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Results for “Transient expression”

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PubMed1992

Neuropeptide Y m-RNA and peptide are transiently expressed in the developing rat spinal cord.

The distribution of neuropeptide Y (NPY) m-RNA and peptide were studied during the ontogeny of the rat spinal cord, by means of in situ hybridization and immunohistochemistry. NPY expressing neurons were visualized by both techniques, although by in situ hybridization they appeared slightly more abundant than by immunohistochemistry. In the ventral horn, a few neurons transiently expressed NPY from the embryonic day 15 to birth, but not in the adult animals. In the embryonic dorsal horn, NPY expressing neurons were numerous and seen as early as embryonic day 15 (E15). In contrast, in the postnatal and the adult dorsal horn, the number of neurons expressing the NPY phenotype was dramatically lower.

Animals↗

PubMed1991

Patterns of transient expression of the arenavirus nucleocapsid protein gene in transfected cells.

The cloned genes for the nucleocapsid proteins N of Junín and LCM (lymphocytic choriomeningitis) arenaviruses were inserted into the SV40-derived expression vector designated pKG4. When BHK-21 (baby hamster kidney fibroblasts) and CV-1 (African green monkey kidney fibroblasts) cell lines were transfected using these constructions, the transient expression yielded a polypeptide that could not be distinguished either by size nor by immunoreactivity from the N protein synthesized during the viral infection. The immunofluorescence analysis showed a pattern of intracellular localization similar to that observed in virus infected cells, i.e. varying from a diffuse cytoplasmic staining to granules, either distributed throughout the cytoplasm or concentrated in the perinuclear region. The association of the N protein with basophilic granules is similar to that observed in the cytopathic effect caused by arenaviruses, and could be related to the physicochemical properties of this polypeptide containing numerous basic amino acid sequences, that would allow for the interaction with cellular RNAs.

Animals↗

PubMed1994

Transient expression of bile-duct-specific cytokeratin in fetal mouse hepatocytes.

The differentiation of hepatocytes and biliary epithelial cells has been histochemically analyzed with anti-calf cytokeratin antiserum in the fetal mouse liver. Almost all young fetal hepatocytes transiently express bile-duct-specific cytokeratin; subsequently, the strong staining of the cytokeratin is confined to progenitor cells of intrahepatic biliary epithelial cells around portal veins. These results suggest that all fetal hepatocytes are bi-potent in terms of the differentiation of mature hepatocytes and intrahepatic bile-duct cells, and that the microenvironment around portal veins plays an important role in bile-duct differentiation. Large periportal hepatocytes continue to stain weakly for cytokeratin until 2 weeks after birth, although the number of positive hepatocytes decreases with development. The differentiation of bile ducts from periportal hepatocytes may continue for 2 weeks after birth.

Albumins↗

PubMed1991

Transient expression of the cloned mouse c-Ha-ras 5' upstream region in transfected primary SENCAR mouse keratinocytes demonstrates its power as a promoter element.

The mouse Ha-ras oncogene is activated by point mutation and overexpressed in developing papillomas during two-stage skin carcinogenesis in SENCAR mice. One of our research aims is to characterize the factors regulating Ha-ras gene expression at the transcriptional level in SENCAR mouse epidermis. Towards this goal, we sequenced 1400 bp of the 5' upstream region of the mouse Ha-ras gene so as to characterize various cis-regulatory elements present in the gene. We identified seven sites with the proper consensus sequence for binding the SP1 transcription factor and three potential binding sites for the CTF-1 factor. In addition, we located a 13-base sequence with 92% homology to the consensus sequence for an estrogen response element and two hexamers with consensus sequences identical to the core sequence of the glucocorticoid response element. A series of transient gene expression vectors was constructed in which various regions of the mouse Ha-ras 5' upstream region were fused to the chloramphenicol acetyltransferase (CAT) gene. These expression plasmids were transfected into newborn and adult primary SENCAR epidermal cells, the epidermal cell population that presumably contains the stem cells involved in two-stage skin tumorigenesis. Transient gene expression assays carried out after 48-72 h indicated that a 2.3-kb Ha-ras 5' fragment produced CAT activity comparable to that produced by pSV2CAT and pdolCMVCAT, both of which are plasmids with strong viral promoters and enhancers driving CAT gene expression. Maintenance of transfected keratinocytes under both nondifferentiating (0.05 mM calcium) and differentiating (1.2 mM calcium) culture conditions demonstrated that the mouse Ha-ras upstream region was relatively unresponsive to changes in calcium concentration in transient expression assays carried out in either newborn or adult keratinocytes. Our results demonstrated the power of the cloned mouse Ha-ras promoter and upstream region in driving transient gene expression after transfection into primary keratinocytes.

