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Results for “Transient expression”

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PubMed1987

Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA.

An in vitro system for the production of hepatitis B virus (HBV) particles was established by the transient expression of transfected HBV DNA using a human hepatocellular carcinoma cell, HuH-7, as a recipient. The 3.6- and 2.2-kilobase transcripts observed were similar to those in virus-infected liver cells. Both transcripts revealed the microheterogeneity of their 5' ends. The formation of virus-related particles subsequent to the RNA transcription was demonstrated. The core particles observed in the cytoplasm and the virus particles secreted in the culture medium contained the replicative intermediates of HBV DNA and banded at densities of 1.35-1.36 g/cm3 and 1.22-1.24 g/cm3, respectively. Furthermore, the in vitro mutagenesis of the template HBV DNA demonstrated that the P gene as well as the C gene products were essential for the production of HBV particles.

Amino Acid Sequence↗

PubMed2001

A macrophage protein, Ym1, transiently expressed during inflammation is a novel mammalian lectin.

Oral infections of mice with Trichinella spiralis induce activation of peritoneal exudate cells to transiently express and secrete a crystallizable protein Ym1. Purification of Ym1 to homogeneity was achieved. It is a single chain polypeptide (45 kDa) with a strong tendency to crystallize at its isoelectric point (pI 5.7). Co-expression of Ym1 with Mac-1 and scavenger receptor pinpoints macrophages as its main producer. Protein microsequencing data provide information required for full-length cDNA cloning from libraries constructed from activated peritoneal exudate cells. A single open reading frame of 398 amino acids with a leader peptide (21 residues) typical of secretory protein was deduced and later deposited in GenBank (accession number M94584) in 1992. By means of surface plasmon resonance analyses, Ym1 has been shown to exhibit binding specificity to saccharides with a free amine group, such as GlcN, GalN, or GlcN polymers, but it failed to bind to other saccharides. The interaction is pH-dependent but Ca2+ and Mg2+ ion-independent. The binding avidity of Ym1 to GlcN oligosaccharides was enhanced by more than 1000-fold due to the clustering effect. Specific binding of Ym1 to heparin suggests that heparin/heparan sulfate may be its physiological ligand in vivo during inflammation and/or tissue remodeling. Although it shares approximately 30% homology with microbial chitinases, no chitinase activity was found associated with Ym1. Genomic Southern blot analyses suggest that Ym1 may represent a member of a novel lectin gene family.

Amino Acid Sequence↗

PubMed2006

A homologue of snail is expressed transiently in subsets of mesenchyme cells in the sea urchin embryo and is down-regulated in axis-deficient embryos.

Vertebrate members of the zinc finger transcription factor family related to Drosophila snail are expressed in neural crest and paraxial mesoderm along the left-right axis of the embryo. As simple deuterostomes, echinoderms are an important sister phylum for the chordates. We have identified populations of patterned, nonskeletogenic mesenchyme in the sea urchin Lytechinus variegatus by their expression of a sea urchin member of the snail family (Lv-snail). Lv-snail mRNA and protein are detectable at the midgastrula stage within the archenteron. At the late gastrula stage, a contiguous cluster of cells on the left side of the tip of the archenteron is Lv-snail-positive. At the early prism stage, two small clusters of mesenchyme cells near the presumptive arm buds are also Lv-snail-positive. At the pluteus stage, staining is detectable in isolated mesenchyme cells and the ciliated band. Based on fate mapping of secondary mesenchyme cells (SMCs) and double-label immunostaining, these patterns are consistent with expression of SNAIL by novel subsets of SMCs that are largely distinct from skeletogenic mesenchyme. In radialized embryos lacking normal bilateral symmetry, mesenchymal expression of Lv-SNAIL is abolished. These results suggest that transient expression of Lv-snail may be important for the differentiation of a subset of axially patterned nonskeletogenic mesenchyme cells and suggest conserved functions for snail family members in deuterostome development.

Animals↗

PubMed1999

Characterization of the regulatory elements of the maize P-rr gene by transient expression assays.

