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Results for “Transient expression”

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PubMed1987

Transient and stable complementation of ultraviolet repair in xeroderma pigmentosum cells by the denV gene of bacteriophage T4.

In this paper we report both transient and stable complementation of pyrimidine dimer repair in xeroderma pigmentosum cells by the denV gene of bacteriophage T4, coding for endonuclease V, a dimer-specific DNA glycosylase. Cotransfection with pRSVdenV in SV40-transformed XP12RO(M1) cells (complementation group A) restored transient expression of an indicator plasmid (pRSVcat) bearing a UV-inactivated chloramphenicol acetyltransferase (cat) gene. In addition, XP12RO(M1) clones stably transformed by pRSVdenV-SVgpt expressed transient chloramphenicol acetyltransferase activity when transfected with UV-inactivated pRSVcat plasmid. These clones also showed partial restoration of colony forming ability and excision repair synthesis after UV irradiation. Immunofluorescence, using an endonuclease V polyclonal antibody, showed the presence of the phage glycosylase in stably transformed xeroderma pigmentosum cells. The cotransfection assay affords a rapid, sensitive procedure to screen for functional cloned DNA repair genes and to test mutant cells for the deficiency of specific steps in DNA repair, such as incision.

Cells, Cultured↗

PubMed1994

Formation of heteromeric gamma-aminobutyric acid type A receptors containing two different alpha subunits.

The functional properties of recombinant gamma-aminobutyric acid (GABA) receptors expressed transiently in human embryonic kidney 293 cells were examined. Combinations of alpha 1 beta 2 gamma 2, alpha 3 beta 2 gamma 2, and alpha 1 alpha 3 beta 2 gamma 2 subunits were transiently expressed and the properties of the resulting receptors were studied with patch-clamp electrophysiology. Each subunit combination produced receptors having a unique set of functional properties. Concentration-response experiments showed that receptors composed of alpha 1 beta 2 gamma 2 subunit combinations were more sensitive to GABA (EC50 = 17.4 microM) than were either alpha 3 beta 2 gamma 2 (EC50 = 103 microM) or alpha 1 alpha 3 beta 2 gamma 2 (EC50 = 55.8 microM) receptors. Consistent with its action at native GABA-A receptors, diazepam (1 microM) potentiated the effect of GABA by shifting the GABA concentration-response curve to the left. The magnitude of the diazepam shift also differed between subunit combinations. The apparent potency of GABA was increased 2-fold by diazepam with alpha 1 beta 2 gamma 2 receptors, 3-fold with alpha 3 beta 2 gamma 2 receptors, and 5-fold with alpha 1 alpha 3 beta 2 gamma 2 receptors. Brief applications (6-25 msec) of 3 mM GABA to outside-out patches revealed that currents decayed predominantly with double-exponential time courses. The decay time courses of currents mediated by alpha 1 beta 2 gamma 2 and alpha 1 alpha 3 beta 2 gamma 2 receptors were similar, whereas the alpha 3 beta 2 gamma 2 receptor response decayed more slowly. The distinct properties observed in cells expressing each of these subunit combinations suggest that the subunits form unique receptors. The possibility that some neuronal GABA receptors contain two different alpha subunits is discussed.

Cell Line↗

PubMed1998

[Analysis of a novel delta gene during the formation of cortical layers].

The preplate is composed of earliest-generated neurons, including Cajal-Retzius cells and prospected subplate neurons which together play a critical role in the formation of cortical layers. Additionally, Notch and Delta have been identified as key components that mediate the lateral specification and fate of numerous cells during development. Here, we demonstrate that a divergent type of Delta (D113) is expressed transiently and specifically in a set of earliest generated neurons in the neural tube. Moreover, in developing cerebrum, D113 is specifically and transiently expressed in prospected subplate neurons. Thus, our results suggest that D113 mediates the specification of cell fate in the earliest stage of neurogenesis including the neurons that compose preplate, leading to the mechanism of genesis of cortical layers.

Animals↗

PubMed1984

Transient and differential expression of aspects of the catecholaminergic phenotype during development of the fetal bowel of rats and mice.

