OBJECTIVETo examine the potential function of NMDA receptor NR2A subunit C-terminus in assembling and surface expression of the receptor in HEK293 cells.

METHODSFive vectors GFP- NR2ADeltaC1- DeltaC5 were constructed for expressing N-terminally GFP-tagged NR2A with C-terminal deletion at different regions by using conventional techniques of molecular cloning. The deleted region for NR2ADeltaC1-Delta C5 was 897L-1017S, 1024D-1142P, 1149D-1347G, 1354S-1464V, and 897L-1464V. These plasmids were transfected alone or co-transfected with NR1-1a into HEK293 cells. The surface NMDA receptors were immuno-stained using rabbit antibody against GFP and Cy3 conjugated secondary antibody in living cells.

RESULTThe vectors GFP-NR2ADeltaC1-DeltaC5 were generated and all of them expressed GFP fluorescence in the transfected cells. Surface NMDA receptors were detected by immuno-labeling with anti-GFP in the cells co-transfected by NR1-1a and any one of GFP-NR2ADeltaC1-DeltaC5. However, no surface expression of NR2A proteins was found in the transfected cells with any one of these plasmids alone.

CONCLUSIONWithin the region downstream from the 897L of NR2A subunit, neither a particular domain directly interacted with ER retention domain in NR1-1a C1 cassette, nor that determining ER retention of NR2A subunit itself has been found, indicating that more complicated mechanisms might exist in which the subunit assembling and targeting to plasma membrane of NMDA receptors undergo.

Cell Line ; Gene Deletion ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; metabolism ; Mutation ; Receptors, N-Methyl-D-Aspartate ; analysis ; genetics

CRISPR-Cas Systems ; Cell Survival ; Genetic Loci ; genetics ; Genome ; genetics ; HEK293 Cells ; Humans ; Luminescent Proteins ; analysis ; Optical Imaging ; methods

To investigate the transduction efficiency of recombinant adeno-associated virus 2 (rAAV-2) in human umbilical cord blood CD34(+) hematopoietic stem/progenitor cells, the CD34(+) cells sorted by the method of magnetic cell sorting from human cord blood were infected with the rAAV-2 expressing the green fluorescent protein (GFP) gene. After transduction for 19 hours, the expression of GFP was detected under fluorescence microscope. The results showed that 43% CD34(+) cells expressed the GFP gene at a multiplicity of infection of 2 x 10(5). It is concluded that the rAAV-2 can transduce human cord blood CD34(+) hematopoietic stem/progenitor cells efficiently.
Antigens, CD34 ; analysis ; Dependovirus ; genetics ; Fetal Blood ; cytology ; Genetic Therapy ; Green Fluorescent Proteins ; Hematopoietic Stem Cells ; metabolism ; Humans ; Luminescent Proteins ; genetics ; Recombination, Genetic ; Transduction, Genetic

The delivery of transgenes to the central nervous system (CNS) can be a valuable tool to treat CNS diseases. Various systems for the delivery to the CNS have been developed; vascular delivery of viral vectors being most recent. Here, we investigated gene transfer to the CNS by intravenous injection of recombinant adenoviral vectors, containing green fluorescence protein (GFP) as a reporter gene. Expression of GFP was first observed 6 days after the gene transfer, peaked at 14 days, and almost diminished after 28 days. The observed expression of GFP in the CNS was highly localized to hippocampal CA regions of cerebral neocortex, inferior colliculus of midbrain, and granular cell and Purkinje cell layers of cerebellum. It is concluded that intravenous delivery of adenoviral vectors can be used for gene delivery to the CNS, and hence the technique could be beneficial to gene therapy.
Adenoviruses, Human/isolation & purification* ; Animals ; Blood-Brain Barrier ; Brain/virology* ; Cerebellum/cytology ; Cerebellum/virology ; Comparative Study ; Female ; Genes, Reporter ; Genetic Vectors/administration & dosage ; Genetic Vectors/isolation & purification ; Genetic Vectors/pharmacokinetics* ; Hippocampus/virology ; Inferior Colliculus/virology ; Injections, Intravenous ; Luminescent Proteins/analysis ; Luminescent Proteins/biosynthesis ; Luminescent Proteins/genetics ; Mice ; Mice, Inbred BALB C ; Neuroglia/virology ; Neurons/virology* ; Purkinje Cells/virology ; Pyramidal Cells/virology ; Recombinant Fusion Proteins/analysis ; Recombinant Fusion Proteins/biosynthesis ; Recombinant Fusion Proteins/genetics ; Tail/blood supply ; Tissue Distribution

