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Results for “expression efficiency”

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At least 1,243 records · Page 69Linked to original sources
PubMed2000

[In vitro study on transduction of human O(6)-methylguanine-DNA-methyltransferase cDNA into human umbilical cord blood CD34(+) cells].

OBJECTIVE: To explore human umbilical cord blood hematopoietic progenitor cells transduced with human O(6)-methylguanine-DNA-methyltransferase (MGMT) gene increase resistance to 1,3-Bis(2-Chloroethyl)-1-Nitrosourea(BCNU). METHODS: The present authors obtained a full length cDNA fragment encoding the human MGMT from a patient with cholelithiasis liver tissue by RT-PCR method and confirmed by DNA sequencing. The fragment was cloned into pGEM-T vector and further subcloned into G1Na retrovirus vector. Then the G1Na-MGMT was transfected into the packaging cell lines GP+E86 and PA317 by LipofectAMINE method; using the medium containing BCNU for cloning selection and ping-ponging supernatant infection between ecotropic producer clone and amphotropic producer clone, the authors obtained high titer amphotropic PA317 producer clone with the highest titer up to 1.6x10(6) CFU/ml. Cord blood CD34(+) cell were transfected repeatedly with supernatant of retrovirus containing human MGMT cDNA under stimulation of hemopoietic growth factors. RESULTS: PCR, RT-PCR, Southern blot, Northern blot, Western blot and MTT analyses showed that MGMT gene had been integrated into the genomic DNA of cord blood CD34(+) cells and expressed efficiently in the transfected cells. The transgene recipient cells conferred 4 folds stronger resistance to BCNU than that of the non-transduced. CONCLUSION: The retrovirus vector-mediated transfer of MGMT drug resistance gene into human cord blood CD34(+) cells and expression could confer the resistance of transgene cells to BCNU toxicity.

Antigens, CD34↗
PubMed2006

[Construction of prokaryotic expression vector for Trichomonas vaginalis silent information regulator 2 and its expression].

Total RNA was isolated from Trichomonas vaginalis and Tv-Sir2-like cDNA was amplified by RT-PCR and cloned into pGEM-T Easy plasmid. A fragment of Tv-Sir2-like cDNA was subcloned into the expression vector pET-41b and expressed in E.coli BL21 with induction of IPTG. The full-length of Tv-Sir2-like cDNA was cloned and sequenced. The prokaryotic expression system of pET-41b/Tv-Sir2-like was constructed. The fusion protein of Tv-Sir2-like was expressed in E. coli BL21, occupying 30% of the total bacterial protein after being induced by IPTG for 5 h. SDS-PAGE analysis showed that the fusion protein was about Mr 59000. The recombinant protein of Tv-Sir2-like is efficiently expressed in E. coli BL21.

Animals↗
PubMed1988

[Epidemiologic surveillance of congenital abnormalities using the EUROCAT Register].

The EUROCAT programme is a concerted action of the European Economic Community for the epidemiologic surveillance of congenital anomalies. Surveillance is based on a network of regional registries coordinated by a central registry. The programme started in 1979 and by 1987, 23 centres were participating, covering together more than 300,000 births per year. The surveillance process implies the selection of the anomalies possibly associated with environmental teratogens or mutagens, the definition of abnormal variations in the rate of these anomalies (the alarms), the establishment of base-line rate and the continuous monitoring of rate. The programme is evaluated by reference to four criteria: the sensitivity of the system in detecting teratogens or mutagens, the specificity of alarms, the rapidity in warning of alarm and in investigating their causes, and the programme efficiency expressed as its cost-utility ratio.

Congenital Abnormalities↗
PubMed1993

Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter.

Adeno-associated virus type 2 (AAV) vectors have been used for gene expression in respiratory epithelial cells and may be useful in gene therapy for diseases like cystic fibrosis (CF) which affect the airways. The AAV p5 promoter together with the AAV inverted terminal repeat (ITR) forms a 263-base pair cassette which mediated efficient expression in a CF bronchial epithelial cell line. We report here that the ITR itself can mediate gene expression. In stable transfection assays, AAV-CF vectors expressing the full-length cystic fibrosis transmembrane conductance regulator (CFTR) cDNA from either the p5 promoter or the ITR restored cAMP regulation of the chloride efflux characteristic of CFTR function. An AAV-ITR-CF vector deleted for the amino terminus of CFTR was also functional. This vector was packaged into AAV particles and used to transduce cells without selection. Transduced cells also exhibited cAMP-regulated Cl- efflux. The complemented cell lines showed increased levels of CFTR protein immunofluorescence, and the presence of intact AAV-CF vector sequence was confirmed by Southern blot analysis of rescued vector sequences. These studies provide novel insights into AAV gene expression, and this newly described promoter allows for the production of AAV vectors expressing CFTR in those differentiated cells affected in CF.