The maize P-rr gene conditions floral-specific flavonoid pigmentation, especially in the kernel pericarp and cob. We analyzed the P-rr promoter by transient expression assays, in which segments of the P-rr promoter were fused to the GUS reporter gene and introduced into maize cells by particle bombardment. A basal P-rr promoter fragment (-235 to +326) gave low, but significant, levels of GUS reporter gene expression. Interestingly, two widely spaced segments containing enhancer-like activity were found. When tested individually, both the proximal (-1252 to -236) and distal (-6110 to -4842) segments boosted expression of the basal P-rr promoter::GUS construct about five-fold. A 1.6 kb segment of the P-rr promoter (-1252 to +326) containing the proximal enhancer and the 5'-untranslated leader driving the GUS reporter gene showed preferential expression in BMS and embryogenic suspension cell cultures vs. endosperm-derived suspension cell cultures. These results demonstrate the application of transient assay techniques for the identification of regulatory elements responsible for floral-specific regulation of the complex P-rr gene promoter in maize.

DNA, Plant↗

PubMed1985

Transformed Xenopus embryos as a transient expression system to analyze gene expression at the midblastula transition.

The onset of transcriptional activity during embryogenesis in Xenopus laevis is at the 4000- to 8000-cell stage (stage 8-8.5) and is referred to as the midblastula transition (MBT). Most exogenous circular DNA that is microinjected into the fertilized egg also is expressed at the MBT. The transformed Xenopus embryo at these early stages was used as a transient expression system in order to determine the effects of (1) promoter strength, (2) physical conformation, (3) degree of replication, and (4) a regulatory molecule on the expression of an injected gene coding for chloramphenicol acetyl transferase. This gene linked to a relatively strong promoter (SV40 early promoter -pSV2CAT), a weak promoter (adenovirus early promoter -pE3CAT), or in circular or linear form is expressed at stage 8-8.5 following injection into fertilized eggs. pE3CAT coinjected with the E1a protein (enhances the transcription of the E3 promoter) is also expressed at stage 8.5, but expression is enhanced 2-7.6 fold. These data suggest that the inhibition of transcription prior to the MBT could not be perturbed by either the presence of different promoters or a positive regulatory molecule such as the E1a protein.

Acetyltransferases↗

PubMed1989

Transient expression of simple epithelial keratins by mesenchymal cells of regenerating newt limb.

Structural proteins of the intermediate filament family are an early indicator of differentiation before organogenesis becomes apparent. Keratin intermediate filaments are characteristically expressed only by epithelial and not by mesenchymal cells. Here we show, using monoclonal antibodies, a transient expression of the keratin pair 8 and 18 in a population of mesenchymal cells in the regenerating newt limb, specifically in the undifferentiated progenitor cells (blastemal cells) which give rise to the new tissues. These keratins are also expressed in cultured limb cells that can differentiate into muscle. In contrast no reactivity with anti-keratin 8 and 18 antibodies was observed in the newt limb bud at an early stage of development, indicating a molecular difference between the developing and regenerating limb. The molecular weights of the newt proteins detected by these antibodies are very similar to those of human keratins 8 and 18, further supporting the immunocytochemical evidence that the newt homologs of these keratins are expressed in blastemal cells. This is the first demonstration of keratin expression in mesenchymal progenitor cells in an adult animal.

Animals↗

PubMed1999

Growth phase-regulated induction of Salmonella-induced macrophage apoptosis correlates with transient expression of SPI-1 genes.

Invasive Salmonella has been reported to induce apoptosis in a fraction of infected macrophages within 2 to 14 h from the time of infection by a mechanism involving the type III secretion machinery encoded by the Salmonella pathogenicity island 1 (SPI-1). Here, we show that bacteria in the transition from logarithmic to stationary phase cause 90% of the macrophages to undergo phagocytosis-independent, caspase-mediated apoptosis within 30 to 60 min of infection. The ability of Salmonella to induce this rapid apoptosis was growth phase regulated and cell type restricted, with epithelial cells being resistant. Apoptosis induction was also abrogated by disruption of the hilA gene (encoding a regulator of SPI-1 genes) and by the expression of a constitutively active PhoPQ. hilA itself and a subset of SPI-1 genes were transiently expressed during aerobic growth in liquid medium. Interestingly, however, hilA was found to be required only for the expression of the prgH gene, while sipB, invA, and invF were expressed in a hilA-independent manner. The expression of SPI-1 genes and the secretion of invasion-associated proteins correlated temporally with the induction of apoptosis and are likely to represent its molecular basis. Thus, growth phase transition regulates the expression and secretion of virulence determinants and represents the most efficient environmental cue for apoptosis induction reported to date.