Animals↗

PubMed2002

Mnb/Dyrk1A is transiently expressed and asymmetrically segregated in neural progenitor cells at the transition to neurogenic divisions.

The Minibrain (Mnb) gene encodes a new family of protein kinases that is evolutionarily conserved from insects to humans. In Drosophila, Mnb is involved in postembryonic neurogenesis. In humans, MNB has been mapped within the Down's Syndrome (DS) critical region of chromosome 21 and is overexpressed in DS embryonic brain. In order to study a possible role of Mnb on the neurogenesis of vertebrate brain, we have cloned the chick Mnb orthologue and studied the spatiotemporal expression of Mnb in proliferative regions of the nervous system. In early embryos, Mnb is expressed before the onset of neurogenesis in the three general locations where neuronal precursors are originated: neuroepithelia of the neural tube, neural crest, and cranial placodes. Mnb is transiently expressed during a single cell cycle of neuroepithelial progenitor (NEP) cells. Mnb expression precedes and widely overlaps with the expression of Tis21, an antiproliferative gene that has been reported to be expressed in the onset of neurogenic divisions of NEP cells. Mnb transcription begins in mitosis, continues during G(1), and stops before S-phase. Very interestingly, we have found that Mnb mRNA is asymmetrically localized during the mitosis of these cells and inherited by one of the sibling cells after division. We propose that Mnb defines a transition step between proliferating and neurogenic divisions of NEP cells.

Amino Acid Sequence↗

PubMed2000

Improved technique for transient expression and negative strand virus rescue using fowlpox T7 recombinant virus in mammalian cells.

The suitability of recombinant T7 polymerase produced using either the highly attenuated MVA strain of vaccinia (MVA-T7) or fowlpox virus (FP-T7) for transient expression and negative strand virus rescue was compared in two mammalian cell lines (MDBK and Vero) and in primary cells of bovine, ovine and caprine origin. Such primary cells are more permissive for the growth of wild type strains of morbilliviruses, such as Rinderpest virus and Peste des petits ruminants virus. MVA-T7 was found to be highly cytopathic in the primary cells, multiplying rapidly and killing the cells within 3-5 days of infection, even when very low multiplicities of infection (MOI) were used. In contrast, FP-T7, which appeared to express similar amounts of T7 polymerase, was found to be non-cytopathic in a variety of primary and established cell lines of mammalian origin and was suitable for use in virus rescue experiments. MDBK cells and primary cells, unlike Vero cells, could not be efficiently transfected and so were unsuitable for virus rescue. Optimal conditions for rinderpest virus rescue in Vero cells were established using FP-T7 in place of MVA-T7. This system will be suitable for rescuing other viruses which grow in Vero cells.

Animals↗

PubMed1982

Transient expression of the chicken lysozyme gene after transfer into human cells.

