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Results for “Transient expression”

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PubMed1995

Lentil root protoplasts: a transient expression system suitable for coelectroporation of monoclonal antibodies and plasmid molecules.

Protoplasts were isolated from lentil (Lens culinaris) roots and their suitability as a transient expression system was investigated. After transfecting the protoplasts with the beta-glucuronidase (GUS) gene by either electroporation or polyethylene glycol (PEG), the specific activity of the reporter enzyme and the cell viability were determined. Electroporation was more effective than PEG treatment as transfection procedure and its efficiency was affected by the plasmid length. The feasibility of electro-transferring at the same time (coelectroporation) inhibitory anti-lipoxygenase monoclonal antibodies and the GUS-carrying plasmid pBI 221 was investigated as well. The amount of transferred immunoglobulins was quantitated by ELISA and the inhibitory ability of monoclonal antibodies on the intracellular target enzyme was determined. Evidence is presented for the successful coelectroporation of immunoglobulins and plasmid DNA into lentil protoplasts, the two types of macromolecules acting independently of each other in the recipient cells.

Antibodies, Monoclonal↗

PubMed1996

Transient expression of acetylcholinesterase messenger RNA and enzyme activity in developing rat thalamus studied by quantitative histochemistry and in situ hybridization.

The molecular basis for transient expression of acetylcholinesterase in noncholinergic regions of the early postnatal rat brain was studied by in situ hybridization histochemistry. A 33P-labelled 63-mer DNA oligonucleotide was used to probe acetylcholinesterase messenger RNA in the brains of rat pups at one, two, six, nine, 12, 16 and 21 days of age (birth = day 0). Cryostat brain-sections were hybridized with probe and exposed to X-ray film or emulsion coatings. Acetylcholinesterase messenger RNA was quantitated by counting silver grains and by measuring X-ray film density with video imaging and computer-based densitometry. Adjacent sections were stained histochemically for acetylcholinesterase activity, also quantitated by video densitometry. Overall there was a significant correlation between apparent levels of acetylcholinesterase activity and acetylcholinesterase messenger RNA. Increases in message tended to accompany the surges of acetylcholinesterase activity that marked the maturation of thalamocortical sensory relay pathways. Acetylcholinesterase expression in the youngest rats was generally sparse but it increased markedly during the first postnatal week, especially in the sensory relay nuclei of the thalamus. Levels of message and enzyme activity in the medial and dorsolateral geniculate and the ventral posteromedial and ventral posterolateral nuclei rose to a peak, typically about day 9. Beyond this time there was a gradual decline. By day 21 the staining and in situ hybridization patterns resembled those in adult brains, whose thalamic relay nuclei are impoverished in acetylcholinesterase activity and messenger RNA. Thus, acetylcholinesterase expression is strongly modulated in certain thalamic systems as they undergo neural morphogenesis.

Acetylcholinesterase↗

PubMed1988

Analysis of clustered point mutations in the human ribosomal RNA gene promoter by transient expression in vivo.

We have mapped the cis regulatory elements required in vivo for initiation at the human rRNA promoter by RNA polymerase I. Transient expression in COS-7 cells was used to evaluate the transcription phenotype of clustered base substitution mutations in the human rRNA promoter. The promoter consists of two major elements: a large upstream region, composed of several domains, that lies between nucleotides -234 and -107 relative to the transcription initiation site and affects transcription up to 100-fold and a core element that lies between nucleotides -45 and +20 and affects transcription up to 1000-fold. The upstream region is able to retain partial function when positioned within 100-160 nucleotides of the transcription initiation site, but it cannot stimulate transcription from distances of greater than or equal to 600 nucleotides. In addition, we demonstrate, using mouse-human hybrid rRNA promoters, that the sequences responsible for human species-specific transcription in vivo appear to reside in both the core and upstream elements, and sequences from the mouse rRNA promoter cannot be substituted for them.

Animals↗

PubMed1993

Transient expression of PKC gamma mRNA in cerebellar granule cells during rat brain development.

The localization of PKC gamma mRNA expression during the maturation of rat cerebellum has been studied by in situ hybridization. We found a transient expression over the granule cell layer and persistent high expression in the Purkinje cells during postnatal development. Expression in granule cells appeared as early as postnatal day 5 over the external granule cell layer, when Purkinje cells are multiply innervated by climbing fibres in contrast to their mono-innervation in the adult. As the regression of the poly-innervation during the following weeks is known to require granule cell input, our findings suggest that the PKC gamma expression over the migrating granule cell layer is linked to the process of selective stabilization of synapses during the maturation of the cerebellum.

