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PubMed1996

Ontogeny of cholecystokinin binding sites in the hindbrain of the laboratory rat.

Studies in our laboratory have revealed a robust, transient expression of cholecystokinin binding sites in the facial motor nucleus during development in the Brazilian opossum, Monodelphis domestica. To investigate the ubiquity of this phenomenon, we have performed receptor autoradiography on the hindbrains of embryonic and neonatal rat pups. Cholecystokinin binding sites are present at very low levels in the embryonic day-16 rat hindbrain, but binding sites are abundant prior to birth. The greatest increase in labelled nuclei occurs prior to 5 days of postnatal age. Binding levels are heavy in the nucleus of the solitary tract, medial vestibular nucleus, posterior dorsal tegmental nucleus, area postrema, and caudal spinal trigeminal nucleus by 30 days postnatal. Both A-type and B-type receptors are present in the neonatal brainstem, although most labelled areas appear to be B-type. A-type binding sites are present in the ventral cochlear nucleus, the nucleus of the solitary tract, the dorsal motor nucleus of the vagus, the area postrema, the spinal nucleus of the trigeminal, and the cuneate and gracile nuclei by 5 days postnatal. As reported for the Brazilian opossum, cholecystokinin binding sites are expressed in the facial motor nucleus of neonatal rats and are transient. In this study of the brainstem in laboratory rats, a transient expression is also observed in the rubrospinal tract, parvocellular reticular nucleus, raphe obscurus, cuneate and gracile nuclei, and the ventral median fissure of the spinal cord. As vasopressin binding sites and estrogen receptors have also been shown to be expressed transiently in the laboratory rat facial motor nucleus, the physiological and developmental significance of transient binding site expression remains to be elucidated.

Animals↗

PubMed2003

Glutamic acid decarboxylase (GAD)67, but not GAD65, is constitutively expressed during development and transiently overexpressed by activity in the granule cells of the rat.

gamma-Aminobutyric acid (GABA)-mediated neurotransmission from the granule cells to CA3 is transiently expressed during the first 3 weeks of age in the rat. In the adult, seizures provoke this inhibitory signaling to reappear. To gain insight into the origin of GABA in these cells, we explored the expression of both isoforms of glutamic acid decarboxylase (GAD, 65 and 67 kDa), during development and after seizures in the adult rat. We found that GAD(67), but not GAD(65), is expressed in the mossy fibers of developing rats. In adults, GAD(67) is no longer detectable, unless seizures are induced. By contrast, GAD(65) is neither expressed in granule cells nor in their mossy fibers at any age nor after seizures, despite the presence of GAD(65) mRNA, confirmed by reverse transcription-polymerase chain reaction in situ.

Animals↗

PubMed1992

Factors controlling variability in measurements of the transient gene expression in electroporated protoplasts of wheat callus.

Factors controlling variability in enzyme transient expression assays have been investigated in electroporated protoplasts isolated from wheat embryogenic and nonembryogenic calli. The level of variation was reduced to a minimum through the optimization of the beta-glucuronidase measurements in the pellet and the supernatant of the homogenized protoplasts, by expressing the data on the basis of the number of protoplasts found to be viable immediately before the assay and on the amount of protein in the pellet and supernatant. Protoplast separation on the basis of size was also useful in eliminating some of the variation resulting from the heterogeneity of the callus used. Efforts to partially synchronize the callus tissue by auxin starvation have not resulted in a significant decrease of this variation. Our results indicate that the level of variation in enzyme transient activity in protoplasts resulting from calli can be reduced by implementation of the experimental techniques presented here.

Cell Membrane Permeability↗

PubMed1995

Effects of poliovirus 2A(pro) on vaccinia virus gene expression.