Aerobiosis↗

PubMed1999

Transient expression of a tumor-specific single-chain fragment and a chimeric antibody in tobacco leaves.

To evaluate the expression of different forms of a tumor-specific antibody in plants, we adapted a recently described Agrobacterium-mediated transient expression system. A recombinant single-chain Fv antibody (scFvT84.66) and a full-size mouse/human chimeric antibody (cT84.66) derived from the parental murine mAb T84. 66 specific for the human carcinoembryonic antigen were engineered into a plant expression vector. Chimeric T84.66 heavy and light chain genes were constructed by exchanging the mouse light and heavy chain constant domain sequences with their human counterparts and cloned into two independent plant expression vectors. In vivo assembly of full-size cT84.66 was achieved by simultaneous expression of the light and heavy chains after vacuum infiltration of tobacco leaves with two populations of recombinant Agrobacterium. Upscaling the transient system permitted purification of functional recombinant antibodies from tobacco leaf extracts within a week. His6-tagged scFvT84.66 was purified by immobilized metal affinity chromatography and cT84.66 by protein A affinity chromatography. Sufficient amounts of recombinant antibodies were recovered for detailed characterization by SDS/PAGE, Western blotting, and ELISA.

Animals↗

PubMed2005

Transient expression and insertional mutagenesis of Puccinia triticina using biolistics.

The fungal genus Puccinia contains more than 4,000 species. Puccinia triticina, causal agent of wheat leaf rust, is an economically significant, biotrophic basidiomycete. Little is known about the molecular biology of this group, and tools for understanding gene function have not yet been established. A set of parameters was established for the transient transformation of urediniospores. The expression of three heterologous promoters (actin, elongation factor 1-alpha, and Hss1, Heat Shock 70 protein), derived from Puccinia graminis, was evaluated along with the potential for insertional mutagenesis. The UidA (GUS) gene was used as a marker for transient expression. When transferred into P. triticina urediniospores, transient expression was observed across four helium pressures using one size of gold and three sizes of tungsten microprojectiles. Each of the three promoters displayed strong transient expression in germinated urediniospores; however, higher numbers of GUS-positive urediniospores were observed when either the actin or Hss1 promoters were used. Possible concomitant insertional mutagenesis of several avirulence genes was selected in wheat cultivars harboring the cognate resistance genes. Using a linearized cloning plasmid, stable integration into the genome was achieved as demonstrated by PCR and sequencing analysis.

Base Sequence↗

PubMed1996

Transient expression of neuropeptide Y (NPY) immunoreactivity in the developing hamster paraventricular thalamic area is due to apoptosis.

1. A new population of neurons with transient expression of NPY immunoreactivity was described in the developing hamster paraventricular thalamic area. The present study was performed to discover whether this phenomenon is due to programmed cell death or apoptosis. 2. Toward this aim, immunocytochemical and electron microscopic examination of the paraventricular thalamic region, as well as DNA electrophoresis of tissue extracted from the described area, was performed on different stages of embryonic and postnatal development. 3. A sudden increase in neuropeptide Y immunoreactivity (NPY-IR) in the paraventricular thalamic area at embryonic day 14 (E14) was the first symptom of neuronal degeneration. 4. Electron microscopy revealed many neurons with large masses of condensed chromatin within nuclei and extracellular bodies. The affected cells had a convoluted shape and condensed cytoplasm. 5. DNA electrophoresis revealed a ladder of bands between 150 and 1000 bp that is specific for internucleosomal DNA fragmentation. 6. The data strongly suggest that developmental disappearance of NPY-IR neurons within the hamster dorsal thalamic area is due to apoptosis.

Animals↗

PubMed1992

Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells.