Aging↗

PubMed1986

Analysis of homologous recombination in cultured mammalian cells in transient expression and stable transformation assays.

Recombination between plasmid molecules, each containing a nonoverlapping deletion mutation in the hamster adenine phosphoribosyltransferase gene, was measured after coinjection into rat cells. Using these two plasmids, as linear or circular molecules, the recombination efficiency was measured soon after injection in a transient expression assay or after selection for stable transformants. The transient assay revealed that linear molecules were a better substrate for recombination, with double strand breaks within the region of homology stimulating recombination more than breaks outside the region of homology. A 20 to 70-fold increase in the efficiency of recombination was observed when two linear molecules were coinjected as compared to two circular molecules. Linear molecules were found to not only stimulate recombination but also to facilitate stable integration of the recombinant molecule into the host genome.

Animals↗

PubMed1996

Astrovirus ribosomal frameshifting in an infection-transfection transient expression system.

Different regions of the human astrovirus frameshift signal were cloned into the rhesus rotavirus VP4 gene and evaluated in an infection-transfection transient expression cell culture system. BHK-21 cells, infected with a vaccinia virus that expresses T7 RNA polymerase (vTF7-3), were transfected with the various astrovirus-VP4 constructs. All constructs were driven by a T7 promoter and contained an internal ribosome entry site. Frameshifted and nonframeshifted protein products were immunoprecipitated with VP4 amino- and carboxy-terminal-specific monoclonal antibodies, and their ratios were determined by PhosphorImager analysis. The efficiency of frameshifting was 25 to 28%, significantly greater than the 5 to 7% efficiency reported previously in a cell-free translation system. Coupling of transcription and translation in a cell-free system yielded frameshifting efficiencies threefold greater than that of the uncoupled in vitro system. The presence of the shifty heptamer was an absolute requirement for frameshifting in both cell-free and intact-cell systems, while deletion of the potential downstream pseudoknot region did not affect the efficiency of frameshifting.

Animals↗

PubMed1987

Transient expression of sialylated glycans during glycoprotein processing by embryonal carcinomas.

Embryonal carcinoma and early embryonic cells express unusually large and complex carbohydrates on their surfaces that are lost during differentiation. These carbohydrates are composed of alternating galactose and N-acetylglucosamine residues and have either linear or branched architectures. Compared to the glycans expressed by many differentiated cells these glycans are poorly sialylated. However, metabolic studies reveal that there is a transient expression of sialylated glycans during the processing of glycoproteins by embryonal carcinomas. After a short pulse with mannose the major complex-type glycan is a biantennary glycan with two sialic acids. During subsequent chase periods this glycan species is replaced by unsialylated glycans that have elongated branches composed of alternating galactose and N-acetylglucosamine residues.

Animals↗

PubMed1990

Transient expression directed by homologous and heterologous promoter and enhancer sequences in fish cells.

In order to construct fish specific expression vectors for studies on gene regulation in vitro and in vivo a variety of heterologous enhancers and promoters from mammals and from viruses of higher vertebrate cells were tested for expression of the bacterial chloramphenicol acetyl transferase reporter gene in three teleost fish cell lines. Several viral enhancers were found to be constitutively active at high levels. The human metallothionein promoter showed inducible expression in the presence of heavy metal ions. A fish sequence was isolated that can be used as a homologous constitutively active promoter for expression of foreign genes. Using the human growth hormone gene with an active promoter in fish cells for transient expression insufficient splicing and lack of translation were observed, pointing to limitations in the use of heterologous genes in gene transfer experiments. On the contrary, some heterologous promoters and enhancers functioned in fish cells as well as in their cell type of origin, indicating that corresponding transcription factors are sufficiently conserved between fish and human over a period of 900 million years of independent evolution.

Animals↗

PubMed1997

Transient expression of GAP-43 within the hippocampus after global brain ischemia in rat.