The chicken lysozyme gene was inserted into an SV40-based plasmid vector, and the recombinants were transfected into the human cell lines HeLa and MCF-7. Correct and efficient transient expression directed by the lysozyme promoter was found in both of these cell lines, as determined by S1 nuclease mapping and Northern blot analysis of the RNAs made. SV40 sequences dramatically enhance the expression of the lysozyme gene. This enhancing effect is only acting in cis and is distance/orientation dependent, since clones containing the lysozyme gene in either orientation produce different amounts of correct lysozyme transcripts. The transfected lysozyme gene was not induced by steroid hormone treatment of the cells.

Breast Neoplasms↗

PubMed1988

Transient expression and sequence of the matrix (M1) gene of WSN influenza A virus in a vaccinia vector.

A cDNA encoding the entire amino acid sequence of the matrix (M1) protein of influenza A/WSN/33 virus was cloned, sequenced, and expressed in a vaccinia virus system consisting of the T7 bacteriophage RNA polymerase and a plasmid carrying the M1 gene flanked by T7 polymerase promoter and terminator sequences. The transiently expressed M1 gene product comigrated on SDS-polyacrylamide gels with the endogenous WSN virus M1 protein and was recognized in Western blot analysis by three epitope-specific monoclonal antibodies directed to the M1 protein. The nucleotide sequence and the predicted amino acid sequence of the cloned WSN virus M1 coding region was found to be more than 97% homologous to that of the M1 gene of influenza virus A/PR/8/34 reported by G. Winter and S. Fields (Nucleic Acids Res. 8, 1965-1974, 1980).

Amino Acid Sequence↗

PubMed1991

Mammalian achaete-scute homolog 1 is transiently expressed by spatially restricted subsets of early neuroepithelial and neural crest cells.

Using monoclonal antibodies, we have examined the expression pattern of MASH1, a basic helix-loop-helix protein that is a mammalian homolog of the Drosophila achaete-scute proteins. In Drosophila, achaete-scute genes are required for the determination of a subset of neurons. In the rat embryo, MASH1 expression is confined to subpopulations of neural precursor cells. The induction of MASH1 precedes, but is extinguished upon, overt neuronal differentiation. MASH1 is expressed in the forebrain by spatially restricted domains of neuroepithelium and in the peripheral nervous system exclusively by precursors of sympathetic and enteric neurons. The features of early and transient expression, in spatially restricted subpopulations of neural precursors, are similar to those observed for achaete-scute. Thus, the amino acid sequence conservation between MASH1 and achaete-scute is reflected in a parallel conservation of cell type specificity of expression, similar to the case of mammalian MyoD and Drosophila nautilus. These data support the idea that helix-loop-helix proteins may represent an evolutionarily conserved family of cell-type determination genes, of which MASH1 is the first neural-specific member identified in vertebrates.

Animals↗

PubMed2007

Transient expression of Nxf, a bHLH-PAS transactivator induced by neuronal preconditioning, confers neuroprotection in cultured cells.

Cortical spreading depression (CSD) induces waves of neuronal depolarization that confer neuroprotection to subsequent ischemic events in the rat brain. To gain insights into the molecular mechanisms elicited by CSD, we used representational difference analysis (RDA) to identify mRNAs induced by potassium depolarization in vivo. Using this approach, we have isolated a cDNA encoding the SIM2-related bHLH-PAS protein Nxf. Our results confirm that Nxf mRNA and protein are rapidly and transiently expressed in cortical neurons following CSD. Reporter assays show that Nxf is a transcriptional activator that associates with the bHLH-PAS sub-class co-factor ARNT2. Adenovirus-mediated expression of epitope-tagged Nxf results in cell death and the direct activation of the Bax gene in cultured cells. However, RNA interference studies show that endogenous Nxf is required for optimal neuroprotection by preconditioning in cultured F-11 cells. Together, our data indicate that Nxf is a novel bHLH-PAS transactivator transiently induced by preconditioning and that its sustained expression is detrimental. The identification of Nxf may represent an important step in our understanding of the molecular mechanisms of brain preconditioning and injury.