A population of proliferating cells in the fetal gut has previously been found to express transiently aspects of a catecholaminergic phenotype (TC cells) during development in both rats and mice. These cells appear to be noradrenergic in rats but dopaminergic in mice. In the current study, the ability of TC cells, identified by the immunocytochemical demonstration of tyrosine hydroxylase (TH), to take up and become radioautographically labeled by [3H]norepinephrine ([3H]NE) was assessed. When TC cells were most numerous in the bowel of rats, no cells were labeled by [3H]NE (days E12 and E13). In rats, but not mice, labeling of larger cell bodies by [3H]NE was found on days E14 and E15. However, no cells showed TH immunoreactivity on day E15, although a few cells were doubly labeled by [3H]NE and TH immunoreactivity on day E14. Therefore, in rats TC cells contain TH immunoreactivity but do not take up [3H]NE prior to day E14, and their disappearance is followed by the appearance of a second population of larger cells that lacks TH immunoreactivity but which does take up [3H]NE. The transient appearance of some cells that express both markers on day E14 suggests, but does not prove, that TC cells change their phenotype and are the precursors of the cells found later in development that lack TH but which take up [3H]NE. The cells that take up [3H]NE are rare or absent in newborn rat gut, indicating that they may also be transient. These results indicate that genes responsible for different aspects of the noradrenergic phenotype need not necessarily be coupled in their expression. Although uptake of [3H] NE into cell bodies was not found on day E13 or later in vivo in mouse gut, it does occur in mouse bowel explanted prior to day E13 and grown for 10 to 12 days in culture. These cultures also contained TH immunoreactive cells. Thus, the potential for development of cells able to take up [3H]NE exists in mice as well as in rats, and the conditions that lead to a loss of catecholaminergic traits in vivo do not exist in vitro.

Animals↗

PubMed2004

Subcellular trafficking of exogenously expressed interferon-beta in Madin-Darby canine kidney cells.

We have recently demonstrated that when IFN-beta was exogenously expressed in epithelial cells, transiently expressed IFN-beta was predominantly secreted from the cell side to which the transfection was performed, while stably expressed one was almost equally secreted to the apical and basolateral sides. In the present study, we analyzed the subcellular transport of IFN-beta using confocal imaging with green fluorescent protein (GFP)-tagged IFN-beta in Madin-Darby canine kidney (MDCK) cells. Stably expressed and transiently expressed human IFN-beta (HuIFN-beta)-GFPs were seen in upper regions of the nucleus. In stable HuIFN beta-GFP-producing transformants, transiently expressed mouse IFN-beta (MuIFN-beta) was apparently co-localized with the bulk of the constitutive HuIFN beta-GFP proteins at TGN, and a significant quantity of them then appeared to pass into distinct post-TGN vesicles, accepting either type of IFN. Meanwhile, when cells were co-transfected with both expression vectors, transiently expressed both IFNs tended to co-localize not only at TGN but in post-TGN vesicles. These results suggest that stably and transiently expressed IFN-betas, albeit co-localized at TGN, were transported through apparently discriminated post-TGN routes.

Animals↗

PubMed1998

A potent cell death activity associated with transient high level expression of BCL-2.

The BCL-2 proto-oncogene contains unusually long untranslated 5' and 3' sequences. Deletion of the sequences flanking the BCL-2 open reading frame dramatically increases the level of protein expression. Transient high level BCL-2 protein expression mediated by plasmid transfection or by infection with recombinant adenovirus results in potent apoptosis of several cell lines. Detailed mutational (deletion and add-back) analysis reveals that both 5'- and 3'-flanking sequences contribute to the negative modulation of protein expression from the BCL-2 open reading frame. It appears that these sequences exert the negative regulatory effect in an orientation-dependent manner. Analysis of BCL-2 RNA levels indicate that elevated levels of mRNA may be the primary cause of elevated levels of protein expression. Apoptosis induced by adenovirus vectors expressing elevated levels of BCL-2 can be readily inhibited by the caspase inhibitor z-VAD-fmk, suggesting that high levels of BCL-2 expression induce apoptosis via the caspase cascade. Mutational analysis of BCL-2 indicates that its pro-apoptotic activity is separable from its anti-apoptosis activity. Our results raise the possibility that oncogenic conversion of BCL-2 may require somatic mutations in the pro-apoptotic activity, in addition to other activating mutations that result in enhanced expression. Consistent with this hypothesis, a somatic mutation of BCL-2 observed in multiple human tumors results in reduced apoptosis activity.