Neuroanatomical methods have been used to study selective vulnerability after global brain ischemia. A consistent pattern of ischemic neuronal damage is found in the rodent hippocampus with loss of CA1 neurons and of some cells in the hilus of the dentate gyrus. Very little is known about plastic changes that would be expected in ischemia-resistant areas such as CA3 neurons and granule cells. Neuronal plasticity after lesions may be indicated by changes in labeling with antibodies to the growth-associated protein 43 (GAP-43). Expression of GAP-43 as a marker for neuronal plasticity was studied here in the hippocampus after global brain ischemia. Halothane-anesthetized rats were subjected to 20 min of transient forebrain ischemia using four-vessel occlusion. In situ hybridization was used to study GAP-43 mRNA at 1, 3, 6, and 12 h and at 1, 3, and 7 days after ischemia. Immunostaining was carried out with two different antibodies to GAP-43 in brains which were perfusion-fixed after 1, 2, 4, and 7/8 days. In the control hippocampus, GAP-43 mRNA was localized to CA1-CA3 and the hilus. Moderate increases in cellular signals were seen in hilar cells and granule cells early after ischemia, and some changes occurred in CA3 at late stages. Hybridization was lost in CA1 due to cell death. With immunostaining, GAP-43 was not seen in the cytoplasm of neurons, whereas dense labeling occurred in a differentiated pattern in the axonal and dendritic layers. At 1 day after ischemia, neurons in the hilus of the dentate gyrus and in the stratum pyramidale and lucidum of CA3 showed strong cytoplasmic labeling for GAP-43. Few cells were labeled in these regions at 2 days, and none at later stages. Pyramidal cells in CA1 and CA3 areas and granule cells were never labeled. These studies demonstrate a transient expression of GAP-43 mRNA and protein in a subset of vulnerable neurons after transient brain ischemia. The cytoplasmic localization in hilar neurons could be due to increased synthesis of GAP-43 or to changes in axoplasmic transport. It is suggested that axonal damage occurs in hilar cells which stimulates GAP-43 expression. The increased production of trophic factors after ischemia in granule cells could also cause plastic changes in hilar cells. Since hilar neurons are in a strategic position to control the excitability of the dentate area, increased expression of GAP-43 may indicate an important pathophysiological process. In seizure experiments, strong expression of GAP-43 mRNA in granule cells was associated with abnormal mossy fiber sprouting and development of chronic epilepsy. The relevance of the minor GAP-43 mRNA upregulation after ischemia must be considered. The changes in CA3 neurons at several days after ischemia might represent a plastic response to a loss of CA1 neurons.

Analysis of Variance↗

PubMed1993

Developmentally transient expression of acetylcholinesterase within cortical pyramidal neurons of the rat brain.

Using a histochemical method for the visualization of cholinesterase activity in neurons, we have observed developmentally transient expression of acetylcholinesterase (AChE) in cortical pyramidal neurons of the rat brain. Depending on the extent of the deposition of AChE reaction product, several types of cortical neurons could be visualized. We designated neurons with moderate-to-high staining intensity as AChEH and neurons with relatively lower staining intensity as AChEL. At birth (P0), very little AChE activity was found within cortical neurons. Between P1-P4, there was a gradual emergence of AChE-stained cortical neurons. At this stage, the majority of these neurons were of the AChEL type. At P5-P7 we observed an abrupt increase in AChE-stained cortical neurons. The number and the staining intensity of these neurons was at a peak at P8-P10. At this age range, the majority of these neurons were of the AChEH variety and displayed morphological characteristics of cortical pyramidal neurons. At P11-P15, there was an abrupt decrease in the number of AChEH neurons. After P15, the density and staining intensity of cortical AChE-positive (cholinergic) axons gradually increased. Nevertheless, AChEL pyramidal neurons were detected through these fibers up to P21. At P21, a dense plexus of AChE-positive axons was observed in all cortical areas while very little AChE reaction product was visible in pyramidal neurons, and this pattern continued into adult life. When the adult cortex was denervated from its AChE-positive axons by lesions of the nucleus basalis magnocellularis, many AChEL pyramidal neurons were uncovered.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗

PubMed1996

Transcriptional activation and transient expression of the human androgen receptor.

