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At least 775 records · Page 43Linked to original sources
PubMed1985

Efficient secretion and purification of human insulin-like growth factor I with a gene fusion vector in Staphylococci.

A novel approach for production of small polypeptides, using a staphylococcal protein A vector, is described. This system is used to express, secrete and purify human insulin-like growth factor I (IGF-I). A fusion protein consisting of protein A and IGF-I is recovered in high yield by passing the culture medium through an IgG affinity column. Using site-specific mutagenesis an acid labile asp-pro cleavage site was introduced at the fusion point between the two proteins. The protein A "tail" can thereby be removed from the affinity purified fusion protein by chemical cleavage releasing biologically active IGF-I molecules.

Escherichia coli↗
PubMed1987

Detection of PDGF-2 homodimers in human tumor cells.

The v-sis oncogene encodes a protein structurally and functionally related to human platelet-derived growth factor (PDGF). In the present studies, we show that the primary translational product of the human sis proto-oncogene is a 26-kd protein, p26c-sis. This product is processed to yield a disulfide-linked homodimer, p56c-sis, which is further processed to a 35,000-dalton dimer, p35c-sis. Like the v-sis gene product, the PDGF-2 precursor undergoes N-linked glycosylation, implying its processing through the endoplasmic reticulum. The PDGF-2 product was shown to possess functional properties of PDGF. Whereas lysates of control COS-1 cells lacked mitogenic activity, lysates of COS-1 cells transfected with a c-sis/PDGF-2 expression vector specifically stimulated DNA synthesis of quiescent fibroblasts. Moreover, this activity was completely inhibitable by PDGF antibody. Identical forms of the sis/PDGF-2 product were identified in human tumor cells that expressed c-sis/PDGF-2 transcripts. These proteins were shown to be specifically associated with the membrane component of the tumor cells and were not detectably secreted into the culture medium. These findings support the concept that expression of the sis/PDGF-2 product in human cells responsive to its proliferative actions can be an important step in the processes leading to malignancy.

Cell Division↗
PubMed2007

Targeting the exogenous htPAm gene on goat somatic cell beta-casein locus for transgenic goat production.

Combining gene targeting of animal somatic cells with nuclear transfer technique has provided a powerful method to produce transgenic animal mammary gland bioreactor. The objective of this study is to make an efficient and reproducible gene targeting in goat fetal fibroblasts by inserting the exogenous htPAm cDNA into the beta-casein locus with liposomes or electroporation so that htPAm protein might be produced in gene-targeted goat mammary gland. By gene-targeting technique, the exogenous htPAm gene was inserted to milk goat beta-casein gene sequences. Fetal fibroblasts were isolated from Day 35 fetuses of Guanzhong milk goats, and transfected with linear gene-targeting vector pGBC4htPAm using Lipefectamin-2000 and electoporation, respectively. Forty-eight gene-targeted cell colonies with homologous recombination were obtained, and three cell colonies were verified by DNA sequence analysis within the homologous recombination region. Using gene-targeted cell lines as donor cells for nuclear transfer, a total of 600 reconstructed embryos had been obtained, and 146 developed cloned embryos were transferred to 16 recipient goats, and finally three goats showed pregnancy at Day 90.

Animals↗
PubMed1987

Regulated overproduction of the GAL4 gene product greatly increases expression from galactose-inducible promoters on multi-copy expression vectors in yeast.