Base Sequence↗
PubMed2005

A novel approach of low-frequency ultrasonic naked plasmid gene delivery and its assessment.

OBJECTIVE: To deliver the naked genes into cells through the bioeffects of cell membrane porous produced by low-frequency ultrasound (US) and to investigate the safety by determining the threshold of cell damage and membrane permeability. METHODS: The suspension of red cells from chickens, rabbits, rats, and S180 cells was exposed to calibrated US field with different parameters in still and flowing state. Laser scanning confocal microscopy, fluorescent microscopy, scanning electron microscopy, flow cytometry and spectrophotometry were used to examine cell morphology, membrane permeability, enzymes, free radicals, naked gene expression efficiency, threshold of cell damage and cell viability. RESULTS: The plasmid of green fluorescent protein (GFP) as a reporter gene was delivered into S180 cells under optimal conditions without cell damage and cytotoxicity. The transfection rate was (35.83 +/- 2.53)% (n = 6) in viable cells, and the cell viability was (90.17 +/- 1.47)% (n = 6). Also, malondialdehyde, hydroxyl free radical, alkaline phosphatase, and acid phosphatase showed a S-shaped growth model (r = 0.98 +/- 0.01) in response to the permeability change and alteration of cell morphology. The constant E of energy accumulation in US delivery at 90% cell viability was an optimal control factor, and at 80% cell viability was the damage threshold. CONCLUSION: US under optimal conditions is a versatile gene therapy tool. The intensity of GFP expression in US group has a higher fluorescent peak than that in AVV-GFP group and control group (P < 0.001). The optimal gene uptakes, expression of gene and safety depend on E, which can be applied to control gene delivery efficiency in combination with other parameters. The results are helpful for development of a novel clinical naked gene therapeutic system and non-hyperthermia cancer therapeutic system.

Animals↗
PubMed1990

Expression in yeast of amino-terminal peptide fusions to hepatitis B core antigen and their immunological properties.

Hepatitis B core protein (HBcAg) is a potent antigen that gives both a T-cell-dependent and a T-cell-independent antibody response. It has been shown that a foreign epitope can be fused to the amino terminus of HBcAg without affecting particle integrity, and that the resulting chimaeric cores retain the immunogenicity of the foreign epitope. Here we describe the efficient expression in yeast of two different chimaeric cores, carrying epitopes of Foot and Mouth Disease Virus (FMDV) or human chorionic gonadotrophin (hCG), which are candidates for FMD and contraceptive vaccines, respectively. These cores could not be produced in E. coli in soluble form but were expressed to high levels in yeast. We constructed a yeast expression vector that allows rapid production of different chimaeric cores by cloning in cassettes encoding foreign epitopes. Both FMDV and hCG-cores were shown to present the epitopes at the surface of the particles. The FMDV-cores produced in yeast were efficient inducers of neutralising antibodies in guinea-pigs after one low dose.

Amino Acid Sequence↗
PubMed2000

Human tumor growth is inhibited by a vaccinia virus carrying the E2 gene of bovine papillomavirus.

