Molecular imaging aims to visualize the cellular and molecular processes occurring in living tissues, and for the imaging of specific molecules in vivo, the development of reporter probes and dedicated imaging equipment is most important. Reporter genes can be used to monitor the delivery and magnitude of therapeutic gene transfer, and the time variation involved. Imaging technologies such as micro-PET, SPECT, MRI and CT, as well as optical imaging systems, are able to non-invasively detect, measure, and report the simultaneous expression of multiple meaningful genes. It is believed that recent advances in reporter probes, imaging technologies and gene transfer strategies will enhance the effectiveness of gene therapy trials.
Animals ; Apoptosis/physiology ; Contrast Media ; Diagnostic Imaging/*methods ; Gene Expression/physiology ; Gene Therapy ; Genes, Reporter/physiology ; Human ; Molecular Biology/*methods ; Neovascularization, Physiologic/physiology ; Rats

PURPOSE: Surfactant protein A(SP-A) is involved in surfactant physiology and structure, and plays a major role in innate host defense and inflammatory processes in the lung. Steroid therapy is widely used for mothers who threaten to deliver prematurely and also used commonly in the management of preterm infants with chronic lung disease. Two SP-A genes(SP-A1, SP-A2) and several alleles have been characterized for each SP-A gene in human. Preliminary evidence indicates that differences may exist among alleles in response to Dexamethasone(Dexa) and that the SP-A 3'UTR plays a role in this process. We studied whether 3'UTR-mediated differences exist among the most frequently found SP-A alleles in response to Dexa. METHODS: Constructs containing the 3'UTR from eight different SP-A alleles were made using luciferase as a the reporter gene. These constructs were driven by the SV40 promotor and were transfected along with a transfection control vector in H441 cells that express SP-A. The activity of the reporter gene in the presence or absence of Dexa(100 nM) treatment was measured. All the experiments for the eight SP-A alleles studied, were performed in triplicate and repeated five times. The results were normalized to the transfection control. RESULTS: Expression of alleles of 6A3, 6A, 1A were significantly decreased in response to Dexa. CONCLUSION: Three UTR mediated differences exist among human SP-A variants both in the basal expression and in response to Dexa. These genotype-dependent differences may point to a need for a careful consideration of individual use of steroid treatment in the prematurely born infant.
3' Untranslated Regions ; Alleles ; Dexamethasone* ; Genes, Reporter ; Humans ; Infant ; Infant, Newborn ; Infant, Premature ; Luciferases ; Lung ; Lung Diseases ; Mothers ; Physiology ; Transfection

Molecular imaging is emerging as an exciting new discipline that deals with imaging of disease on a cellular or genetic level. Nuclear medicine has tra6 tionally focused on noninvasive imaging of in vivo physiology using radiolabeled tracers. As such, molecular imaging has its roots in nuclear medicine and in many ways is a direct extension of this field. The myriad of biological processes that may be targeted for molecular nuclear imaging can be grouped into direct and indirect strategies, depending on the type of imaging probe. The direct strategy uses de novo synthesis of molecular probes targeted to a specific molecular marker such as a receptor, transporter, or enzyme. For each novel target, new radiolabeled compounds are required as well as characterization of their detection sensitivity, interaction specificity, pharmacokinetics of delivery, and signaltonoise ratio. The indirect strategy entails the use of a pretargeting molecule that is subsequently activated upon occurrence of a specific molecular event, which in turn is targeted by a specific molecular radioprobe. Reporter gene imaging falls into this category and provides a rapid and convenient tool to monitor gene expression by yielding a phenotype that is readily imaged upon expression. The remarkable efforts currently focused on the molecular nuclear technology signify its importance and wide range of application. With continued improvements in instrumentation, identification of novel targets, and design of better radioprobes, molecular nuclear imaging promises to play an increasingly important role in disease diagnosis and therapy.
Biological Processes ; Diagnosis ; Gene Expression ; Genes, Reporter ; Molecular Imaging ; Molecular Probes ; Nuclear Medicine* ; Pharmacokinetics ; Phenotype ; Physiology ; Sensitivity and Specificity