High-level, galactose-inducible expression originating from GAL promoters in Saccharomyces cerevisiae is mediated by highly specific interactions between the GAL4-coded protein and nucleotide sequences. The potential utility of recombinant GAL promoter/foreign gene constructions for the regulatable and high-level expression of foreign proteins in yeast is well recognized. However, the utility of this system is limited severely in the case of multiple copies of such constructions due to the very low level of the GAL4-coded protein and to the loss of inducibility which occurs if levels of the GAL4 protein are amplified constitutively. To surmount these limitations, we have constructed a novel yeast strain which overproduces the GAL4 protein in a regulated fashion. This 'integrant' strain contains an integrated copy of a hybrid gene consisting of the galactose-inducible GAL10 promoter fused to the GAL4 structural gene. In the absence of galactose, the integrant strain and the isogenic 'non-integrant' parental strain show only a basal level of transcription from the constitutively active chromosomal GAL4 gene. However, following the addition of galactose to the culture medium, the 'integrant' strain synthesizes at least 20-fold more GAL4 mRNA and substantially more GAL4 protein than the 'non-integrant' strain. A high-copy-number expression vector containing the GAL10 promoter and alpha mating factor pre-pro leader fused to the structural gene for Epstein-Barr virus gp350 was introduced into both types of cells. The resulting transformed 'integrant' cells produced approximately five-fold more gp350 mRNA and ten-fold more gp350-related proteins than the transformed 'non-integrant' cells following galactose induction. This 'integrant' strain should prove generally useful for the maximal, regulated expression in yeast of structural genes driven by a galactose-inducible promoter.

DNA, Recombinant↗
PubMed1986

Gene therapy: current status and future directions.

Of the many techniques which have been used to introduce new genes into cells, four are of particular interest for gene therapy. Transfection has been most extensively studied and has been used to put new genes into mice and men. Homologous recombination offers the promise of curing certain mutations in situ, but is still largely unexplored. Injection of new genes into the nuclei of single cell embryos has had startling successes in animals, but there are many scientific and ethical issues to be resolved before such an approach can be considered for gene therapy. Retroviral vectors currently offer the most exciting prospect for introducing useful gene sequences into defective cells; however, safety issues and problems of low levels of gene expression are still to be resolved. It appears likely that the bone marrow will be the first target of gene therapy for genetic diseases. The marrow is accessible and contains proliferating stem cells which can transmit the new genes to progeny cells. The possibility of treating enzyme deficiency diseases and hemoglobinopathies by gene transfer is attractive. Experiments in two patients with thalassemia major indicate that it is feasible to introduce new genes into bone marrow cells of man and return the modified cells to hematopoietic sites.

Gene Expression Regulation↗
PubMed2002

Upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin-12.

OBJECTIVE: Injection of dendritic cells (DC) engineered with recombinant adenoviral vectors to produce interleukin-12 (IL-12) inside experimental murine tumors frequently achieves complete regressions. In such a system the function of CD8(+) T cells has been shown to be an absolute requirement, in contrast to observations made upon depletion of CD4(+) T cells, which minimally affected the outcome. The aim of this work was to study the possible involvement of natural killer (NK) cells in this setting. MATERIALS, METHODS, AND RESULTS: Depletions with anti-AsialoGM1 antiserum showed only a small decrease in the proportion of complete regressions obtained that correlated with induction of NK activities in lymphatic tissues into which DC migrate, whereas combined depletions of CD4(+) and NK cells completely eliminated the antitumor effects. Likewise in vivo neutralization of interferon-gamma (IFN-gamma) also eliminated those therapeutic effects. Trying to define the cellular role played by NK cells in vivo, it was observed that injection of cultured DC inside the spleen of T- and B-cell-deficient (Rag1(-/-)) mice induced upregulation of NK activity only if DC had been adenovirally engineered to produce IL-12. In addition, identically transfected fibroblasts also activated NK cells, indicating that IL-12 transfection was the unique requirement. Equivalent human DC only activated in vitro the cytolytic and cytokine-secreting functions of autologous NK cells if transfected to express human IL-12. CONCLUSIONS: Overall, these results point out an important role played by NK cell activation in the potent immunotherapeutic effects elicited by intratumoral injection of IL-12--secreting DC and that NK activation under these conditions is mainly, if not only, dependent on IL-12.

Animals↗
PubMed2003

Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector.

We have searched for plasmids in a collection of 55 Bacillus subtilis strains isolated from various natural sources of the territory of Belarus. Twenty percent of the strains contained one or two plasmids of either 6-8 or approximately 90 kb. Small plasmids were shown to carry a rolling circle replicon of the pC194 type. Four out of the eight large plasmids contained a related theta replicon that has no homolog in databases as shown by sequence determination. A B. subtilis/Escherichia coli shuttle vector based on this replicon was constructed. It has a low copy number (6 units per chromosome) and is stably inherited in B. subtilis. It might thus be a useful tool for DNA cloning. These data extend previous observations, indicating that most of the small plasmids of B. subtilis replicate as rolling circles and belong to the pC194 family. On the contrary, large plasmids appear to form a large pool of theta-replicating determinants, since three different replicons have already been isolated from them.

