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Results for “Transient expression”

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At least 433 records · Page 24Linked to original sources

PubMed1992

Genetic manipulation of mammalian dictyate oocytes: factors affecting transient expression of microinjected DNA templates.

Transcription of exogenous DNA templates in mouse ovarian oocytes was investigated by microinjecting constructs encoding for the Escherichia coli lacZ gene under control of promoters from: 1) the mouse hsp68 gene; 2) the human beta-actin gene; and 3) simian virus 40 (SV40) early genes. Various amounts of circular or linear DNA constructs were injected into dictyate oocyte nuclei at different stages of follicle growth, and the beta-galactosidase activity was then cytochemically evaluated in single cells. In middle-sized growing oocytes, expression of circular constructs was observed with amounts of DNA ranging from 50 to 10(3) plasmid copies/nucleus and was first observed 10-12 hr after injection. Maximal expression levels were reached by 17 hr after injection and were specific for the constructs used. Circular constructs containing the hsp68 and early SV40 promoters were expressed at similar levels in small- and middle-sized growing oocytes, while the construct carrying the beta-actin promoter was expressed only in small-sized cells. In contrast to growing oocytes, these constructs were never expressed in fully grown oocytes. DNA linearization depressed construct activity regardless of the site of cleavage. These results show that: 1) lacZ is a valuable reporter gene in the analysis of eukaryotic promoter activity in dictyate mouse oocytes; 2) transient construct expression requires the injection of DNA in circular form; and 3) the expression efficiency of different DNA templates is dependent on the presence of a specific promoter and on the differentiation stage of oocytes analyzed.

Actins↗

PubMed1993

Transient expression of a slow-tonic MHC isoform by extrafusal fibers in the developing rat.

ALD 19, a monoclonal antibody that recognizes the slow-tonic myosin heavy chain (MHC) isoform, has been used extensively as a marker for nuclear bag intrafusal fibers of muscle spindles in developing and adult rats. Extrafusal fibers of adult rat hindlimb muscles do not express slow-tonic MHC. However, while using ALD 19 to trace the fate of intrafusal fibers following neonatal denervation, we noted that some extrafusal fibers of neonates also bound this antibody. The immunolabeled extrafusal fibers were a subset of slow fibers located in the deep axial regions of crural muscles. The same fiber subset transiently displayed a weak affinity for ALD 19 during the first postnatal week in normal muscles. Denervation at birth increased the intensity of ALD 19 immunolabelling by these extrafusal fibers and extended the duration of the slow-tonic immunoreactivity into the 2nd postnatal week, after which expression diminished or ceased. Demonstration that some developing extrafusal fibers have a nerve-independent capacity for transiently expressing slow-tonic MHC, an MHC previously though to be expressed only by intrafusal fibers, raises the possibility that both types of fiber originate from a subset of bipotential slow primary myotubes in rat hindlimbs.

Animals↗

PubMed1996

Host haemocyte inactivation by an insect parasitoid: transient expression of a polydnavirus gene.

Polydnaviruses produced by the hymenopteran endoparasitoid Cotesia rubecula are deposited together with the egg into the lepidopteran host Pieris rapae and are apparently involved in the suppression of the host's defence system. Around 6 h post-parasitization host haemocytes change their surface properties, actin cytoskeleton structure and adhesion properties. Here we show that a single polydnavirus gene is expressed inside the caterpillar haemocytes in a transient fashion between 4 to 8 h post-parasitization.

Amino Acid Sequence↗

PubMed1996

Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors.

Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration.

Amino Acid Transport Systems↗

PubMed1993

Expression of the S1 and S2 subunits of murine coronavirus JHMV spike protein by a vaccinia virus transient expression system.

The spike (S) protein of murine coronavirus JHMV, variant cl-2, comprises two polypeptides, N-terminal S1 (with an N-terminal signal peptide) and C-terminal S2 (with a C-terminal transmembrane domain). In order to express these subunits, we constructed three different vaccinia virus transfer vectors (VV-TVs) containing cDNAs encoding the S1 protein without a transmembrane domain (pSFS1utt), the S1 protein with a C-terminal transmembrane domain derived from S2 (pSFS1tmd) or the S2 protein with an N-terminal signal peptide derived from S1 (pSFssS2). The S1 and S2 proteins were expressed in DBT cells by infection with vaccinia virus and transfection of these VV-TVs. In cells transfected with the pSFS1utt and pSFS1tmd, 96K and 106K proteins, respectively, were detected by Western blotting. The ssS2 protein expressed by pSFssS2 was 96K, which was slightly larger than the authentic S2 protein. The S1utt and S1tmd proteins were shown by binding studies using a panel of monoclonal antibodies to be antigenically indistinguishable from the authentic S1 protein. The S1tmd and ssS2 proteins were detected on the cell surface by immunofluorescence, whereas the S1utt protein was not. However, when the S1utt protein was expressed together with the ssS2 protein, the S1utt was detected on the cell membrane. This suggested that the S1utt was associated with ssS2 on the cell membrane. These observations indicate that the expressed S1 and S2 proteins associated in a similar manner to the authentic S1 and S2 proteins produced in DBT cells infected with cl-2. However, cell fusion was not observed in cells expressing either S1 or S2 nor in cells coexpressing both S1 and S2, although the whole S protein expressed by VV-TV did induce fusion.