Animals↗

PubMed1994

Analysis of the cucumber malate synthase gene promoter by transient expression and gel retardation assays.

Recently it has been demonstrated that the single-copy malate synthase (MS) and isocitrate lyase (ICL) genes from cucumber are regulated by nutritional status in cucumber cell cultures. In this paper a new cucumber mesophyll protoplast transient expression system is described in which electroporated MS promoter-GUS reporter gene constructs exhibit the same pattern of expression as the endogenous MS gene. Both the electroporated MS-GUS constructs and the endogenous gene are expressed when protoplasts are cultured for 48 h on a non-metabolizable carbon source such as mannitol or 3-methylglucose, and repressed when cultured on a utilizable carbon source such as sucrose, glucose or fructose. A series of deletion mutants identified a region from position -248 to -125 relative to the start of transcription that is essential for expression of the MS-GUS construct under the different metabolic conditions. A 191 bp fragment spanning this region was fused, in both orientations, to the CaMV 35S core promoter. A pattern of metabolic regulation similar to that of the intact MS promoter was observed for these promoter fusion constructs which strongly suggests the presence of enhancer element(s) within this region. Comparison of the 191 bp region with other MS and ICL promoter sequences revealed a region of homology, designated RT. A gel retardation assay was used to assess binding of the 191 bp fragment to whole cell protein extracts from cell cultures expressing MS. Both the unlabelled 191 bp fragment and a synthetic oligonucleotide of RT compete specifically for the demonstrated binding activity.

Base Sequence↗

PubMed1995

Regulation of mammalian S-adenosylmethionine decarboxylase as studied in a transient expression system.

Mammalian S-adenosylmethionine decarboxylase (AdoMetDC), which catalyzes a key step in the biosynthesis of polyamines, is regulated by a multitude of mechanisms. The polyamines exert a strong feedback control of the enzyme. In the present study we have used a transient expression system to study the regulation of mammalian AdoMetDC. COS cells were transfected with a SV 40-based expression vector containing a 5'- and 3'-truncated human AdoMetDC cDNA (pSDC:16). The cells were shown to contain high levels of AdoMetDC activity 2 days after expression. This was partly due to an increase in the synthesis of the enzyme. However a marked stabilization of the enzyme against degradation did also contribute to the high AdoMetDC activity seen in the COS cells after the transfection with pSDC:16. The high expression of AdoMetDC was reflected in a marked change in intracellular polyamine levels. The cells were almost depleted of their putrescine, and their spermidine content was decreased to about 35% of that found in the mock-transfected cells. The spermine content, on the other hand, was increased. This change in polyamine levels was most likely attributable to the pSDC:16-induced increase in decarboxylated S-adenosylmethinine, which favors the accumulation of spermine at the expenses of putrescine and spermidine. The effects on the expression of AdoMetDC of polyamine synthesis inhibitors varied dependent on whether the COS cells were transfected with control vector or pSDC:16, in spite of similar effects on cellular polyamine levels, indicating a difference in feedback regulation of 'native' and recombinant AdoMetDC. The construct used in the present study gave rise to an AdoMetDC mRNA without a 5' and devoid of most of the 3' untranslated regions. However, whether these parts of the mRNA are involved in the polyamine-mediated translational control of the enzyme remains to be confirmed.

Adenosylmethionine Decarboxylase↗

PubMed1986

Transient expression of a plasmid gene, a tool to study DNA repair in human cells: defect of DNA repair in Cockayne syndrome; one thymine cyclobutane dimer is sufficient to block transcription.