Amino Acid Sequence↗
PubMed1986

Human adenovirus cloning vectors based on infectious bacterial plasmids.

By making use of the fact that human adenovirus DNA circularizes in infected cells, and that circular forms of the viral genome are infectious, we have developed an improved adenovirus-based cloning system. A deletion mutant of adenovirus type 5 (Ad5) with deletions in early regions 1 (E1) and 3 (E3) was converted to a bacterial plasmid which can regenerate infectious virus following transfection into human 293 cells. A single XbaI recognition site in the deleted E3 region serves as a site for the insertion of foreign DNA. We have used this system to clone a number of genes into the Ad5 genome and describe the insertion of the neomycin/G418 resistance marker into Ad5 as an example.

Adenoviruses, Human↗
PubMed1993

Mouse genetics in the 21st century: using gene targeting to create a cornucopia of mouse mutants possessing precise genetic modifications.

Over 1500 mouse mutants have been identified, but few of the genes responsible for the defects have been identified. Recent developments in the area of gene targeting are revolutionizing the field of mouse genetics and our understanding of numerous genes, including those thought to be involved in cell proliferation and differentiation. Gene targeting was developed as a method for producing a predetermined mutation in a specific endogenous gene. Advances in the design of targeting vectors and in the use of embryonic stem cells have permitted the production of numerous mutant mice with null mutations in specific genes. These mutant mice will be critical for investigating the in vivo functions of many genes that have been cloned in recent years. This review discusses a wide range of new developments in the field of gene targeting with a focus on issues to be considered by those planning to use this new technology. It also examines some of the lessons learned from recent gene targeting studies and discusses different applications of the technology that are likely to generate scores of new animal models for a wide range of human diseases.

Animals↗
PubMed1999

HSV-1-based vectors for gene therapy of neurological diseases and brain tumors: part I. HSV-1 structure, replication and pathogenesis.

The design of effective gene therapy strategies for brain tumors and other neurological disorders relies on the understanding of genetic and pathophysiological alterations associated with the disease, on the biological characteristics of the target tissue, and on the development of safe vectors and expression systems to achieve efficient, targeted and regulated, therapeutic gene expression. The herpes simplex virus type 1 (HSV-1) virion is one of the most efficient of all current gene transfer vehicles with regard to nuclear gene delivery in central nervous system-derived cells including brain tumors. HSV-1-related research over the past decades has provided excellent insight into the structure and function of this virus, which, in turn, facilitated the design of innovative vector systems. Here, we review aspects of HSV-1 structure, replication and pathogenesis, which are relevant for the engineering of HSV-1-based vectors.

Brain Neoplasms↗
PubMed1987

Intermolecular recombination assay for mammalian cells that produces recombinants carrying both homologous and nonhomologous junctions.

We present an intermolecular recombination assay for mammalian cells that does not involve the reconstitution of a selectable marker. It is based on the generation of a shuttle vector by recombination between a bacterial and a mammalian vector. The recombinants can thus be amplified in mammalian cells, isolated by plasmid rescue in an Escherichia coli RecA- host, and identified by in situ hybridization, by using mammalian vector sequences as probes. Since both parental molecules can share defined lengths of homology, this assay permits a direct comparison between homologous and nonhomologous intermolecular recombination. Our results indicate that the dominant intermolecular recombination mechanism is a nonhomologous one. The relative frequency of homologous to nonhomologous recombination was influenced by the length of shared homology between parental molecules and the replicative state of the parental molecules, but not by the introduction of double-strand breaks per se. Finally, almost all of the recombinants with a homologous junction did not have the reciprocal homologous junction but instead had a nonhomologous one. We propose a model to account for the generation of these recombinants.

Animals↗
PubMed2002

The first reporter gene assay on living cells: green fluorescent protein as reporter gene for the investigation of Gi-protein coupled receptors.