A series of cDNAs containing deletions within the open-reading frame of the human androgen receptor (AR) were constructed and transiently expressed in CV1 cells to investigate the effects of these alterations on the level of expression of the protein and on its capacity to activate a model reporter gene (MMTV-luciferase). The levels of AR expression were assayed using immunoblots made using an antibody directed at an epitope (amino acids 1-21) preserved in all of the deletions. Treatment of the transfected cells with androgen increased the level of normal or mutant AR approximately five-fold in all constructs in which the hormone-binding domain was intact. This finding indicates that an intact hormone-binding domain is necessary and sufficient for the androgen-dependent increase in AR levels. Contraction of expansion or the glutamine repeat or deletion of the glycine repeat in the amino terminus diminished the capacity of the mutant ARs to activate the MMTV luciferase gene. The presence of a large-scale deletion within the amino terminus (amino acid residues 96-483), abolished receptor function, and two smaller deletions (bounded by residues 80-93 and 245-485) within the amino terminus substantially impaired receptor function. As previously described, deletion of the hormone-binding domain (amino acids 708-917) resulted in a constitutively active receptor. Unexpectedly, the large-scale deletion within the amino terminus (amino acids 96-483), in combination with deletion of the carboxy terminus also produced a constitutively active receptor that was almost as active as ligand-activated normal AR. None of the alterations in AR function could be explained by changes in the level of AR expression and the function of some mutant receptors was even more defective when the relative levels of mutant ARs expressed was considered. These findings imply that interaction of the sequences within the amino- and carboxy-terminal portions of the AR, or proteins that interact with these segments, is critical for regulation of transcription by the AR.

Animals↗

PubMed1986

Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.

DNA coding for bacteriophage T7 RNA polymerase was ligated to a vaccinia virus transcriptional promoter and integrated within the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and stably expressed T7 RNA polymerase in mammalian cells. Target genes were constructed by inserting DNA segments that code for beta-galactosidase or chloramphenicol acetyltransferase into a plasmid with bacteriophage T7 promoter and terminator regions. When cells were infected with the recombinant vaccinia virus and transfected with plasmids containing the target genes, the latter were expressed at high levels. Chloramphenicol acetyltransferase activity was 400-600 times greater than that observed with conventional mammalian transient-expression systems regulated either by the enhancer and promoter regions of the Rous sarcoma virus long terminal repeat or by the simian virus 40 early region. The vaccinia/T7 hybrid virus forms the basis of a simple, rapid, widely applicable, and efficient mammalian expression system.

Acetyltransferases↗

PubMed1983

Transient expression of a mouse alpha-fetoprotein minigene: deletion analyses of promoter function.

The constitutive transcription of a mouse alpha-fetoprotein (AFP) minigene was examined during the transient expression of AFP-simian virus 40-pBR322 recombinant DNAs introduced into HeLa cells by Ca3(PO4)2 precipitation. We tested three constructs, each of which contains the AFP minigene and pBR322 DNAs inserted in the late region of simian virus 40 and found that the relative efficiency of AFP gene expression was dependent on the arrangement of the three DNA elements in the vector. The transcripts begin at the authentic AFP cap site and are properly spliced and polyadenylated. To define a sequence domain in the 5' flanking region of the AFP gene required for constitutive expression, sequential 5' deletion mutants of the AFP minigene were constructed and introduced into HeLa cells. All AFP deletion mutants which retained at least the TATA motif located 30 base pairs upstream from the cap site were capable of directing accurate and efficient AFP transcription. However, when the TATA sequence was deleted, no accurately initiated AFP transcripts were detected. These results are identical to those obtained from in vitro transcription of truncated AFP 5' deletion mutant templates assayed in HeLa cell extracts. The rate of AFP transcription in vivo was unaffected by deletion of DNA upstream of the AFP TATA box but was greatly affected by the distance between the simian virus 40 control region and the 5' end of the gene. The absence of any promoter activity upstream of the TATA box in this assay system is in contrast to what has been reported for several other eucaryotic structural genes in a variety of in vivo systems. A sequence comparison between the 5' flanking region of the AFP gene and these genes suggested that the AFP gene lacks those structural elements found to be important for constitutive transcription in vivo. Either the AFP gene lacks upstream promoter function in the 5' flanking DNA contained within the minigene, or the use of a viral vector in a heterologous system precludes its identification.

Animals↗

PubMed1997

Mutant endoglin in hereditary hemorrhagic telangiectasia type 1 is transiently expressed intracellularly and is not a dominant negative.

Endoglin (CD105), a component of the TGF-beta 1 receptor complex, is the target gene for the dominantly inherited vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1). We have identified a novel endoglin splice site mutation, leading to an in-frame deletion of exon 3, in a new-born from a family with HHT. Expression of normal and mutant endoglin proteins was analyzed in umbilical vein endothelial cells from this baby and in activated monocytes from the affected father. In both samples, only normal dimeric endoglin (160 kD) was observed at the cell surface, at 50% of control levels. Despite an intact transmembrane region, mutant protein was only detectable by metabolic labeling, as an intracellular homodimer of 130 kD. In monocytes from three clinically affected HHT1 patients, with known mutations creating premature stop codons in exons 8 and 10, surface endoglin was also reduced by half and no mutant was detected. Overexpression into COS-1 cells of endoglin cDNA truncated in exons 7 and 11, revealed their intracellular expression, inability to be secreted and to form heterodimers at the cell surface. These results indicate that mutated forms of endoglin are transiently expressed intracellularly and not likely to act as dominant negative proteins, as proposed previously. A reduction in the level of functional endoglin is thus involved in the generation of HHT1, and associated arteriovenous malformations.