Transfected recombinant DNA with regulatory elements such as eukaryotic promoter and termination sites is transiently expressed in human fibroblast cells. Utilizing an expression vector containing the simian virus 40 (SV 40) early control region followed by the E. coli chloramphenicol acetyltransferase (CAT) gene, we investigated the ability of normal, Xeroderma pigmentosum and Cockayne Syndrome cells to repair UV lesions in transfected DNA. Fibroblasts from Xeroderma pigmentosum patients which cannot excise pyrimidine cyclobutane dimers were unable to restore expression of UV irradiated CAT gene. An UV dose inducing one thymine cyclobutane dimer in the transcribed strand of the CAT gene blocked its transcription in these repair deficient cells. Normal cell were able to repair the lesions in transfected DNA during an incubation period of about 40 h and in this way could overcome the UV block. In several fibroblast cell lines from patients suffering from Cockayne Syndrome expression of UV damaged CAT gene was restored significantly less than in normal fibroblasts, indicating that Cockayne Syndrome is associated with a UV repair defect.

Alkylating Agents↗

PubMed2000

Transient expression of bacterial gene fragments in eukaryotic cells: implications for CD8(+) T cell epitope analysis.

CD8(+) T cells are potent effectors of acquired immunity against some viruses and intracellular bacterial pathogens. Antigens recognized by CD8(+) T cells are small, 8-9 amino acid peptides derived from proteins produced by the pathogen. These peptides are presented by MHC class I molecules on the surface of the infected cell. When characterizing the CD8(+) T cell response to a bacterial or viral pathogen, it is often necessary to express an antigenic protein in a eukaryotic host cell that is capable of processing and presenting peptide epitopes to antigen-specific CD8(+) T cells. We describe a system designed to transiently express bacterial polypeptides and MHC class I molecules in eukaryotic cells. Recognition of these peptide-MHC complexes stimulates TNF production by antigen-specific CD8(+) T cell lines. This system should be useful for analysis of CD8(+) T cell epitope-containing bacterial gene fragments when expression of the entire bacterial protein is detrimental to the eukaryotic cell, or when overexpression of the bacterial gene is detrimental to the bacterial cloning strain. Furthermore, this system can be used for the rapid mapping of CD8(+) T cell epitopes within a protein.

Animals↗

PubMed1999

Transient expression of GABAA receptor subunit mRNAs in the cellular processes of cultured cortical neurons and glia.

In this study, we have studied by in situ hybridisation histochemistry the expression and intracellular distribution of the GABAA receptor subunit mRNAs in cultured neurons obtained from postnatal day 1-3 rats in order to determine how neurotransmitter receptor expression may be regulated during development of the nervous system. In postnatal cortical cells, we found that GABAA receptor subunit mRNAs coding for alpha2, alpha5, beta2, beta3 and gamma2 subunits were transiently expressed in the cellular processes and growth cones after 1-3 days in culture. These observations indicate that GABAA receptor subunit mRNAs are transported (or trafficked) into the cellular processes of early postnatal cortical cells. These selective localisations were rarely observed after 5 days in culture and only in cells which had not made cell-to-cell contact. The localisation of subunit mRNAs in the processes was more effectively maintained up to 5 days or even longer if cell-to-cell contact was avoided by culturing the cells at low density or by inhibiting neurite sprouting pharmacologically with the GABA receptor channel antagonist TBPS. Finally, immunocytochemistry revealed the expression of GABAA receptors in the growth cones of pyramidal neurons in culture. Thus, the expression of mRNA correlates to the expression of protein. These results suggest that the selective trafficking of GABAA receptor subunit mRNAs during synaptogenesis may be regulated by synapse formation and/or glial-neural communication.

Animals↗

PubMed1992

Transient expression from cab-m1 and rbcS-m3 promoter sequences is different in mesophyll and bundle sheath cells in maize leaves.