Amino Acid Sequence↗

PubMed1995

Transient expression of genes transferred in vivo into heart using first-generation adenoviral vectors: role of the immune response.

Gene therapy for heart diseases requires availability of an efficient vector for gene transfer into myocardium. Recombinant adenovirus expressing the Escherichia coli beta-galactosidase (beta-Gal) gene was shown to infect rat cardiocytes efficiently in vivo. However, a time course of gene expression showed that transgene expression was maximal during the first week following injection, then declined and disappeared by day 21. An immunosuppressive treatment prolonged beta-Gal expression for at least 21 days. On the contrary, a preimmunization of the animals by two intraperitoneal injections of the vector led to a decreased transgene expression 48 hr after intramyocardial injection and to a barely detectable expression at the sixth day. Appearance of adenovirus neutralizing antibodies in preimmunized animals could have contributed to such a refractoriness to further adenoviral infection. Finally, a neonatal intrathymic injection of the vector was able to induce long-term LacZ expression for more than 2 months after heart injection, although neutralizing as well as anti-beta-Gal antibodies were detected in sera of the animals. These results indicate that an immune response against first-generation replication-defective adenoviral vectors is a major cause of transient transgene expression, a cellular response being most probably responsible for ablation of transgene expression in immunocompetent animals.

Adenoviridae↗

PubMed1994

Restoration of aryl hydrocarbon (Ah) responsiveness in MDA-MB-231 human breast cancer cells by transient expression of the estrogen receptor.

MDA-MB-231 human breast cancer cells express the aryl hydrocarbon (Ah) receptor; however, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) does not induce CYP1A1 gene expression or chloramphenicol acetyltransferase (CAT) activity in cells transiently transfected with pRNH11c, and Ah-responsive plasmid derived from the 5'-flanking region of the human CYP1A1 gene. However, when MDA-MB-231 cells were treated with 10 nM TCDD and co-transfected with pRHN11c and a human estrogen receptor (hER) expression plasmid (delta hER), there was approximately a 10-fold increase in CAT activity. The restoration of Ah-responsiveness in MDA-MB-231 cells by expression of nuclear hER was highly specific since parallel studies in which plasmids that express the progesterone receptor and Jun nuclear proteins did not restore Ah-responsiveness to this cell line. Moreover, in cells transiently transfected with the pRNH11c and delta hER plasmids and 10 nM TCDD, overexpression of the Jun protein inhibited the effects of the hER on Ah-responsiveness. Plasmids that express truncated forms of the hER were also active in MDA-MB-231 cells but were not as effective as the complete hER. These studies reveal a unique function for the ER in MDA-MB-231 cells in which expression of this protein results in restoration of Ah-responsiveness.

Adenocarcinoma↗

PubMed2003

Transient expression of PU.1 commits multipotent progenitors to a myeloid fate whereas continued expression favors macrophage over granulocyte differentiation.

OBJECTIVES: The Ets-family transcription factor PU.1 is expressed specifically in the hematopoietic system, in which it is absolutely required for the generation of B lymphocytes and macrophages. In contrast, overexpression of PU.1 blocks terminal differentiation of the erythroid lineage, in which it can act as an oncogene. In this study we used a multipotential progenitor cell line to examine the effects of PU.1 overexpression on myeloerythroid commitment within a single model system. MATERIALS AND METHODS: PU.1 cDNA was introduced transiently and stably into the multipotent, nonleukemic hemopoietic cell line FDCPmix. Transiently transfected cells were isolated by fluorescence-activated cell sorting within 18 hours of transfection. Stable transfectants were selected by antibiotic resistance over a number of weeks. The effects of short- and long-term overexpression of PU.1 on self-renewal, proliferation, and differentiation were investigated. RESULTS: A transient pulse of expression in multipotent progenitor cells eliminated the options of self-renewal and erythroid differentiation, resulting in commitment to the myeloid lineage. However, this transient pulse of expression did not affect the subsequent lineage choice of bipotent granulocyte/macrophage progenitors. In contrast, continuous expression of PU.1 resulted in a strong bias toward macrophage rather than granulocyte differentiation. CONCLUSIONS: These results demonstrate promyeloid effects of PU.1 at two distinct stages of hematopoiesis.