Adenoviridae↗

PubMedDate not supplied

Engineering mRNA translation initiation to enhance transient gene expression in chinese hamster ovary cells.

To increase transient expression of recombinant proteins in Chinese hamster ovary cells, we have engineered their protein synthetic capacity by directed manipulation of mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser(51)Ala site-directed mutant of eIF2alpha, a subunit of the trimeric eIF2 complex that is implicated in regulation of the global rate of mRNA translation initiation in eukaryotic cells. Phosphorylation of eIF2alpha by protein kinases inhibits eIF2 activity and is known to increase as cells perceive a range of stress conditions. Using single- and dual-gene plasmids introduced into CHO cells by electroporation, we found that transient expression of the eIF2alpha Ser(51)Ala mutant with firefly luciferase resulted in a 3-fold increase in reporter activity, relative to cells transfected with reporter only. This effect was maintained in transfected cells for at least 48 h after transfection. Expression of the wild-type eIF2alpha protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2alpha phosphorylation in cells transfected with the mutant eIF2alpha construct. Transfection of CHO cells with the luciferase-only construct resulted in a marked decrease in the global rate of protein synthesis in the whole cell population 6 h post-transfection. However, expression of the mutant Ser(51)Ala or wild-type eIF2alpha proteins restored the rate of protein synthesis in transfected cells to a level equivalent to or exceeding that of control cells. Associated with this, entry of plasmid DNA into cells during electroporation was visualized by confocal microscopy using a rhodamine-labeled plasmid construct expressing green fluorescent protein. Six hours after transfection, plasmid DNA was present in all cells, albeit to a variable extent. These data suggest that entry of naked DNA into the cell itself functions to inhibit protein synthesis by signaling mechanisms affecting control of mRNA translation by eIF2. This work therefore forms the basis of a rational strategy to generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering.

Animals↗

PubMed1999

Towards molecular farming in the future: transient protein expression in plants.

Molecular farming in plants can be achieved by stable or transient expression of a recombinant protein. Transient expression of recombinant proteins in plants can rapidly provide large amounts of the proteins for detailed characterization. It is fast, flexible and can be carried out at field scale using viral vectors, but it lacks the increases in production volume that can be achieved easily with stable transgenic crops. This review article focuses on discussing the applications of transient expression using viral vectors, biolistic methods or agroinfiltration.

Antibodies↗

PubMed2000

Transient high altitude expressive aphasia.

Transient focal neurological deficits have been described in sojourners to high altitude. We present two cases of transient expressive aphasia in well-acclimatized high altitude climbers. We speculate that this type of transient focal neurological impairment may represent migraine aura, and we discuss other reports of transient focal neurological deficit at high altitude.

Adult↗

PubMed1996

Steroidogenic factor-1 expression is transiently repressed and c-myc expression and deoxyribonucleic acid synthesis are induced in rat granulosa cells during the periovulatory period.