The effects of transient expression of poliovirus 2A(pro) on p220 cleavage in COS cells have been analyzed. When 2A(pro) was cloned in plasmid pTM1 and transiently expressed in COS cells, efficient cleavage of p220 occurred after infection of these cells with a recombinant vaccinia virus bearing phage T7 RNA polymerase. High numbers of COS cells were transfected with pTM1-2A, as judged by p220 cleavage, thereby allowing an analysis of the effects of poliovirus 2A(pro) on vaccinia virus gene expression. A 40-50% cleavage of p220 by transfected poliovirus 2A(pro) was observed ten hours post infection and cleavage was almost complete (80-90%) 20-25 hours post infection with vaccinia virus. Profound inhibition of vaccinia virus protein synthesis was detectable ten hours post infection and was maximal 20-25 hours post infection. This inhibition resulted from neither a blockade of transcription of vaccinia virus nor a lack of translatability of the mRNAs present in cells that synthesize poliovirus 2A(pro). Addition of ara-C inhibited the replication of vaccinia virus and allowed the continued synthesis of cellular proteins. Under these conditions, 2A(pro) is expressed and blocks cellular translation. Finally, p220 cleavage by 2A(pro) did not inhibit the translation of a mRNA encoding poliovirus protein 2C, as directed by the 5' leader sequences of encephalomiocarditis virus. Therefore, these findings show a correlation between p220 cleavage and inhibition of translation from newly made mRNAs. Our results are discussed in the light of present knowledge of p220 function, and new approaches are considered that might provide further insights into the function(s) of initiation factor eIF-4F.

Amino Acid Sequence↗

PubMed2000

A rapid and efficient system of Agrobacterium infection-mediated transient gene expression in rice Oc cells and its application for analysis of the expression and antisense suppression of preprophytosulfokine, a precursor of phytosulfokine-a, encoded by OsPSK gene.

A rapid and efficient system for Agrobacterium infection-mediated transient gene expression in rice has been developed. Using this system, transient expression of preprophytosulfokine, a precursor of phytosulfokine-a, encoded by OsPSK gene was analyzed. The results suggest that the Agrobacterium infection-mediated transient gene expression system is as efficient in rice Oc cells as in tobacco BY-2 cells and might be useful for rapid analysis not only of foreign gene expression, but also of antisense gene suppression.

Actins↗

PubMed2000

Amino acid determinants of alpha 7 nicotinic acetylcholine receptor surface expression.

Transient transfection has not been a successful method to express the alpha7 nicotinic acetylcholine receptor such that these receptors are detected on the cell surface. This is not the case for all ligand-gated ion channels. Transient transfection with the 5-hydroxytryptamine type 3 subunit cDNA results in detectable surface receptor expression. Cell lines stably expressing the alpha7 nicotinic acetylcholine receptor produce detectable, albeit variable, levels of surface receptor expression. alpha7 nicotinic acetylcholine receptor surface expression is dependent, at least in part, on cell-specific factors. In addition to factors provided by the cells used for receptor expression, we hypothesize that the surface expression level in transfected cells is an intrinsic property of the receptor protein under study. Employing a set of alpha7-5-hydroxytryptamine type 3 chimeric receptor subunit cDNAs, we expressed these constructs in a transient transfection system and quantified surface receptor expression. We have identified amino acids that control receptor distribution between surface and intracellular pools; surface receptor expression can be manipulated without affecting the total number of receptors. These determinants function independently of the cell line used for expression and the transfection method employed. How these surface expression determinants in the alpha7 nicotinic acetylcholine receptor might influence synaptic efficacy is discussed.

Amino Acid Sequence↗

PubMed1992

High level transient gene expression in human lymphoid cells by SV40 large T antigen boost.

High level transient gene expression in lymphoid cells has always been challenging because of the difficulty to efficiently transfect such cells. This has precluded any attempt to clone cDNA encoding proteins by means of their specific biological function in lymphoid cells. We have developed a very efficient transient eukaryotic expression system analogous to the well-known expression system in COS cells. Firefly luciferase and human CD2 genes were used as reporter genes and cloned into the eukaryotic shuttle vector pCDM8 which contains the strong cytomegalovirus promoter and the SV40 origin of replication for autonomous plasmid replication in permissive host cells that express the large SV40 T Antigen. Co-transfection of the reporter plasmids together with an SV40 T Ag expressing plasmid resulted in the several fold amplification of either the Luc activity or the cell surface expression of the CD2 marker in a transient assay. The level of amplification was dependent on the strength of the promoter used to drive the SV40 T Ag expression and was correlated with the extent of autonomous replication of the reporter plasmid in transfected cells. This highly efficient transient gene expression by SV40 T Ag boost was suitable to several human cell lines, making this system of general interest for expression cloning strategies or other gene transfer application that need high level expression.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1990

Vanadate inhibits expression of the gene for phosphoenolpyruvate carboxykinase (GTP) in rat hepatoma cells.