Animals↗

PubMed1988

Follicle-stimulating hormone induces transient expression of the protooncogene c-fos in primary Sertoli cell cultures.

The expression of the protooncogene c-fos has been associated with the transduction of cell surface stimuli into changes in nuclear function. To evaluate the possibility that this protooncogene plays a role in the gonadotropin-dependent gene regulation, the effect of FSH on the expression of c-fos was studied in primary Sertoli cell cultures. Sertoli cells were stimulated for different time intervals with FSH and c-fos mRNA levels measured by Northern RNA blot analysis. FSH treatment increased c-fos mRNA transiently with a maximal stimulation reached in 1 h. The level of c-fos mRNA returned to basal level within 4-6 h. The induction of c-fos mRNA was dependent on the concentration of FSH used with an ED50 of 3-5 ng/ml ovine FSH-16. A similar increase in c-fos expression was induced with highly purified hFSH. The c-fos mRNA was also elevated after treatment of the Sertoli cell with (Bu)2cAMP and forskolin. (Bu)2cAMP treatment led to a sustained induction of c-fos mRNA, with increased mRNA levels being maintained after 12 h. The FSH-dependent induction of c-fos mRNA was still present in cells treated for 3 h with cycloheximide, but it was greatly reduced by actinomycin D pretreatment. These data indicate that FSH induces a transient expression of c-fos in cultured Sertoli cells. This induction is probably mediated by cAMP and likely involves an increased transcription of the c-fos gene. Early expression of this gene might be an intermediate step required for gonadotropin-dependent regulation of expression of other genes.

Animals↗

PubMed1989

Biphasic survival curves for XRS radiosensitive cells: subpopulations or transient expression of repair competence?

Four of the most radiosensitive xrs variants of CHO-K1 cells, obtained after mutagenizing treatment with EMS, have been studied in detail over three to five decades of cell survival. Although these lines were initially reported to have very steep exponential survival curves, and to vary in sensitivity between themselves by a factor of two, we found in each case a similar biphasic response. The initial sensitivity was similar for all four lines, with a D0 of 0.5-0.7 Gy. A subpopulation, representing between 0.4 and 12 per cent of the cells, showed a resistant response, characterized by a D0 of 1.5-2.0 Gy. The previously reported variation in sensitivity seems to result from differences in the fraction of resistant cells rather than from differences in the D0. The consequence of such phenotypic variants within each cloned line is considerable, both for radiobiological studies of repair, and for molecular biology studies of the repair genes. Attempts were made to clone the sensitive and resistant subpopulations from each xrs cell line. Simple cloning from an untreated population was expected to yield pure sensitive cells, but these cells also gave biphasic responses in most cases. Only the cell line with the lowest resistant fraction (xrs5) gave a completely sensitive response in two of its subclones. Cells selected as survivors after high radiation doses were expected to yield resistant populations. However, for xrs4, 5 and 7 these subclones all gave biphasic responses. Three of the subclones from xrs6 gave biphasic responses but others gave a resistant response close to the wild type. We present a model in which transient gene expression may be seen in each individual cell if the silent copy of the xrs repair gene is temporarily hemimethylated. This transient gene transcription should occur during DNA synthesis, in the interval between synthesis of the gene and maintenance methylation. This interval may vary from cell line to cell line, resulting in different fractions of resistant cells.

Animals↗

PubMed2000

In vivo gene electroporation confers strong transient expression of foreign genes in the chicken testis.

To attain foreign gene expression in vivo in the testis of living chickens, chloramphenicol acetyltransferase (CAT) and firefly luciferase reporter genes were transfected by electroporation (EP). Bioluminescence imaging indicated clear expression of the luciferase reporter gene localized in and around the injection site of the chicken testis. The CAT activity decreased sharply from 7 to 14 d posttransfection (P < 0.01) and remained low until 28 d. The presence of the self-replication sequence of Epstein-Barr virus did not give significantly higher CAT gene expression over the 28-d posttransfection. The results suggest that in vivo gene EP confers strong, likely transient, foreign gene expression in the testis of living chickens.

Animals↗

PubMed2006

On the effect of transient expression of mutated eIF2alpha and eIF4E eukaryotic translation initiation factors on reporter gene expression in mammalian cells upon cold-shock.