The expression of steroidogenic factor 1 (SF-1), cytochrome P450 aromatase (P450arom), and cytochrome P450 cholesterol side-chain cleavage (P450scc) was examined during the periovulatory period. Immature rats were injected with eCG to induce development of ovarian follicles to the preovulatory stage. At 48 h after the eCG injection, the LH surge was simulated by an injection of an ovulatory dose of hCG, and RNA was isolated at 0, 2, 4, 6, 8, and 24 h after hCG injection. The mRNA levels for SF-1, P450arom, and P450scc were relatively high in total ovarian RNA samples from eCG-treated rats. Levels of SF-1 and P450arom mRNA decreased within 2 h after injection of hCG. The SF-1 mRNA levels gradually increased from 4 to 24 h. Aromatase levels remained undetectable until 24 h after hCG. P450scc mRNA levels increased throughout the 24-h period after hCG. Levels of SF-1 and P450arom, but not P450scc, mRNA were also reduced in RNA samples from isolated granulosa cells at 4 h after hCG treatment relative to those from eCG-treated rats. In situ hybridization analysis also revealed that hCG uniformly suppressed SF-1 mRNA levels an all granulosa cells compared to those of eCG-treated controls. The relationship of SF-1 expression to immediate/early gene expression and cell cycle traverse was also examined. C-myc mRNA levels were induced by up to 10-fold at 4 h after hCG injection. Similarly, DNA synthesis, as measured by the percentage of granulosa cells that incorporated 5'-bromodeoxyuridine, was increased from 16 +/- 4% in eCG-treated rats to 61 +/- 7% at 4 h after hCG treatment (p < 0.05). This study provides the novel finding that SF-1 expression is transiently repressed to very low levels in response to the LH surge. Further, these studies suggest that granulosa cells traverse the cell cycle before becoming terminally differentiated luteal cells.

Animals↗

PubMed1992

Potato granule-bound starch synthase promoter-controlled GUS expression: regulation of expression after transient and stable transformation.

Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the beta-glucoronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient to result in tissue-dependent GUS expression: levels in stably transformed microtubers exceeded levels in corresponding leaves by orders of magnitude. GBSS-GUS constructs could be transiently expressed in leaf protoplasts from wild-type and amylose-free potato lines, etuberosum Solanum brevidens, Nicotiana tabacum and Arabidopsis thaliana. Transient expression levels in potato leaf protoplasts were clearly lower than in corresponding suspension cell protoplasts. This lower expression in leaf protoplasts could not be elevated by increasing DNA concentrations during transfection. Light incubation of electroporated suspension cell protoplasts reduced transient GBSS-GUS expression, whereas incubation of transfected protoplasts in media with different sucrose concentrations did not affect transient expression levels. However, electroporated protoplasts, isolated from suspensions, which had been grown on media with increasing amounts of sucrose showed a sucrose concentration-dependent transient expression profile. This indicates that studying GBSS regulation by transient expression experiments needs pre-treatment of the protoplast source. Sequence data of the GBSS promoter were compared to those of two other potato alleles.

Alleles↗

PubMed1995

Replication-deficient vaccinia virus encoding bacteriophage T7 RNA polymerase for transient gene expression in mammalian cells.

The vaccinia virus/bacteriophage T7 hybrid transient expression system employs a recombinant vaccinia virus that encodes the T7 RNA polymerase gene, a plasmid vector with a gene of interest regulated by a T7 promoter, and any cell line suitable for infection and transfection. Although high expression in a majority of cells is achieved, the severe cytopathic effects of vaccinia virus and the safety precautions required for use of infectious agents are undesirable features of the system. Here, we report the construction of a highly attenuated and avian host-restricted vaccinia virus recombinant that encodes the T7 RNA polymerase gene (MVA/T7 pol) and demonstrate the use of the virus for transient expression in mammalian cells. MVA/T7 pol has reduced cytopathic effects compared to the previously used replication-competent vaccinia virus, while providing a high level of gene expression in multiple mammalian cell lines.

Animals↗

PubMed1993

Identification, sequence, and transcriptional mapping of lef-3, a baculovirus gene involved in late and very late gene expression.

A trans-acting gene required for late viral gene expression in transient expression assays was identified in the genome of Autographa californica nuclear polyhedrosis virus. A genomic library of A. californica nuclear polyhedrosis virus DNA lacking a clone spanning the region from 43 to 48 map units (mu) was unable to activate gene expression from a reporter plasmid when the reporter gene was driven by a baculovirus late or very late promoter in transient expression assays. The genomic region responsible for activating reporter gene expression was further mapped to 43.4 to 45.2 mu of the viral genome. The nucleotide sequence of this region was determined and shown to contain several small open reading frames (ORFs) and one major ORF, named lef-3, for late expression factor 3. The lef-3 ORF was predicted to encode a polypeptide of 385 amino acids and with a molecular mass of 44,529 daltons. No homolog to the lef-3 ORF was found in existing data bases. Further analysis showed that only the lef-3 ORF in the region from 43.4 to 45.2 mu was necessary for late gene activation in the assays used. The temporal regulation of lef-3 transcription was studied by Northern (RNA) blot hybridization; lef-3 was found to be an early gene that was transcribed primarily as a 2.0-kb mRNA. Primer extension analysis and S1 nuclease protection assays revealed that lef-3 transcription initiated about 280 bp upstream of the first ATG codon and terminated near a polyadenylation signal, 130 bp downstream of the last codon of the lef-3 ORF.