OBJECTIVETo develop a system for quick screening of efficient siRNA targeted HBx mRNA.

METHODSUsing recombination DNA technique, the fusion expression plasmid of HBx and EGFP was constructed, and siRNA expression cassettes (SECs) containing U6+1, H1 or tRNA(Val )promoter were prepared via one-step overlapping extension PCR. By co-transfection with recombinant plasmid and SECs into AD293 cell, the inhibition effects on the transient expression of HBx-EGFP fusion protein were analyzed by FACS and semi-quantitated RT-PCR analysis.

RESULT(1)HBx-EGFP fusion protein expression plasmid pHBx-EGFP was constructed successfully, which expressed green fluorescence in cell mainly located at plasma or the periphery of nucleus in granules. (2) Co-transfection with recombinant plasmid and SECs into AD293 cells resulted in inhibition of HBx-EGFP expression. SEC-siHBx388 showed significant inhibition effect on HBx-EGFP expression compared with SEC-siHBx271, indicating that siHBx388 is effective siRNA site and could be screened out with our screening system. In addition,the results of that U6+1-, tRNA(Val) and H1-siHBx388 reduced HBx-EGFP expression by 21.7%, 12.9% and 12.4% of control respectively indicated that both tRNAVal and H1 promoter was high efficient in driving effect of siHBx388.

CONCLUSIONCombination of the HBx expression carrying reporter gene and PCR-based multi promoter SECs may develop a useful system to be applied in identification of optimal HBx- siRNA and its matching promoter.

Base Sequence ; Cells, Cultured ; Genetic Therapy ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; genetics ; Molecular Sequence Data ; Plasmids ; RNA, Small Interfering ; analysis ; Recombinant Fusion Proteins ; biosynthesis ; Trans-Activators ; antagonists & inhibitors ; genetics ; Transfection

To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-kappa B were labelled with DIG by terminal transferase. After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8% nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded specifically to the NF-kappa B consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-kappa B in PMA group was more than that in PMA + PDTC group. It is suggested that detection of NF-kappa B by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories.
Animals ; DNA-Binding Proteins ; analysis ; genetics ; Electrophoretic Mobility Shift Assay ; Luminescent Measurements ; NF-kappa B ; analysis ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trans-Activators ; analysis ; genetics ; Transcription, Genetic ; Transcriptional Activation

Visualizing living cells growing on co-cultured biomaterials is ideal for material biocompatibility evaluation in vitro. In this experiment, mouse fibroblasts L929 were labeled by introducing the gene coding enhanced green fluorescent protein (EGFP) marker into the cells. Morphology as well as proliferation of labeled cells surrounding or on the surface of co-cultured denture base resin slides were observed by use of phase-contrast microscope and fluorescent microscope directly. It was found that residual methyl methacrylate (MMA) in the denture base resin exhibited transient cytotoxicity to fibroblasts and this transient cytotoxicity could be eliminated by pre-extracting the resin with ddH2O for a short time. This fact demonstrated that even slight cytotoxicity of materials could be detected through imaging of living cells near material or material touched. And it was suggested that imaging of living cells co-cultured with biomaterial is helpful to understanding biocompatibility of materials more accurately.
Acrylic Resins ; Animals ; Biocompatible Materials ; Cell Division ; Cells, Cultured ; Culture Media ; Dental Materials ; Denture Bases ; Fibroblasts ; cytology ; Green Fluorescent Proteins ; Indicators and Reagents ; analysis ; Luminescent Proteins ; genetics ; Mice ; Polymethyl Methacrylate ; Transfection

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

OBJECTIVETo explore the in vitro methods of isolation and culture of human fetal epidermal stem cells (HFESCs) and the feasibility of the cultured cells as the target cells for gene transfection.