Reporter gene assay systems are important tools for the investigation of G-protein coupled receptors and their interaction with ligands. Here, we describe a novel reporter gene assay system for the investigation of Gi-protein coupled receptors in living cells. For the first time green fluorescent protein (GFP) was used as reporter gene under the transcriptional control of cAMP-response elements (CREs). Three different reporter gene vectors with increasing numbers of CREs were cloned and GFP expression was investigated after forskolin stimulation. Furthermore, the novel reporter system was successfully applied to the neuropeptide Y (NPY) rY5 receptor subtype, a Gi-protein coupled receptor. Our data clearly demonstrate dose-dependent GFP expression for NPY. Furthermore, receptor subtype selectivity of well characterized NPY analog could be proven for the NPY rY5 receptor. The great advantage of the method is that no cell lysis is required and assays can be performed on living cells. Accordingly, in vitro testing of agonist gets faster and significantly more convenient.

Animals↗
PubMed2002

[Development of gene-viral vector and its anti-tumor effect, a primary study].

OBJECTIVE: To develop a new kind of vector system, named as gene-viral vector, which combines the advantages of the gene therapy and virus therapy. METHOD: An anti-tumor gene was inserted into the genome of the replicative virus specific for the tumor cells by virus recombination technology. The killing effect, report gene expression of the green fluorescence protein, expression of the anti-tumor gene of mouse IL12, and the replication of the virus were observed respectively by cell pathology, fluorescence microscopy, ELISA and electron microscopy. RESULTS: A new kind of gene-viral vector system, in which the E1b-55 000 gene is deleted but the E1a gene of adenovirus is preserved, was constructed. The vector system possessed the same characteristics as the replicative virus ONYX-015, replication and proliferation in the tumor cells but not in the normal cells, thus specifically killing the tumor cells. Besides, it carried many kinds of anti-tumor genes. When carrying the report gene of the green fluorescence protein it made the expression of this gene in tumor cells far more effectively than the adenovirus vector employed in the traditional gene therapy did. However in the normal cells the expression of green fluorescence protein caused by this vector system was as little as or even less than that by the traditional adenovirus system. The similar result was also observed in the experiments of this vector system carrying the anti-tumor gene, gene of mouse IL12. The replication and proliferation of the virus carrying the gene of mouse IL12 in the tumor cells were confirmed by electron microscopy. CONCLUSION: Gene-viral vector is a new kind of vector in which the anti-tumor gene is inserted into the genome of the replicative virus specific for the tumor cells. It increases the expression of the anti-tumor gene by hundreds even tens of thousand times. It posseses all the advantages of gene therapy and virus therapy, thus further enhancing the curative effect and it overcomes such disadvantages as low transfer rate, low expression, lack of target tropism and low anti-tumor activity. It will become one of the most promising means in tumor treatment.

Adenoviridae↗
PubMed1989

Expression of a multidrug resistance-adenosine deaminase fusion gene.

A novel fusion gene has been created in which the expression of a dominant selectable marker, the human multidrug resistance gene, is directly linked to the expression of human adenosine deaminase cDNA. The chimeric gene was inserted between the long terminal repeats of a Harvey murine sarcoma virus expression vector and used to transfect drug-sensitive human KB carcinoma cells. Transfectants were selected in increasing concentrations of colchicine and found to contain multiple copies of the intact fusion gene, which is stably and efficiently expressed. A membrane-associated 210-kDa human P-glycoprotein-adenosine deaminase fusion protein is synthesized which retains function of the multidrug transporter and also exhibits adenosine deaminase activity. The data indicate that the human multidrug resistance gene may be used as a dominant selectable marker to introduce other genes in the form of gene fusions into cultured cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Construction and application of a prokaryotic vector which expresses the protein that can be quickly purified by IMAC.

A vector was constructed by inserting a pair of complementary oligo nucleotides encoding 6 histidine residues into the polylinker's upstream of the prokaryotic high expression vector pBV220. The resultant vector is named pBV222. Proteins expressed by this vector will have a 6-histidine tail as an affinity handle fused to their N-terminus and can be quickly purified by one-step immobilized metal affinity chromatography (IMAC). This plasmid was verified by restriction mapping and DNA sequencing. When GM-CSF and IL-2 cDNA were closed into pBV222, expressed proteins in the inclusion body showed the predicted molecular weight and biological activity. The expressed bacteria were dissolved in 6 mol/L guanidine.HCl and the supernatant was loaded directly to IMAC. IL-2 and GM-CSF fusion proteins were eluted by the pH gradient, and over 90% purity was achieved.