BACKGROUND: Papillomavirus is the etiologic agent associated with cervical carcinoma. The papilloma E2 protein is able to regulate negatively the expression of E6 and E7 papilloma oncoproteins. Therefore, a new, highly attenuated vaccinia virus known as modified vaccinia virus Ankara (MVA), which carries the papillomavirus E2 gene, was used for the treatment of tumors associated with human papillomavirus. METHODS: Analysis of expression of the E2 gene from the recombinant vaccinia virus was performed by reverse transcription-polymerase chain reaction of RNA isolated from infected cells. Detection of the E2 protein was done by immunoprecipitation from proteins labeled with [(35)S]-methionine, isolated from infected cells. The therapeutic effect of the MVA E2 recombinant virus over human tumors was tested in nude mice bearing tumors generated by inoculation of HeLa cells. Series of 10 nude mice with tumors of different sizes were injected with MVA, MVA E2, or phosphate-buffered saline. Tumor size was monitored every week to assess growth. RESULTS: The MVA E2 recombinant virus efficiently expressed the E2 protein in BS-C-1 cells. This protein was able to repress, in vivo, the papillomavirus P105 promoter, which controls the expression of the E6 and E7 oncoproteins. In nude mice the MVA E2 virus reduced tumor growth very efficiently. In contrast, tumors continued to grow in mice treated with MVA or PBS. The life expectancy of MVA E2-treated mice was also increased three- to fourfold compared with that of animals that received MVA or PBS. CONCLUSIONS: The growth of human tumors was efficiently inhibited by the MVA E2 recombinant vaccinia virus. The absence of side effects in treated animals suggested that the MVA E2 virus is a safe biologic agent that could in the future be used in humans for the treatment of cervical carcinoma.

3T3 Cells↗
PubMed2000

Cloning and characterization of the 5'-flanking region for the mouse phospholipase C-delta1 gene.

To date, little is known about the molecular mechanisms controlling the regulation of phospholipase C-delta1 (PLC-delta1) gene expression. To understand the mechanisms responsible for the regulation of PLC-delta1 gene expression, the 5'-flanking region of the mouse PLC-delta1 gene was isolated from a mouse genomic DNA library. Primer extension analysis revealed that there is a single transcriptional start site located at 127 bases upstream from the translation start codon in the mouse PLC-delta1 gene. DNA sequence analysis showed that the sequence around the transcriptional start site is very GC-rich and has no TATA or CAAT boxes. Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the 160-base-pair region from -622 to -462 upstream of the transcriptional start site includes a positive cis-acting element(s) for the efficient expression of the PLC-delta1 gene. Gel retardation analysis suggests that multiple transcription factors bind to separate sites on the promoter region. Based on these results, our study suggests that the minimal essential region located at -622 to +70 is fully sufficient to confer high-level transcriptional activity and contains high-affinity binding elements for multiple transcription factors.

Animals↗
PubMed2005

Genetic transformation of Ascochyta rabiei using Agrobacterium-mediated transformation.

In order to study pathogenic mechanisms of the plant pathogen Ascochyta rabiei, conditions for efficient transformation using Agrobacterium-mediated transformation were investigated. Hygromycin B resistance (hph) was superior to geneticin resistance (nptII) for selecting transformants, and the hph gene was more efficiently expressed by the Aspergillus nidulans trpC promoter than by the Cauliflower mosaic virus 35S promoter CaMV35S. Co-cultivation on solid media for 72 h was optimal for generating transformants, but increasing the ratio of bacterial cells to conidia did not affect transformation efficiency. All hygromycin B-resistant transformants carried transfer-DNA (T-DNA) as determined by polymerase chain reaction (PCR) and the T-DNA integrations appeared to be random and in single copy as detected by Southern hybridization. Transformants remained resistant to hygromycin B in the absence of selection. Variations in colony morphology were observed in the presence of hygromycin B under different culture conditions, and a variety of altered phenotypes including reduced virulence were observed among 550 transformants. Inverse PCR was more efficient than TAIL-PCR in identifying flanking genomic sequences from T-DNA borders, and the possible causes are discussed. This transformation technique and recovery of flanking DNA using inverse PCR will provide a useful tool for genetic studies of A. rabiei.

Ascomycota↗
PubMed2006

Secreted expression and purification of dengue 2 virus full-length nonstructural glycoprotein NS1 in Pichia pastoris.

The dengue 2 virus (DEN-2) RNA (NGC strain) was used as a substrate to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA followed by polymerase chain reaction amplification of the NS1 region. Products were cloned into pPICZalphaB vector for sequencing and into Pichia pastoris for expression. A recombinant protein with a molecular size of approximately 80 KDa was secreted into the supernatant from the yeast cells when induced with methanol. The expressed protein was able to bind with mouse polyclonal antibody or NS1-specific monoclonal antibody of dengue 2 virus. Purified NS1-poly(His)-tagged fusion protein was obtained from the expressed product by passing through a metal-chelating affinity chromatographic (MCAC) column. The study also verified that our purified rNS1 protein retained its antigenicity. High-level production of the rNS1 protein up to 70 mg/l indicates that P. pastoris is an efficient expression system for dengue virus full-length NS1 glycoprotein.