METHODSThe HFESCs were isolated by means of type IV collagen rapid adhering method. The culture medium for HFESCs was prepared according to that for human fetal fibroblasts. The cultured cells were identified by immunohistochemistry staining of keratin-19 and integrin-beta1, cell cycle analysis and clone forming rate determination. Then the cultured cells were gene transfected in vitro by liposome mediating method in which eukaryon expression vector pcDNA3.1/VEGF165 containing vascular endothelial growth factor 165 (VEGF165) were transfected into cultured cells, or by virus vector mediating method in which recombinant adenovirus accompanied vector (raav) containing green fluorescent protein (GFP) (raav/GFP) were transfected into the cultured cells, respectively. The results of in vitro gene transfection of HFESCs were observed by immunohistochemisty staining and fluorescence microscope.

RESULTSHFESCs grew well and formed large clones with higher cloning efficiency and higher ratio of G1 cells than keratinocytes. The cultured cells were strongly positive with immunohistochemistry staining of keratin-19 and integrin-beta1. After being gene-transfected by pcDNA3.1/VEGF165, the VEGF165 of HFESCs showed positive immunohistochemistry staining property, while the HFESCs transfected by raav/GFP exhibited strong fluorescence.

CONCLUSIONHFESCs could be isolated and cultured in vitro by means of rapid adherence to type IV collagen. It seemed feasible that HFESCs were gene transfected with liposome or adeno-associated virus as the vector.

Cell Adhesion ; Cell Cycle ; physiology ; Cells, Cultured ; Endothelial Growth Factors ; genetics ; metabolism ; Epidermis ; Fetus ; G1 Phase ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry ; Integrin beta1 ; analysis ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Keratinocytes ; cytology ; Keratins ; analysis ; Luminescent Proteins ; genetics ; metabolism ; Lymphokines ; genetics ; metabolism ; Microscopy, Fluorescence ; Plasmids ; genetics ; Stem Cells ; chemistry ; cytology ; metabolism ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo identify and characterize laryngeal cancer related novel genes located on chromosome 6q25.

METHODSElectric hybridization was performed in human genome database using EST (expression sequence tag) as probe. Novel genes were deduced by software from positive DNA clones and their cDNAs were amplified by RT-PCR using primers designed according to the sequence of the putative genes.

RESULTSA novel gene was cloned successfully. The full length of this gene was about 21 kb. It contained two exons and produced a 1006 bp transcript coding a protein with 235 amino acid residues. It's 5'flanking sequence contained two binding sites of oncoprotein c-Myc, thus it was named MTLC (c-Myc target from laryngeal cancer cells). Homologous assay showed that MTLC exhibited little overall homology to known human proteins but it exhibited good overall homology to mouse MT-MC1 protein with an identity of 78%. The primary structure of MTLC protein contained a nuclear location signal motif, but it did not have other conserved domains. The results of subcellular location experiment showed that MTLC expressed in nuclei of human hepatocellular carcinoma cell line Bel7402 cells, while a wide distribution of MTLC in various tissues was demonstrated by Northern blotting.

CONCLUSIONMTLC may play an important role as a target gene of c-Myc and as a transcription factor in keeping the normal physiological process of cells.

Amino Acid Sequence ; Base Sequence ; Blotting, Northern ; Chromosomes, Human, Pair 6 ; genetics ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Gene Expression ; Green Fluorescent Proteins ; Humans ; Laryngeal Neoplasms ; genetics ; Luminescent Proteins ; genetics ; metabolism ; Microscopy, Fluorescence ; Molecular Sequence Data ; Nuclear Proteins ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Sequence Alignment ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Tumor Cells, Cultured