There are a growing number of reports on the beneficial effects of subphysiological temperature in vitro culturing (27-35 degrees C) of mammalian cells on recombinant protein yield. However, this effect is not conserved across cell lines and target products, and our understanding of the molecular mechanism(s) responsible for increased recombinant protein yield upon reduced temperature culturing of mammalian cells is poor. What is known is that mammalian cells respond to cold-shock by attenuating global cap-dependent translation. Here, we have investigated the hypothesis that the cap-dependent attenuation of mRNA translation upon cold-stress of in vitro-cultured mammalian cells can be prevented, or at least alleviated, by overexpressing mutant translation initiation factors in Chinese hamster ovary and HeLa cells. We have shown that the transient coexpression of either an eIF2alphaSer51 Ala51 mutant or an eIF4ESer209 Glu209 mutant with firefly luciferase affects luciferase expression levels in a cell line and temperature dependent manner. Further, regardless of the coexpression of initiation factors, transient reporter gene expression was enhanced at subphysiological temperatures (<37 degrees C), suggesting that reduced temperature cultivation can be used to improve the yield of recombinant protein during transient expression. The implications of these results upon cell engineering strategies involving manipulation of the translational apparatus for the enhancement of recombinant protein synthesis upon cold-shock are discussed.

Amino Acid Substitution↗

PubMed1993

Rhodopsin phosphorylation by transiently expressed human beta ARK1: a new method for drug development.

Receptor phosphorylation is a key step in the process of rapid desensitization of the beta-adrenergic and other related G-coupled receptors. A specific kinase (called beta-adrenergic receptor kinase, beta ARK) has been identified, which phosphorylates the agonist-occupied form of these receptors. We have cloned the cDNA for human beta ARK1. The full-length cDNA was inserted in an expression vector (pBJI neo) and used for the transfection of eukaryotic cells (COS7). The kinase activity of the cytosolic fraction of COS7 cells was assayed 72 hours after beta ARK1 transfection. A 40-70 fold increase in cytosolic beta ARK1 activity was observed. To validate this approach we demonstrated a different degree of kinase inhibition by various types of heparin. Our system, based on transient gene expression and in vitro phosphorylation of rhodopsin, represents a new method to screen for pharmacological agents acting on this kinase.

Animals↗

PubMed2001

Myf-5 is transiently expressed in nonmuscle mesoderm and exhibits dynamic regional changes within the presegmented mesoderm and somites I-IV.

Myf-5 is one of four myogenic regulatory factors that play important roles in skeletal muscle development. This study provides detailed analysis of Myf-5 expression during early chick development using an in situ hybridization technique that has been optimized to detect low level Myf-5 transcripts. This facilitated detection of heretofore unrecognized dynamic changes in Myf-5 expression patterns. Myf-5 expression is first detected at stage 3 in the primitive streak and exhibits transient low-level expression in nonmyogenic mesoderm. Myf-5 is later expressed in the presegmented mesoderm (psm) in a reiterating pattern that is coordinated with somitogenesis and also colocalizes with the Notch ligand C-Delta-1. In somites (S) I-IV, Myf-5 expression exhibits dynamic regional changes, and in somites rostral to S IV, Myf-5 is expressed at higher levels in muscle precursors in the dorsomedial somite. Semiquantitative comparison of Myf-5 mRNA levels in the psm and in myotome-containing somites indicates about a 10-fold difference. The expression pattern of Myf-5 differs from that of MyoD, which we find is expressed only in the dorsomedial somite. These data reveal that Myf-5 is expressed at low levels several stages before muscle differentiation occurs and suggest that only a subset of cells that initially express Myf-5 will upregulate its expression and differentiate as muscle.

Animals↗

PubMed1988

Tumor promoters induce a transient expression of tumor-associated genes in both basal and differentiated cells of the mouse epidermis.

The effect of tumor promoters on the in vivo expression of tumor-associated, overexpressed genes was studied. Two of the tumor-associated genes, mal 1 and mal 2 were overexpressed already in the benign papilloma stage of mouse skin carcinogenesis. Overexpression of the other two genes, mal 4 and transin, was specific for the malignant state. Treatment of the normal adult epidermis with the complete tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the incomplete, second-stage promoter 12-O-retinylphorbol-13-acetate (RPA) enhanced transiently the expression of the mal sequences and transin. Fractionation of the adult epidermis on Percoll gradients into basal cells and differentiated, suprabasal cells showed that expression of the mal sequences was enhanced by TPA in both basal and differentiated cells. In contrast, transin expression, which was undetectable in cells of the normal epidermis, was enhanced in only the basal cells of the TPA-treated epidermis. The non-tumor-promoting hyperplastic agent, ethylphenyl propiolate (EPP), applied to the skin at a hyperplastic dose level did not enhance the expression of the mal 4 or transin sequences in the epidermis and had only a slight enhancing effect on the levels of mal 1 and mal 2 transcripts in the epidermis. Our results suggest that the observed stimulated expression of mal 1 and mal 2 is related to proliferative processes, whereas stimulated expression of mal 4 and transin reflects tumor-promoter-specific responses.