Cell-specific and light-regulated expression of the beta-glucuronidase (GUS) reporter gene from maize cab-m1 and rbcS-m3 promoter sequences was studied in maize leaf segments by using an in situ transient expression microprojectile bombardment assay. The cab-m1 gene is known to be strongly photoregulated and to be expressed almost exclusively in mesophyll cells (MC) but not in bundle sheath cells (BSC). Expression of GUS from a 1026-base-pair 5' promoter fragment of cab-m1 is very low in dark-grown leaves; GUS expression is increased about 10-fold upon illumination of dark-grown leaves. In illuminated leaves, the ratio of GUS expression in MC vs. BSC is about 10:1. The cab-m1 region between 868 and 1026 base pairs 5' to the translation start confers strong MC-preferred expression on the remainder of the chimeric gene in illuminated leaves, but a region between -39 and -359 from the translation start is required for photoregulated expression. Transcripts of rbcS-m3 are found in BSC but not in MC and are about double in BSC of greening dark-grown seedlings. In contrast to the behavior of the cab-m1-GUS construct, GUS expression driven by 2.1 kilobase pairs of the rbcS-m3 5' region was about twice as high in MC as in BSC of unilluminated dark-grown maize leaves. The number of BSC, but not MC, expressing GUS nearly doubled upon greening of bombarded etiolated leaves. These data suggest that the 5' region of rbcS-m3 used here could be responsible for most of the light-dependent increase in rbcS-m3 transcripts observed in BSC of greening leaves and that transcriptional or posttranscriptional mechanisms are responsible for the lack of rbcS-m3 transcripts in MC.

Base Sequence↗

PubMed2006

Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development.

When aequorin is microinjected into cleavage-stage zebrafish embryos, it is largely used up by ~24 hours. Thus, it is currently not possible to image Ca(2+) signals from later stages of zebrafish development using this approach. We have, therefore, developed protocols to express apoaequorin, i.e., the protein component of aequorin, transiently in zebrafish embryos and then reconstitute intact aequorin in vivo by loading the coelenterazine co-factor into the embryos separately. Two types of apoaequorin mRNA, aeq-mRNA and aeq::EGFP-mRNA, the latter containing the enhanced green fluorescent protein (EGFP) sequence, were in vitro transcribed and when these were microinjected into embryos, they successfully translated apoaequorin and a fusion protein of apoaequorin and EGFP (apoaequorin-EGFP), respectively. We show that aeq::EGFP -mRNA was more toxic to embryos than equivalent amounts of aeq-mRNA. In addition, in an in vitro reconstitution assay, apoaequorin-EGFP produced less luminescence than apoaequorin, after reconstitution with coelenterazine and with the addition of Ca(2+). Furthermore, when imaging intact coelenterazine-loaded embryos that expressed apoaequorin, Ca(2+ )signals from ~2.5 to 48 hpf were observed, with the spatio-temporal pattern of these signals up to 24 hpf, being comparable to that observed with aequorin. This transient aequorin expression approach using aeq-mRNA provides a valuable tool for monitoring Ca(2+ )signaling during the 2448 hpf period of zebrafish development. Thus, it effectively extends the aequorin-based Ca(2+) imaging window by an additional 24 hours.

Aequorin↗

PubMed1992

Early transient expression of somatostatin (SRIF) immunoreactivity in dorsal root ganglia during ontogenesis in the rat.

Immunofluorescence was used in the rat to study the early ontogenetic expression of somatostatin (SRIF) in the dorsal root ganglia (DRGs) from gestational day 10.5 to day 15.5. SRIF-immunoreactivity (IR) was not detectable in day-10.5 embryos, was first observed in DRGs at day 11.5, reached a peak in intensity and distribution at around day 13.5 and thereafter decreased to become undetectable by day 15.5 in the DRGs of the trunk region. The dynamic expression of SRIF-IR in DRG perikarya could be correlated with its expression in nerve fibers located in the limbs and the abdominal mesenchyme. Thus, SRIF-IR is expressed at a time when sensory fibers could have established connections with their embryonic targets and when DRG neurons could have undergone their final mitotic phase. These data showing the earliest and transient expression of a neuropeptide in developing DRGs confirm and extend the notion that SRIF plays an important role in developmental processes.

Animals↗