIVETo detect the effect of resistin on the transcription of insulin receptor promoter.

METHODSLuciferase reporter gene was fused downstream of human insulin receptor promoter and the enzymatic activity of luciferase was determined in the presence or absence of resistin. The resistin expressed with plasmid was stained with antibody against Myc tag which was in frame fused with resistin coding sequence, and then imaged with confocal microscopy.

RESULTSThe treatment of pIRP-LUC transfected cells with recombinant resistin did not result in significant difference in the enzymatic activity of luciferase compared to the untreated cells. Cell staining showed that green fluorescence could be observed in the cytoplasm, but not in the nucleus.

CONCLUSIONThe results suggest that the endogenous resistin may functionally locate in the cytoplasm, but does not enter the nucleus and not down-regulate the transcription of insulin receptor promoter.

Cell Line ; Cell Nucleus ; metabolism ; Cytoplasm ; metabolism ; Down-Regulation ; Gene Expression Regulation ; Humans ; Luciferases ; metabolism ; Microscopy, Confocal ; Models, Biological ; Plasmids ; metabolism ; Promoter Regions, Genetic ; Receptor, Insulin ; biosynthesis ; genetics ; Resistin ; pharmacology ; Transcription, Genetic ; Transfection

Lentiviral vectors were powerful gene delivery tools for gene therapy. We developed a new lentiviral vector pBobi-MIL that constitutively expressed O6-methylguanine-DNAmethyltransferase (MGMT) and Luciferase, linked by the internal ribosomal entry site (IRES), to realize drug tolerance and real time monitoring in vivo. All results from RT-PCR, drug treating clones forming, immunofluorometric assay and chemiluminescence detection showed that cells infected by recombinant lentivirus L-MIL simultaneously expressed these two genes. This lays the foundation for the further research in gene therapy and can also help identify lentivirus titer.
DNA Modification Methylases ; biosynthesis ; genetics ; DNA Repair Enzymes ; biosynthesis ; genetics ; Drug Resistance ; genetics ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Luciferases ; biosynthesis ; genetics ; Tumor Suppressor Proteins ; biosynthesis ; genetics