Animals↗

PubMed2004

Transient expression of hypoxia-inducible factor-1 alpha and target genes in peripheral nerves from diabetic rats.

Decreased blood flow is one of the earliest physiological changes observed after the onset of either clinical or experimental diabetes. The reduction in blood flow is believed to lead to nerve hypoxia, which in conjunction with other metabolic alterations and degenerative processes in different nerve compartments, results in the dysfunction known as diabetic neuropathy. The transcriptional regulator hypoxia-inducible factor-1 alpha (HIF-1alpha) accumulates rapidly under hypoxic conditions and modulates the expression of several target genes that protect tissues against ischemia and infarction. At present it is unclear whether diabetic nerve injury results from an abnormal response of HIF-1alpha and its protective target genes. In the present study we have analyzed the expression and activity of HIF-1alpha and its target genes in diabetic nerves as a first step to determine their possible contribution to the development or maintenance of diabetic neuropathy. We observed a transient increase in the expression of HIF-1alpha that peaked between 4 and 6 weeks and declined 8 weeks after induction of experimental diabetes in rats. The increase in HIF-1alpha in diabetic nerves coincided with a similarly transient increase in the expression of several HIF-1alpha target genes including vascular endothelial growth factor, lactate dehydrogenase and erythropoietin, which subsided 8-10 weeks after induction of diabetes. These results suggest that the transient activation of neurotrophic and angiogenic genes, as opposed to a more sustained effect in response to the chronic injury, may be responsible for the alterations in nerve function and regeneration that characterize the diabetic neuropathy.

Analysis of Variance↗

PubMed1996

Immunohistochemical investigation of gamma-aminobutyric acid ontogeny and transient expression in the central nervous system of Xenopus laevis tadpoles.

The ontogeny of the gamma-aminobutyric acid (GABA)-positive neurons in the brain of Xenopus laevis tadpoles was investigated by means of immunohistochemistry, using specific antibodies both against GABA and its biosynthetic enzyme, glutamate decarboxylase (GAD). The results obtained with the two antisera were comparable. The GABA system differentiates very early during development. At stages 35/36, numerous GABA-positive neurons were seen throughout the prosencephalon and formed two main bilateral clusters within the lateral walls of the forebrain that ran caudally toward the hindbrain. Other GABA-immunolabeled cell bodies, together with a conspicuous network of GABAergic fibers, were seen in the posterior hypothalamus. In the spinal cord, the lateral marginal zone was GABA-positive, as were Rohon-Beard neurons, interneurons, and Kolmer-Agdhur cells. A very rich GABA innervation was observed in the pars intermedia of the pituitary. At stage 50, plentiful immunopositive neurons and fibers were found in the telencephalic hemispheres, the diencephalon, and the mesencephalon (optic tectum and tegmentum). By stage 54, the number of GABA-immunoreactive neurons in the posterior hypothalamus had decreased, so that, at stage 58, there were very few GABA-labeled cell bodies in the dorsolateral walls of the infundibulum, despite a strong GABAergic innervation within the median eminence and the pars intermedia. From stage 58 to stage 66, the distribution pattern was very similar to that described in the adult X. laevis and in other amphibian species. These results point to transient GABA expression within the hypothalamus, possibly related to either 1) a naturally occurring cell death or 2) a phenotypic switch.

Animals↗

PubMed1985

Comparison of the transient late region expression of SV40 DNA and SV40-based shuttle vectors: development of a new shuttle vector that is efficiently expressed.

Transient expression of an SV40-based shuttle vector similar in design to commonly used vectors is shown to be inefficient when compared to expression of SV40 viral DNA. To eliminate this problem, we have designed and constructed a new vector, pSVPiC, which contains a minimal noninterfering plasmid component, the 885-bp plasmid PiAN7, and two SV40 promoter/origin regions. Transient expression of the SV40 late region from pSVPiC is much more efficient than that from previously used vectors and even more efficient than that from SV40 viral DNA. When the gene for rabbit beta-globin is placed in the late region of pSVPiC, it is also expressed at high levels, indicating that this shuttle vector is generally useful for expressing eukaryotic genes.

DNA Replication↗