Vanadate, at concentrations between 0.5 and 2 mM, rapidly decreased the basal level of P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) mRNA and blocked the dibutyryl cyclic AMP (Bt2cAMP)-induced increase in enzyme mRNA in both FTO-2B and H4IIE rat hepatoma cells. The concentration of vanadate necessary to inhibit the expression of this gene was similar to that required for the vanadate-mediated activation of the insulin receptor tyrosine kinase. To determine whether vanadate could inhibit PEPCK gene transcription, a series of chimeric genes containing several deletions in the P-enolypyruvate carboxykinase promoter between -550 and -68 was linked to the structural genes for either amino-3-glycosyl phosphotransferase (neo) or chloramphenicol acetyltransferase and introduced into hepatoma cells using three methods: (a) infection with a Moloney murine leukemia virus-based retrovirus, (b) transfection and stable selection for neo expression, or (c) transient expression of chloroamphenicol acetyltransferase. In FTO-2B hepatoma cells infected with retrovirus, vanadate rapidly (within 1 h) inhibited transcription of the PEPCK-neo gene and blocked induction of gene expression caused by the addition of either Bt2cAMP or dexamethasone to the cells. Vanadate was not a general transcription inhibitor since, it like insulin, stimulated the expression of the c-fos gene. Also, the inhibitory effect of vanadate was rapidly reversible in FTO-2B cells since PEPCK gene expression could be stimulated by Bt2cAMP and dexamethasone after removal of vanadate. A series of 5' deletions in the P-enolpyruvate carboxykinase promoter (-550 to +73) was ligated to the structural gene for neo and stably transfected into hepatoma cells. Sequences responsive to vanadate were detected between -109 and -68. This result was confirmed using H4IIE hepatoma cells transiently expressing the PEPCK-CAT gene. The most likely target for vanadate in that region of the P-enolpyruvate carboxykinase promoter is cAMP regulatory element 1 which maps from -91 to -84. A comparison of the inhibitory effects of insulin and vanadate in this system indicated a major difference in the site of action of these two compounds on PEPCK gene transcription.

Animals↗

PubMed1999

Transient gene expression in mammalian and mosquito cells using a recombinant Semliki Forest virus expressing T7 RNA polymerase.

In this report, we describe a novel recombinant Semliki Forest virus (SFV) expressing T7 RNA polymerase (T7-RP), which was shown to drive transient expression of the chloramphenicol acetyltransferase (cat) gene in mammalian and mosquito cells after transfection of plasmids carrying the reporter gene under the control of the T7 promoter. To our knowledge, this is the first description of a T7-RP-based expression in mosquito cells. Expression of the cat gene was significantly enhanced in mammalian cells by inserting the sequence of the encephalomyocarditis virus internal ribosome entry site between the T7 promoter and the 5' end of the cat gene. In mosquito cells, the level of chloramphenicol acetyltransferase activity was increased by flanking the cat gene with the Autographa californica baculovirus p10 gene 5' and 3' untranslated regions. SFV expressing T7-RP appears, therefore, to be an alternative to other virus-based gene-expression systems in mammalian cells and a powerful tool for protein expression in mosquito cells.

Aedes↗

PubMed1996

Monitoring the transfection efficiency of the human follicle-stimulating hormone receptor cDNA in COS-7 cells: evaluation of the growth hormone transient gene expression assay system.