Amino Acid Sequence↗

PubMed1997

Optimization of plasmid vectors for high-level expression in lung epithelial cells.

Nonviral gene therapy approaches use a plasmid vector to express the desired transgene. We have systematically examined several regulatory elements within plasmid vectors that govern gene expression, e.g., the promoter, enhancer, intron, and polyadenylation signal, by constructing a series of plasmids that differed only in the particular sequence element being evaluated. Of the several promoters and polyadenylation signal sequences that were tested, the human cytomegalovirus (CMV) immediate early gene promoter and the addition of polyadenylation signal sequences from the bovine growth hormone (BGH) gene or rabbit beta-globin gene produced the highest levels of expression in vitro. The inclusion of a hybrid intron 3 to the promoter further increased expression 1.6-fold. The addition of a region of the CMV enhancer 5' to several weak promoters increased expression 8- to 67-fold, and co-transfection with a second plasmid encoding a chimeric transcription factor also enhanced expression. On the basis of these results, the CMV promoter, the hybrid intron, and the BGH polyadenylation signal were selected for consistent high level expression in vitro and in the mouse lung. However, expression was transient, with greater than 60% loss of activity in the first 7 days. This transient expression was not specific to CMV promoter-containing plasmids, because plasmids containing other heterologous promoters showed a similar profile of transient expression in vivo. These comparative analyses begin to provide a basis for the development of optimized expression plasmids for gene therapy of lung diseases.

Animals↗

PubMed1996

Quantitative analysis of transient gene expression in mammalian cells using the green fluorescent protein.

The green fluorescent protein (Gfp) has been used as a reporter, along with flow cytometric analysis, to follow the dynamics of gene expression in transiently transfected mammalian cells. Gene transfer conditions for lipofection were optimized. The highest fraction of transfectants were obtained when lipid-DNA complexes were formed with 6 microliters lipid and 1 microgram DNA for chinese hamster ovary (CHO) cells and with 9 microliters lipid and 2 micrograms DNA for NIH/3T3 cells. Chinese hamster ovary cells were monitored for Gfp expression and growth for 6 days following transfection. An initial decrease in viability for 36 h was observed after which cell growth followed exponential kinetics with increasing viability. Intracellular accumulation of recombinant protein peaked at 24 h post-transfection and then decreased with first order kinetics at a rate comparable to the specific growth rate. It appears that dilution by growth accounts for the decrease of Gfp in the biomass. Immunofluorescent staining of Gfp and subsequent flow cytometric analysis of transfected cells revealed a linear correlation between the green fluorescence and immunofluorescence. This indicates that green fluorescence is a quantitative measure of intracellular Gfp in single cells in spite of the dynamics of post-translational modifications involved in the conversion of expressed protein into its fluorescent form. A structured model has been formulated to describe the observed kinetics of gene expression and fluorophore formation. The model accurately predicts experimental trends and suggests that the fraction of non-fluorescent Gfp is significant only during the initial period of gene expression.

3T3 Cells↗

PubMed1995

Mutational analysis of varicella-zoster virus major immediate-early protein IE62.