The present study was aimed at investigating the effect of activin on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the underlying molecular mechanism. The method of luciferase reporter gene was used. We firstly established a stable GH3 cell line which contains hGH gene promoter (-484 to 30 bp) and luciferase reporter gene by transfecting pGL3-484-Luc2 luciferase expression plasmid into GH3 cells using Lipofectamine transfection reagent. After treating these cells with activin or activin plus various signaling transduction activators, the concentration of GH in the medium and lysate of GH3 cells and luciferase activities in GH3 cells were measured. The results showed that activin (5 nmol/L, 50 nmol/L) decreased the secretion and synthesis of GH. The amounts of GH content in GH3 lysate and medium treated with 50 nmol/L activin were 82% and 59% of the control, respectively. Furthermore, activin (5, 50 nmol/L) reduced the luciferase expression in stable GH3 cells, with the expression being 77% and 69% of the control (P<0.001). Among the activators of intracellular signaling transduction pathways, mitogen-activated protein kinases kinase (MAPKK/MEK) activators C(6) ceramide (1 micromol/L) abolished completely the inhibitory effect of activin. Western blot analysis further confirmed the inhibition of phosphorylated MEK in GH3 cells. The inhibitory effect of activin was abrogated following the deletion of the fragment from -132 to -66 bp within the hGH gene promoter. These results indicate that activin decreases the activity of hGH gene promoter in rat pituitary GH3 cells. The intracellular MEK dependent signaling pathway and the promoter sequence that spans the -132 to -66 bp fragment of hGH gene are involved in the inhibitory effect of activin.
Activins ; physiology ; Animals ; Cell Line ; Cells, Cultured ; Genes, Regulator ; Genes, Reporter ; genetics ; Human Growth Hormone ; genetics ; Humans ; Luciferases ; genetics ; Promoter Regions, Genetic ; genetics ; Rats ; Somatotrophs ; cytology ; metabolism ; Transfection

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

The convergence of molecular and genetic disciplines with non-invasive imaging technologies has provided an opportunity for earlier detection of disease processes which begin with molecular and cellular abnormalities. This emerging field, known as molecular imaging, is a relatively new discipline that has been rapidly developed over the past decade. It endeavors to construct a visual representation, characterization, and quantification of biological processes at the molecular and cellular level within living organisms. One of the goals of molecular imaging is to translate our expanding knowledge of molecular biology and genomic sciences into good patient care. The practice of molecular imaging is still largely experimental, and only limited clinical success has been achieved. However, it is anticipated that molecular imaging will move increasingly out of the research laboratory and into the clinic over the next decade. Non-invasive in vivo molecular imaging makes use of nuclear, magnetic resonance, and in vivo optical imaging systems. Recently, an interest in Positron Emission Tomography (PET) has been revived, and along with optical imaging systems PET is assuming new, important roles in molecular genetic imaging studies. Current PET molecular imaging strategies mostly rely on the detection of probe accumulation directly related to the physiology or the level of reporter gene expression. PET imaging of both endogenous and exogenous gene expression can be achieved in animals using reporter constructs and radiolabeled probes. As increasing numbers of genetic markers become available for imaging targets, it is anticipated that a better understanding of genomics will contribute to the advancement of the molecular genetic imaging field. In this report, the principles of non-invasive molecular genetic imaging, its applications and future directions are discussed.
Animals ; Biological Processes ; Gene Expression ; Genes, Reporter ; Genetic Markers ; Genomics ; Molecular Biology ; Molecular Imaging* ; Optical Imaging ; Patient Care ; Physiology ; Positron-Emission Tomography