Segments of the human immunodeficiency virus (HIV) type 1 gag and pol genes and mutants thereof were transiently expressed in mammalian cells. Expression was dependent on the presence of the rev responsive element in cis and the rev protein in trans and was readily detected by indirect immunofluorescence or Western blotting. Transfection of constructs encoding the entire gag and pol open reading frames yielded efficient release of particles banding at a density of 1.16 g of sucrose per milliliter and consisting mainly of processed gag proteins. In addition, these particles contained the p66/p51 heterodimer of reverse transcriptase (RT), had associated RT activity, and contained RNA. Electron micrographs revealed immature retrovirus-like particles budding primarily from the plasma membrane and extracellular particles with morphological characteristics of HIV. Particle production was independent of the pol open reading frame or an active HIV proteinase (PR) but without active PR, cell-associated and particle-associated proteins remained completely uncleaved and budding occurred primarily into intracellular vacuoles. A mutation preventing myristoylation of the viral polyproteins abolished particle release but did not interfere with polyprotein synthesis and did not prevent processing. Expression of gag and PR in the same reading frame yielded complete processing of polyproteins but no budding and led to increased cell toxicity. A mutation of the PR active site in this construct prevented cytotoxicity and restored particle release indicating that the observed phenotype was caused by the overexpression of PR. These particles were aberrant in size and morphology when analyzed on sucrose density gradients and by electron microscopy. Budding was arrested at an early stage and extracellular particles appeared to be released by a different mechanism. Only short C-terminal extensions were compatible with this release mechanism since expression of a similar mutant construct encoding the entire gag-pol open reading frame did not yield particles.

Animals↗

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↗

PubMed2001

Frizzled 2 is transiently expressed in neural crest-containing areas during development of the heart and great arteries in the mouse.

Frizzled 2 acts as a 7-transmembrane receptor in the Wnt-Dishevelled signal transduction cascade. Among others, this cascade has been associated with neural crest cell proliferation and early migration during development in mammals. The genes for some components of this cascade are located in chromosomal regions that are deleted in human syndromes associated with neural crest cell defects, like DiGeorge and Velo-Cardio-Facial Syndrome. These syndromes are often accompanied by abnormalities in cardiac morphology. Furthermore, we have reported in previous studies the upregulation of the tissue polarity gene frizzled 2 in myofibroblasts during their migration into the necrotic area after myocardial infarction in the adult heart. It is known that genes that are upregulated during cardiac remodeling due to pathology often play a role during development. To investigate whether frizzled 2 can be associated with the process of cardiac morphogenesis we studied its expression in the thoracic arterial system and heart of mouse embryo's of 10, 12, 14, 16 and 18 days after conception by means of in situ hybridization. At day 10 after conception signal could be found in the pharyngeal arches and arch arteries. The outflow tract, the ascending aorta and the pulmonary trunk were positive for frizzled 2 from day 12 on. This expression decreased with time and at day 18 only some signal could be detected in the aorta and pulmonary trunk. In contrast, in coronary and pulmonary arteries no expression was observed at any time point. Minor myocardial expression was observed in the ventricular septum at days 12 and 14. Atrial expression, although considerably lower than ventricular expression, could be detected somewhat later at days 14 and 16. Our results indicate that there is transient expression of frizzled 2 in areas that are invested by neural crest cells. This expression is downregulated upon neural crest cell differentiation. The frizzled 2 expression supports a role for the Wnt-frizzled pathway in neural crest-related disorders.

Actins↗

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↗

PubMed2005

Transient expression in tobacco leaves of an aglycosylated recombinant antibody against the epidermal growth factor receptor.

When generating stably transformed transgenic plants, transient gene expression experiments are especially useful to rapidly confirm that the foreign molecule of interest is correctly assembled and retains its biological activity. TheraCIM(R) (CIMAB S.A., Havana) is a recombinant humanized antibody against the Epidermal Growth Factor receptor (EGF-R), now in clinical trials for cancer therapy in Cuba and other countries. An aglycosylated version (Asn 297 was mutated for Gln 297) of this antibody was transiently expressed in tobacco leaves after vacuum-mediated infiltration of recombinant Agrobacterium tumefaciens that carried a binary plasmid bearing the antibody heavy and light chain genes and plant regulation signals. Protein extracts from "agroinfiltrated" leaves were tested by ELISA and Western blot, showing that the fully assembled antibody was accumulated in plant tissues. The absence of plant specific glycans did not interfere in the assembling or in the activity of the plantibody, as demonstrated in this work. Indirect immunofluorescence demonstrated that the aglycosylated antibody expressed in plants recognizes the EGF-R expressed on the surface of A431 human tumor culture cells.

Antibodies, Monoclonal↗