The human growth hormone (GH) transient gene expression assay system is frequently used to monitor transfection efficiency in transient transfection experiments. In this paper, we analyzed the suitability of the GH reporter gene to monitor transfection efficiency in COS-7 cells of an expression vector carrying the cDNA for the normal and mutated human follicle-stimulating hormone receptor (FSHR). The FSHR cDNA was cloned in the pSG5 expression vector and mutagenized (Ala307-->Thr) by oligonucleotide-mediated, site-directed mutagenesis. The expression plasmid pXGH5, carrying the structural gene for human GH, was used to monitor transfection efficiency. Different concentrations of pXGH5 and pSG5 containing normal or mutated FSHR cDNA were transfected in COS-7 cells by lipofection. The results showed: 1) The expression of pXGH5 was constant within individual experiments, but only in culture wells cotransfected with the same type of FSHR construct. On the contrary, the GH values normalized by the cell densities changed consistently depending on the type of FSHR construct. 2) The expression of the GH plasmid was influenced by type and concentration of the cotransfected plasmid. 3) The expression of pXGH5 cotransfected with the same FSHR construct was quite variable between experiments, without any relationship to the type of FSHR construct. These data show that the GH secretion is not a good parameter to monitor the transfection efficiency of the FSHR in pSG5 in COS-7 cells. Nor are other parameters such as semiquantitative mRNA determination or ligand binding to the transfected receptor ideal when mutations resulting in changes in receptor function are expected.

Cell Line↗

PubMed1984

An assay for transient gene expression in transfected Drosophila cells, using [3H]guanine incorporation.

We have developed an assay for transient gene expression using a dominant-selectable marker previously employed to transform Drosophila cultured cells. Drosophila hydei cells transfected with a functional Escherichia coli xanthine guanine phosphoribosyl transferase gene (gpt), under the control of the long terminal repeats (LTRs) of the copia transposable element, rapidly incorporate guanine into acid-precipitable counts. Autoradiographic analysis in situ shows that approximately 20% of cells take up, and express, the gpt gene. This transient gpt expression depends on the Drosophila promoter sequences since vectors with the gpt gene in reverse orientation to the copia LTRs fail to incorporate guanine. Deletion analysis confirms that the LTRs are essential for gpt gene expression. Similarly, cells transfected with gpt controlled by the Drosophila 70 000 mol. wt. heat-shock (hsp 70) promoter show regulated guanine incorporation when heat shocked. The efficiency of the copia LTRs varies considerably between the cell lines we tested, whereas that of the hsp 70 promoter does not. The heterologous promoters of the Rous sarcoma virus (RSV) and simian virus 40 (SV40) function poorly in these cells.

Animals↗

PubMed2006

Oestrogens inhibit interleukin 1beta-mediated nitric oxide synthase expression in articular chondrocytes through nuclear factor-kappa B impairment.

OBJECTIVES: To investigate the presence and functionality of oestrogen receptor alpha (ERalpha) in interleukin (IL)1beta-treated rabbit articular chondrocytes in culture, and to determine the mechanisms of 17beta oestradiol (E2) effects on IL1beta-induced inducible nitric oxide synthase (iNOS) expression. METHODS: The presence and functionality of ERalpha were investigated by immunocytochemistry and transient expression of an E2-responsive reporter construct. iNOS expression and production were determined by transient expression of a chimeric iNOS promoter-luciferase construct and protein immunoblotting. Nitric oxide (NO) production was determined by the Griess reaction. DNA-binding activities of nuclear factor-kappaB (NF-kappaB) and activated protein 1 were determined by electrophoretic mobility shift assay (EMSA)-ELISA assays. Nuclear translocation of p65 was studied by immunocytochemistry. RESULTS: ERalpha was identified in the nucleus of chondrocytes. ERalpha efficiently transactivated a transiently expressed E2-responsive construct. On IL1beta treatment, ERalpha partially diffused from its nuclear localisation into the cytoplasm and its transactivation ability was impaired. Nevertheless, E2, tamoxifen and raloxifene efficiently inhibited IL1beta-induced NO production (-34%, -31% and -36%, respectively). E2 decreased IL1beta-induced iNOS protein expression (-40%). Transient expression of an iNOS promoter construct strongly suggested that iNOS expression was inhibited at the transcriptional level, and EMSA-ELISA assays showed that E2 reduced (-60%) the IL1beta-induced p65 DNA-binding capacity. Finally, the p65 nuclear translocation induced by IL1beta was also strongly decreased by E2. CONCLUSIONS: Our data support a reciprocal antagonism between oestrogens and IL1beta, ultimately resulting in the decrease of cytokine-dependent NO production through transcriptional inhibition of iNOS expression. This effect was associated with selective inhibition of p65 DNA binding and nuclear translocation.

Animals↗

PubMed2001

Methylation of episomal plasmids as a barrier to transient gene expression via a synthetic delivery vector.