Aedes↗
PubMed1996

Variegation and silencing of the Heat Shock Cognate 80 gene are relieved by a bipartite downstream regulatory element.

Transgenes are important tools for plant molecular biologists. However, some aspects of their regulation are not completely understood. First, the contribution of elements separate from the promoter has been documented for several genes, but a general requirement for such elements is still unclear. Second, transgene expression can be subject to variability from chromosomal position effects and from epigenetic effects, often due to the presence of repeated sequences. While studying the regulation of the developmentally expressed Heat Shock Cognate 80 (HSC80) gene, several peculiarities were found which involve both of these questions. First, HSC80 transgenes are subject to silencing and variegation at a high frequency. Second, a bipartite element downstream of the transcriptional start is necessary to achieve efficient expression from the HSC80 promoter in stable transformants. Finally, this bipartite element is not required for expression in a transient system, where the promoter alone is sufficient. These results together support a role for chromatin structure in the regulation of HSC80.

Arabidopsis↗
PubMed1999

Conserved extracellular cysteine pair in the M3 muscarinic acetylcholine receptor is essential for proper receptor cell surface localization but not for G protein coupling.

Most G protein-coupled receptors contain a conserved pair of extracellular cysteine residues that are predicted to form a disulfide bond linking the first and second extracellular loops. Previous studies have shown that this disulfide bond may be critical for ligand binding, receptor activation, and/or proper receptor folding. However, the potential importance of the two conserved cysteine residues for proper receptor cell surface localization has not been investigated systematically. To address this issue, we used the rat M3 muscarinic receptor as a model system. Most studies were carried out with a modified version of this receptor subtype (lacking potential N-glycosylation sites and the central portion of the third intracellular loop) that could be readily detected via western blot analysis. Cys-->Ala mutant receptors were generated, transiently expressed in COS-7 cells, and then examined for their subcellular distribution and functional properties. ELISA and immunofluorescence studies showed that the presence of both conserved cysteine residues (corresponding to C140 and C220 in the rat M3 muscarinic receptor sequence) is required for efficient expression of the M3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor-mediated G protein activation (studied in second messenger assays). These results shed new light on the functional role of the two extracellular cysteine residues present in most G protein-coupled receptors.

Alanine↗
PubMed2005

The destabilization of polyplexes facilitates intranuclear transcription efficiency.

We have been focusing on the last step of transfection, that is, the intranuclear recognition of polyplexes by transcription factors. In this work, we studied about the relationship between the intracellular destabilization of polyplexes and the transcription efficiency. Polypeptides containing specific sequences which are digested by intracellular proprotein convertase (PC), furin (Fur-polypeptide) were synthesized as novel carriers. When Fur-polypeptide/plasmid DNA polyplexes were incubated with furin in the cell-free transcription/translation system, the sequence specific expression was observed as time. Additionally, Fur-polypeptides/plasmid DNA polyplexes led to site-specific effective transgene expression even in COS-1 cells. These results indicate that shortened carriers lose their abilities to form polyplexes in response to furin activity in the cells, and it seems to enhance the destabilization of polyplexes, the recognition of transgene in polyplexes by transcription factors, and the expression efficiency.

Animals↗
PubMed2004

Protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants.

Vaccines produced in transgenic plants constitute a promising alternative to conventional immunogens, presenting the possibility of stimulating secretory and systemic immunity against enteric pathogens when administered orally. Protection against enteric pathogens affecting newborn animals requires, in most cases, the stimulation of lactogenic immunity. Here, the group presents the development of an experimental immunogen based on expression of an immunorelevant peptide, eBRV4, of the VP4 protein of bovine rotavirus (BRV), which has been described as harbouring at least one neutralizing epitope as well as being responsible for the adsorption of the virus to epithelial cells. The eBRV4 epitope was efficiently expressed in transgenic alfalfa as a translational fusion protein with the highly stable reporter enzyme beta-glucuronidase (betaGUS), which served as a carrier, stabilized the synthesized peptide and facilitated screening for the higher expression levels in plants. Correlation of expression of the eBRV4 epitope in plants with those presenting the highest betaGUS activities was confirmed by a Western blot assay specific for the BRV peptide. The eBRV4 epitope expressed in plants was effective in inducing an anti-rotavirus antibody response in adult female mice when administered either intraperitoneally or orally and, more importantly, suckling mice born from immunized female mice were protected against oral challenge with virulent rotavirus. These results demonstrate the feasibility of inducing lactogenic immunity against an enteric pathogen using an edible vaccine produced in transgenic plants.