Chromatography, Affinity↗
PubMed1988

The production of foreign proteins in mammalian cells.

Expression systems for producing foreign proteins in mammalian cells are built from two components. One component is the DNA expression vector and the other is the mammalian host-cell line. Functional elements of DNA are better understood and generally easier to manipulate than complex mammalian cells. The standard approach, therefore, has been to manipulate the expression vectors to work well in convenient host cell lines. A wide variety of DNA regulatory signals for efficient transcription and translation have been tested in mammalian-cell-expression vectors. Many of the most successful and widely used regulatory signals are derived from eukaryotic viral DNAs. In addition to optimizing the vectors to give efficient transcription and translation of the foreign protein, higher expression levels can be achieved by increasing the number of foreign gene copies per cell. High gene copy number is usually attained by including an amplifiable gene, such as dhfr, in the expression vector, introducing the vector DNA into the host-cell lines, and then using a toxic agent to select for resistant cell lines containing high copy numbers of the amplifiable gene. Cells with amplified copy numbers of the selected gene generally also contain high copy numbers of the foreign gene and thus produce elevated levels of the foreign protein product. Although gene amplification has been most successful in creating cell lines producing high levels of foreign proteins, there are inherent instability problems with cell lines forced to carry extremely high copy numbers of foreign genes. One means of avoiding instability problems due to continuous high gene copy and continuous high foreign protein production, is to develop regulatable expression systems. The regulatable expression systems being developed are based either on regulating the gene copy number by regulating DNA replication or on regulating the level of transcription by using a regulatable promoter to transcribe the foreign protein coding cDNA. In addition to designing a good expression vector, it is important to consider the mammalian host cell. Although most potential mammalian host-cell lines are capable of post-translational processing and secretion, certain processing steps, such as gamma-carboxylation, may be done efficiently only in specialized cell types. It is also important to estimate how much of the foreign protein will be needed and to decide whether the proposed host-cell line can be easily and economically grown to produce that amount. Regulatory considerations are also important in choosing a host-cell line for commercial production. Many of the potential host-cell lines are tumorigenic and carry retroviruses.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

Intravenous administration of replication-incompetent adenovirus to rhesus monkeys induces thrombocytopenia by increasing in vivo platelet clearance.

A replication-incompetent adenovirus vector was administered to rhesus macaques at 1, 3 and 6 x 1012 particles/kg doses to investigate its toxicity. Platelet count decrements of 28%, 82% and 90%, respectively, were observed, with corresponding platelet half-lives of 69.0, 25.2 and 22.2 h (compared with 111 h in untreated animals). The platelet decline was equivalent for all three doses for 8 h, and platelet count recovery began as early as 8 h after infusion for low-dose recipients, or as late as 24 h for the medium and high dose recipients. These observations suggest that thrombocytopenia is a saturable, reversible consumptive process.

Adenoviridae↗
PubMed2002

[Delivery of secreted placental alkaline phosphatase (SEAP) gene in vitro and in vivo as a component of recombinant avian adenovirus (CELO)].

Recombinant adenoviruses capable of expressing the gene of secreted placentary alkaline phosphatase (SEAP) under control of CMV-promoter was obtained on the basis of CELO avian adenovirus and human adenovirus-5 (Ad5) genomes. The efficiency of the CELO vector was determined in experiments with transduction of human (293, A549, and H1299), mouse (B16), and avian (LMH) cell cultures. It was shown in C57BL/6 mice in vivo that SEAP gene is expressed under conditions of intravenous, intranasal, and intratumoral application of recombinant adenovirus CELO-SEAP. The duration of expression of the alkaline phosphatase CELO = SEAP gene in immunocompetent mouse body was 21 days. The level of SEAP gene expression was measured in the allantois fluid of chicken embryo infected with recombinant adenovirus CELO-SEAP.

Alkaline Phosphatase↗