Efficient and sustained transgene expression are desirable features for many envisioned gene therapy applications, yet synthetic vectors tested to date are rarely successful in achieving these properties. Substantial research efforts have focused on protection of plasmid DNA from nuclease attack as well as increasing nuclear transport of plasmids, resulting in significant but still limited gains. We show here that a further barrier to efficient and sustained expression exists for synthetic vectors: plasmid DNA methylation. We have investigated this barrier for transient expression of a green fluorescent protein (GFP) transgene delivered via Lipofectamine, by testing the effects of culturing C3A human hepatoblastoma cells with 5-Azacytidine (AzaC), an irreversible inhibitor of DNA methyltransferase. To control for loss of plasmids by dilution during mitosis, transfected cells were growth-arrested for 1 week and their subsequent GFP expression quantified by FACS. In the presence of AzaC, a significantly greater fraction of transfected cells remained GFP-positive and possessed higher levels of GFP production relative to AzaC-untreated cells. Additionally, we have applied a Methyl-Assisted PCR (MAP) assay to quantify a subset of methylated CpG sites in the GFP gene. When MAP was performed on plasmids isolated from transfected cells, the extent of methylation was found to be inversely related to the level of GFP expression.

Adolescent↗

PubMed1993

Drosophila nervous system muscarinic acetylcholine receptor: transient functional expression and localization by immunocytochemistry.

The pharmacological properties of a cloned Drosophila muscarinic acetylcholine receptor (mAChR) were investigated using two independent transient expression systems. The binding characteristics of the expressed receptor were determined using transfected COS-7 cells, whereas the mAChR functional properties were analyzed using nuclearly injected Xenopus oocytes. Competition displacement studies with transfected COS-7 cell membranes showed that N-[3H]methylscopolamine binding was displaced most effectively by atropine, followed by 4-diphenylacetoxy-N-methylpiperidine methiodide, pirenzepine, and AFDX-116. This same order of effectiveness (4-diphenylacetoxy-N-methylpiperidine methiodide > pirenzepine > AFDX-116) was observed in oocytes expressing Dm1 when carbamylcholine-induced currents were inhibited by the same antagonists. Thus, the expressed Drosophila mAChR (Dm1) exhibits a pharmacology that broadly resembles that of the vertebrate M1 and M3 mAChR subtypes. To determine the anatomical localization of the Drosophila mAChR, polyclonal antiserum was raised against a peptide corresponding to the predicted carboxyl-terminal domain of the receptor. Immunocytochemistry on fly sections demonstrated that the mAChR gene product was found in the nervous system and was not seen in skeletal muscle. The most intense staining was localized to the glomeruli of the antennal lobes, an area of the insect brain where first-order synaptic processing of olfactory information occurs.

Animals↗

PubMed1993

Effects of promoter, intron and enhancer elements on transient gene expression in sugar-cane and carrot protoplasts.

Various chimaeric promoter regions coupled to the uidA beta-glucuronidase gene were evaluated for transient expression strength following electroporation into sugar-cane (monocot) and carrot (dicot) protoplasts. Multiple enhancer elements increased expression in sugar-cane, by up to 400-fold for the artificial Emu promoter relative to the CaMV 35S promoter. The relative expression strengths of promoters varied substantially between the species. Sugar-cane also differed in some respects from previously tested species in the family Poaceae. For example, in sugar-cane the nopaline synthase and CaMV 35S promoters were of equivalent strength, and insertion of Adh1 intron 1 into the 5' transcribed region decreased expression strength.

Enhancer Elements, Genetic↗

PubMed1995

Preprotachykinin-A gene expression occurs transiently in the developing rat endocrine pancreas and can be regulated in RINm5F cells.