Adult↗

PubMed1986

[Stimulation of human lymphocytes by phorbol acetate and a calcium ionophore induces an early transient expression of the c-fos oncogene followed by c-myc].

After stimulation of human lymphocytes by 12-0 tetradecanoyl phorbol acetate (TPA) and by the calcium ionophore A23187 an early transient expression of proto-oncogene fos followed by an expression of c-myc is observed, as has been described in rodent fibroblast stimulated by growth factors. This observation suggests a direct role of protein Kinase C and of calcium flux an the induction of cellular oncogenes fos and myc that may be associated with the early steps of activation and proliferation of lymphocytes.

Calcimycin↗

PubMed1992

Binding of typical and atypical antipsychotic agents to transiently expressed 5-HT1C receptors.

We determined the affinities of clozapine and 21 other typical and atypical antipsychotic agents for the cloned 5-hydroxytryptamine-1C (5-HT1C) receptor. For these studies, 5-HT1C receptors were transiently expressed in COS-7 cells using the vector pSVK3-5HT1C. We discovered that clozapine and several other putative typical and atypical antipsychotic agents (loxapine greater than tiosperone greater than SCH23390 greater than fluperlapine greater than rilapine greater than chlorpromazine) had relatively high affinities (7-30 nM) for the cloned 5-HT1C receptor. Other antipsychotic agents (risperidone greater than tenilapine greater than mesoridazine greater than thioridazine greater than cis-fluphenthixol) had intermediate affinities (30-100 nM), whereas many other antipsychotics (fluphenazine greater than spiperone greater than amperozide greater than melperone greater than thiothixene greater than haloperidol, metoclopramide, pimozide, domperidone, sulpiride) had low affinities (greater than 500 nM) for the cloned 5-HT1C receptor. The results indicate that although several putative atypical antipsychotic agents have high affinities for the cloned rat 5-HT1C receptor, the spectrum of drug binding does not correlate with the atypical nature of these compounds.

Antipsychotic Agents↗

PubMed1997

Transient expression of the serotonin transporter in the developing mouse thalamocortical system.

The serotonin transporter was visualized in sections through the developing mouse thalamus by autoradiography of [3H]citalopram binding. In late gestation, a high density of transporter expression appeared in the ventrobasal thalamic complex and medial geniculate body. During the first postnatal week, binding in these areas decreased to low levels. A similar pattern of transient [3H]citalopram binding was observed in the somatosensory cortex, although the rise and decline of labeling occurred some days later. The density of the serotonergic innervation in the ventrobasal thalamic complex is known to be very low during the entire developmental period. Therefore, these data suggest that the serotonin transporter may be expressed transiently by thalamic neurons projecting to the cerebral cortex (as a "heterocarrier") which are capable of taking up serotonin in the somatosensory cortex. We propose that serotonin may act temporarily as a "false" transmitter in thalamocortical axons.

Animals↗

PubMed1993

Transient expression of the intermediate filament nestin during skeletal muscle development.

It has previously been established that skeletal muscle development is accompanied by changes in the composition of intermediate filaments: vimentin is expressed predominantly in myoblasts and desmin in adult myotubes. We show that the intermediate filament transitions during muscle development are more complex, and involve a transient expression of the recently discovered intermediate filament nestin. Nestin RNA is expressed predominantly early, in a biphasic pattern, and is markedly downregulated in adult rat muscle, whereas desmin RNA becomes more abundant throughout development. Nestin protein was found up to the postnatal myotube stage, where it colocalized with desmin in Z bands. The intracellular distribution of nestin, vimentin and desmin was analysed in the human myogenic cell line G6 before and after in vitro differentiation. Despite its more distant evolutionary and structural relationship to the other two intermediate filaments, nestin formed a cytoplasmic filamentous network indistinguishable from that of desmin and vimentin, both in undifferentiated myoblasts and after differentiation to multinuclear myotubes. In conclusion, our data suggest that nestin is an integrated component of the dynamic intermediate filament network during muscle development and that nestin copolymerizes with desmin and vimentin at stages of coexpression.

Animals↗