Ebola virus, the cause of severe and fatal hemorrahagic fever in humans, belongs to filovirus family. This study was designed to establish a cell-based screening and evaluation system in the pharmacological study of antivirus compounds. Three reporter systems were established with recombinant pseudoviral luciferase of HIV core (pNL4-3.Luc.R(-)E(-)) packed with filovirus glycoprotein (EBOV-Zaire GP/HIV-luc, EBOV-Sudan GP/HIV-luc and Marburg GP/HIV-luc), which are required for virus entry of cells. The level of filovirus entry was determined by the expression of luciferase reporter gene in the infected cells. For screening of filovirus entry inhibitors, the vesicular stomatitis G packed pseudovirions (VSVG/HIV-luc) was used to determine the compound specificity. The results of known filovirus entry inhibitors demonstrated successful establishment of the new model systems, which would be useful in high throughput screening of anti-filovirus drugs in the future.
Antiviral Agents ; pharmacology ; Drug Evaluation, Preclinical ; methods ; Ebolavirus ; drug effects ; physiology ; Genes, Reporter ; Glycoproteins ; genetics ; Hemorrhagic Fever, Ebola ; Humans ; Luciferases ; Viral Proteins ; genetics ; Virus Internalization ; drug effects

Animals ; Electrochemical Techniques ; methods ; Gene Silencing ; Genes, Reporter ; Humans ; MicroRNAs ; blood ; genetics ; metabolism ; physiology ; Microarray Analysis ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo evaluate the possibility of hepatitis B virus (HBV) as a vector in liver-targeting gene therapy.

METHODSA fragment containing the small envelope gene of HBV was replaced with the reporter gene green fluorescent protein (GFP) to construct the recombinant HBV vector, which was transfected into HepG2 cells with liposome. The expression of GFP was observed with fluorescence microscope. The HBV cccDNA was testified using semi-nest PCR. The viral particles of the recombinant HBV in culture medium were detected by PCR as well as Southern blot.

RESULTSThe HBV vector carrying the interesting gene of GFP could express the functional protein in the transfected hepatocytes. However, the recombinant HBV vector was replication-deficient, which could not be packed and replicated in the hepatocytes to secrete mature recombinant HBV particles carrying the interesting gene of GFP when transfected solely but could when cotransfected with the recombinant and helper construct which lacked part of 5'-proximal HBV RNA packaging signal epsilon.

CONCLUSIONIt is possible that HBV is reconstructed as a liver-targeting vector for gene therapy.

Cell Transformation, Viral ; Cells, Cultured ; Gene Transfer Techniques ; Genes, Reporter ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; physiology ; Hepatitis B virus ; genetics ; physiology ; Hepatocytes ; cytology ; virology ; Humans ; Liver ; cytology ; virology ; Recombinant Proteins ; genetics ; Transfection ; Virus Replication

OBJECTIVETo explore the potential reporter gene assay for the detection of sodium channel-specific toxins in shellfish as an alternative for screening harmful algal bloom (HAB) toxins, considering the fact that the existing methods including HPLC and bioassay are inappropriate for identifying HAB toxins which poses a serious problem on human health and shellfish industry.

METHODSA reporter plasmid pEGFP-c-fos containing c-fos promoter and EGFP was constructed and transfected into T24 cells using LipofectAMINE 2000. Positive transfectants were screened by G418 to produce a pEGFP-c-fos-T24 cell line. After addition of increasing neurotoxic shellfish poison (NSP) or GTX2,3, primary components of paralytic shellfish poison (PSP), changes in expression of EGFP in the cell line were observed under a laser scanning confocal microscope and quantified with Image-pro Plus software.

RESULTSDose-dependent changes in the intensity of green fluorescence were observed for NSP in a range from 0 to 10 ng/mL and for GTX2,3 from 0 to 16 ng/mL.

CONCLUSIONpEGFP-c-fos-T24 can be applied in detecting HAB toxins, and cell-based assay can be used as an alternative for screening sodium channel-specific HAB toxins.

Animals ; Biological Assay ; Cell Line, Tumor ; Genes, Reporter ; physiology ; Green Fluorescent Proteins ; Harmful Algal Bloom ; physiology ; Humans ; Plasmids ; Proto-Oncogene Proteins c-fos ; genetics ; metabolism ; Shellfish ; analysis ; Sodium Channels ; Toxins, Biological ; chemistry ; toxicity