We have reported previously that the rat insulinoma cell lines, RINm5F and RINr1046-38, express the preprotachykinin(PPT)-A gene, which encodes the tachykinin peptides, substance P and neurokinin A. Because endocrine cells of the adult rat pancreas do not appear to express PPT-A, we investigated whether the gene is expressed by rat pancreatic endocrine cells during development. We used immunohistochemistry, employing different substance P and neurokinin A antibodies, to show that many endocrine cells of the fetal and neonatal rat pancreas synthesise these products of PPT-A gene expression. Colabeling experiments revealed that a significant number of both insulin-containing and non-insulin-containing cells express tachykinins. After postnatal day 20, the number of tachykinin-immunoreactive pancreatic islet cells declines and, as already reported, none were detected in the adult rat pancreas. The transient expression of PPT-A by the developing endocrine pancreas is a novel finding. Substance P and neurokinin A are known to have trophic actions and may serve as growth factors during pancreatic islet development. PPT-A gene expression by RINm5F and RINr1046-38 cells is further evidence that these cells resemble developing pancreatic endocrine cells. They are potentially valuable as unique models for studying the regulation of tachykinin biosynthesis. We provide evidence in this study, using quantitative PPT-A messenger RNA analysis, that PPT-A expression in RINm5F cells may be up-regulated by activation of protein kinase C, down-regulated by activation of glucocorticoid receptors, and is not significantly affected by changes in intracellular cAMP levels.

Animals↗

PubMed2001

Quantitative analysis by flow cytometry of abscisic acid-inducible gene expression in transiently transformed rice protoplasts.

BACKGROUND: Quantifying plant gene expression by flow cytometry (FCM) would allow multidimensional cell-parameter analysis on a per-cell basis, thereby providing insight into the cellular mechanisms of plant gene regulation. Here we sought to establish quantitation by FCM of plant hormone (abscisic acid, ABA)-inducible green fluorescent protein (GFP) expression and to compare the method directly with traditional reporter enzyme assays. MATERIALS AND METHODS: GFP, beta-glucuronidase, and luciferase reporter genes driven by ABA-inducible or constitutive promoter constructs were expressed in transiently cotransformed rice protoplasts and reporter activities quantified by FCM (for GFP) or traditional enzyme assays. Treatments included cotransformations with specific ABA signaling effector cDNA constructs (encoding VIVIPAROUS-1, an ABA transcription factor, and ABA-INSENSITIVE1-1, a dominant-negative protein phosphatase regulator) and the ABA agonist lanthanum chloride. Dual-color FCM was also performed on GFP-expressing cells immunodecorated with an mAb recognizing a rice cell surface epitope. RESULTS: Quantitative analysis of ABA-inducible gene expression by FCM using GFP as reporter gave comparable results to traditional reporter enzyme assays, although the signal-to-noise ratio was less for FCM, which can be a limitation of the method at low promoter strengths. Multiparameter-correlated analysis of ABA-inducible GFP expression with a plasma membrane marker showed no apparent correlation between ABA sensitivity, marked by GFP, and presence of a cell surface arabinogalactan glycoprotein. CONCLUSIONS: Quantitative FCM of GFP-expressing plant cells is a rapid, robust, reproducible, and value-added method relative to traditional enzymatic reporter gene assays.

Abscisic Acid↗

PubMedDate not supplied

Transient gene expression in suspension HEK-293 cells: application to large-scale protein production.

Recent advances in genomics, proteomics, and structural biology raised the general need for significant amounts of pure recombinant protein (r-protein). Because of the difficulty in obtaining in some cases proper protein folding in bacteria, several methods have been established to obtain large amounts of r-proteins by transgene expression in mammalian cells. We have developed three nonviral DNA transfer protocols for suspension-adapted HEK-293 and CHO cells: (1) a calcium phosphate based method (Ca-Pi), (2) a calcium-mediated method called Calfection, and (3) a polyethylenimine-based method (PEI). The first two methods have already been scaled up to 14 L and 100 L for HEK-293 cells in bioreactors. The third method, entirely serum-free, has been successfully applied to both suspension-adapted CHO and HEK-293 cells. We describe here the application of this technology to the transient expression in suspension cultivated HEK-293 EBNA cells of some out of more than 20 secreted r-proteins, including antibodies, dimeric proteins, and tagged proteins of various complexity. Most of the proteins were expressed from different plasmid vectors within 5-10 days after the availability of the DNA. Transfections were successfully performed from the small scale (1 mL in 12-well microtiter plates) to the 2 L scale. The results reported made it possible to establish an optimized cell culture and transfection protocol that minimizes batch-to-batch variations in protein expression. The work presented here proves the applicability and robustness of transient transfection technology for the expression of a variety of recombinant proteins.

Bioreactors↗