Animals↗
PubMed2000

Vectors to achieve selective expression of vaccine antigens within eukaryotic cells using Salmonella spp. as carrier strains.

A set of expression vectors was constructed which allows the expression of recombinant antigens under the control of Salmonella typhi promoters that are selectively activated after infection of eukaryotic cells. The pUC18Not derivatives contain a promoter downstream of the early transcriptional terminator from phage T7 and followed by a multiple cloning site, whereas the pBluescript II S/K derivatives contain the ribosomal RNA T(1) transcriptional terminator and also the strong translation signals of the Escherichia coli atpE gene. The expression cassettes are flanked by NotI or PacI sites to simplify their subcloning where required. The resulting vectors were validated using the S1 subunit of pertussis toxin as a model antigen and Salmonella typhimurium aroA SL7207 as a carrier. The S1 subunit was efficiently expressed by recombinant Salmonella within Henle 407 cells.

Antigens, Bacterial↗
PubMed2003

[Expression of Chinese sturgeon cystatin in yeast Pichia pastoris and its proteinase inhibitory activity analysis].

Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher than that of CPI (cysteine proteinase inhibitor) from seeds of corn, that about 1 microg purified CIP could inhibited the activity of 0.278 microg papain. But the heat stability of recombinant cystatin is lower than that of the corn CPI. The expression level and the activity of recombinant cystatin from yeast Pichia pastoris were higher than those from E. coli. Moreover, recombinant cystatin from Pichia pastoris was easier to separate and purify. This paper reported that recombinant fish cystatin was produced in a highly efficient expression system based on the methylotrophic yeast, further work will focus on the function of recombinant Chinese sturgeon cystatin to resist fish disease and explore the value of cystatin as a food additive to inhibit cysteine proteinases during surimi processing.

Animals↗
PubMed2006

[Cloning and expression of murine Toll-like receptor-2 N terminal and preparation of its antibody].

OBJECTIVE: To prepare the recombinant murine Toll-like receptor-2 N-terminal (mTLR-2N) fusion protein and obtain anti-mTLR-2N polyclonal antibody. METHODS: The gene encoding 153 amino acids of mTLR-2N was amplified by PCR and cloned into pET32A vector with sequence verification. The recombinant fusion protein was expressed in E. coli and purified by Probond resin column. Rabbits were immunized with fusion protein to obtain the polyclonal anti-sera, and the antibodies were identified by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. RESULTS: The recombinant fusion protein was efficiently expressed and purified. The polyclonal antibodies could bind to the fusion protein expressed in different vectors as the antigens in ELISA, and also bind with RAW264.7 cells expressing mTLR-2 and CHO cells transfected with full-length mTLR-2 gene. CONCLUSION: The recombinant mTLR-2N fusion protein is obtained and the anti-mTLR-2N polyclonal antibody can recognize natural mTLR-2 on the cell surface.

Animals↗
PubMed1994

Expression of the human glycine receptor alpha 1 subunit in Xenopus oocytes: apparent affinities of agonists increase at high receptor density.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel, which mediates post-synaptic inhibition in spinal cord and other brain regions. Heterologous expression of the ligand binding alpha subunits of the GlyR generates functional agonist-gated chloride channels that mimic most of the pharmacological properties of the receptor in vivo. Here, nuclear injection into Xenopus oocytes of a human alpha 1 subunit cDNA, engineered for efficient expression, was used to create GlyR channels over a wide density range, resulting in whole-cell glycine currents of 10 nA to 25 microA. Notably, the pharmacology of these channels changed at high expression levels, with the appearance of a novel receptor subpopulation of 5- to 6-fold higher apparent agonist affinity at current values > 4 microA. The low-affinity receptors were readily blocked by nM concentrations of the competitive antagonist strychnine, whereas the high-affinity receptors were more resistant to antagonism by this alkaloid. Picrotoxinin, a chloride channel blocker, inhibited both GlyR populations with equal potency. Our data suggest that receptor interactions, occurring at high receptor density, modify the agonist response of the GlyR. This phenomenon may contribute to neurotransmitter efficacy at fast synapses.

Animals↗