The varicella-zoster virus (VZV) open reading frame 62 encodes an immediate-early protein (IE62) that transactivates expression of various VZV promoters and autoregulates its own expression in transient expression assays. In Vero cells, IE62 was shown to transactivate the expression of all putative immediate-early (IE) and early (E) genes of VZV with an up-regulating effect at low intracellular concentrations. To define the functional domains involved in the regulatory properties of IE62, a large number of in-frame insertions and deletions were introduced into a plasmid-borne copy of the gene encoding IE62. Studies of the regulatory activities of the resultant mutant polypeptides in transient expression assays allowed to delineate protein regions important for repression of its own promoter and for transactivation of a VZV putative immediate-early gene (ORF61) promoter and an early gene (ORF29) promoter. This mutational analysis resulted in the identification of a new functional domain situated at the border between regions 4 and 5 which plays a crucial role in the IE62 regulatory functions. This domain turned out to be very well conserved amongst homologous alphaherpesvirus regulatory proteins and appeared to be rich in bulky hydrophobic and proline residues, similar to the proline-rich region of the CAAT box binding protein CTF-1. By immunofluorescence, a nuclear localization signal has been mapped in region 3.

Amino Acid Sequence↗

PubMed1998

Transient developmental expression of monoamine transporters in the rodent forebrain.

Neurons in first-order sensory thalamic nuclei have been shown to express functional plasma membrane serotonin (SERT) and vesicular monoamine (VMAT2) transporters during early postnatal development. In the present study, we provide an extensive description of the spatial and the temporal patterns of VMAT2 and SERT expression, during early embryonic development and postnatal life, by using in situ hybridization and immunocytochemistry. VMAT2 and SERT genes are transiently expressed in a wide population of non-monoaminergic neurons in the central and peripheral nervous system with a large overlap in the temporal and spatial pattern of expression of both genes. A selective pattern of expression of both genes was observed in the thalamus with expression limited to the dorsal thalamus and more particularly to primary sensory relay nuclei that convey point to point projection maps. Transient expression of the transporters was also observed in sensory cranial nerves, in the hippocampus, cerebral cortex, septum, and amygdala. VMAT2 and SERT gene expression was not necessarily linked, as some neural populations expressed only VMAT2, while others only contained SERT. Since VMAT2 serves to transport catecholamines besides serotonin, we examined the developmental expression of the plasma membrane dopamine and norepinephrine transporters but found no transient expression of these genes. Despite minor temporal disparities, VMAT2 and SERT extinguished almost simultaneously during the second and third weeks of post-natal life. These expressions did not seem to be dependent on peripheral neural inputs, since monocular enucleations and infraorbital nerve cuts effected on the day of birth, did not modify the period of transporter expression or of extinction.

Animals↗

PubMed1989

Neonatal enucleations reduce number, size, and acetylcholinesterase histochemical staining of neurons in the dorsal lateral geniculate nucleus of developing rats.

Previous studies have demonstrated that transient patterns of acetylcholinesterase (AChE) activity are characteristic of geniculo-recipient regions of rat cortical area 17 during the second and third postnatal weeks of life. Neonatal enucleation results in a marked reduction of this transiently expressed cortical AChE. Parallel studies have demonstrated that the dorsal lateral geniculate nucleus (dLGN) also expresses AChE transiently during development. The present study examines neuronal number and size as well as AChE histochemical staining in the dLGN of normal and neonatally enucleated rat pups to determine whether changes in dLGN neurons could account for the decreased visual cortical AChE staining that results from neonatal enucleation. Changes in 4 parameters in dLGN were noted after neonatal enucleation. First, a 26-37% shrinkage in the volume of dLGN occurred contralateral to enucleation. Second, enucleation resulted in a loss of 16-30% of AChE-stained neuronal somata. Third, remaining AChE-positive neuronal somata appeared shrunken by approximately 40%. Fourth, intensity of AChE histochemical staining of individual dLGN neurons was reduced by approximately 24% following neonatal enucleation. These data suggest that loss of transient AChE activity in cortical area 17 consequent to neonatal enucleation is secondary to enucleation-induced alterations in the dLGN; these alterations include loss of neurons, shrinkage of neurons, and an apparent decrease in the ability of neurons to synthesize AChE. These data support the hypothesis that geniculocortical projection neurons express AChE transiently during development of geniculocortical connectivity and indicate that normal afferent connections and/or activity are important for the transient expression of AChE by these neurons.

Acetylcholinesterase↗