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Results for “Transient expression”

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PubMed1991

Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge.

The ovarian steroid progesterone affects reproductive physiology by regulating the expression of specific genes in target tissues. In an attempt to address the question of whether the ovary itself is a target tissue for progesterone action, we have examined the localization and regulation of progesterone receptor (PR) mRNA in the rat ovary. We used the polymerase chain reaction to clone the steroid-binding domain of the rat PR from uterine cDNA and used this as a probe to isolate a larger cDNA from a rat placental cDNA library. We used RNA filter hybridization, a quantitative reverse transcription-polymerase chain reaction amplification assay, and in situ hybridization to detect PR mRNA in the rat ovary. Expression of the PR gene was initially studied in an immature animal model; 23-day-old rats were treated with either PMSG or PMSG followed by hCG. We found little or no PR mRNA in the ovaries of control or PMSG-treated animals; however, the mRNA was highly expressed in the granulosa cells of large follicles in the ovaries of animals treated with PMSG followed by hCG. Other cell types, including thecal and interstitial cells, did not express detectable levels of PR mRNA. The PR mRNA was induced more than 20-fold in the immature ovary 5 h after hCG administration and was down-regulated to near-basal levels by 12 h after hCG administration. In a subsequent series of experiments, we examined PR gene expression in adult rats during the estrous cycle. The expression of PR mRNA was transient and was tightly coupled to the preovulatory LH surge on proestrous evening. PR mRNA was localized to the granulosa cells of mature ovarian follicles during the estrous cycle. In cycling animals treated with pentobarbital to block the preovulatory LH surge, no induction of PR mRNA on proestrous evening was observed. This transient, hormonally regulated, and cell-specific expression of the PR gene in the rat ovary strongly suggests an important intraovarian function for progesterone during the rat reproductive cycle.

Amino Acid Sequence↗

PubMed1994

Coupling of the alpha 2A-adrenergic receptor to multiple G-proteins. A simple approach for estimating receptor-G-protein coupling efficiency in a transient expression system.

It is now widely appreciated that G-protein-coupled cell-surface receptors can modulate distinct signal transduction pathways via coupling to different GTP-binding proteins. In the present study, we have used a transient co-expression approach to study the coupling of a single alpha 2-adrenergic receptor (alpha 2AAR) population to three different G protein subtypes (Gi, Gq, and Gs) acting on two different cellular effectors in HEK 293 cells. In all cases, the affinity of the receptor for the alpha 2A-adrenergic agonist, UK14304, is unchanged (KD approximately equal to 670 nM). However, there is a dramatic difference in the EC50 of UK14304 in eliciting inhibition of endogenous adenylyl cyclase via endogenous Gi (0.09 nM) versus activation of phospholipase C via co-transfected Gq (50 nM) or stimulation of endogenous adenylyl cyclase via co-transfected Gs (70 nM) in HEK 293 cells. These findings are consistent with the interpretations that the alpha 2AAR preferentially interacts with Gi rather than Gs or Gq. When the alpha 2AAR was mutated at Asp79, a residue highly conserved among G-protein-coupled receptors, the mutant D79N alpha 2AAR lost the ability to couple to Gq and Gs and, although it was able to couple to inhibition of cyclase via pertussis toxin-sensitive pathways (Gi), it did so with a lower potency than observed for the wild-type alpha 2AAR (EC50 = 7.2 nM). The most straightforward interpretation of these data is that the D79N mutation in the alpha 2AAR reduces the efficiency of coupling of the alpha 2AAR to all G-proteins, thus eliminating signal transduction through those pathways less efficiently coupled to the alpha 2AAR. Since the transient expression assays described permit manipulation of the structure of both the receptor or the G-protein, the present strategies could be exploited to delineate the complementary domains specifying the affinity and/or efficacy of receptor coupling to distinct GTP-binding proteins.

Adenylyl Cyclase Inhibitors↗

PubMed2002

Transient expression of Ym1, a heparin-binding lectin, during developmental hematopoiesis and inflammation.

Ym1, a secretory protein transiently produced by activated peritoneal macrophages elicited by parasitic infections, has been identified as a novel heparin-binding lectin. X-ray crystallography study revealed that Ym1 has a beta/alpha barrel structure with a carbohydrate-binding cleft similar to that of triose-phosphate isomerases. To further delineate the physiological significance of Ym1, we examined its expression patterns during mouse embryonic development and inflammation states elicited by agents other than parasitic infections in the peritoneal cavity and brain. This is the first report revealing prominent expression of Ym1 in early myeloid precursor cells of hematopoietic tissues-initially in the yolk sac and subsequently in fetal liver, spleen, and bone marrow. In nonhematopoietic systems, Ym1 was not detected in most of the tissues examined, with the exception of lung. Although no expression was detected up to gestation day 16.5 (E16.5), an increasing level of Ym1 expression in lung was detected from E18.5 on and persisted through adulthood. While most resident macrophages in various tissues examined are Ym1-negative, transient expression of Ym1 may be induced in their activated counterparts during inflammation in response to different stimuli in vivo, ranging from various chemical agents to brain injuries. The temporal and spatial expression in myeloid precursors and its transient induction in activated macrophages support the notion that Ym1 may be involved in hematopoiesis and inflammation. In addition, its putative functional association with heparin/heparan sulfate is discussed.

Animals↗

PubMed1997

The revised 8307 base pair coding sequence of human thyroglobulin transiently expressed in eukaryotic cells.

We developed a transient transfection system for human thyroglobulin (TG) cDNA in both human thyroid cells and in COS-1 cells. Four overlapping TG cDNA fragments were amplified by reverse transcription-PCR from RNA of normal thyroid tissue. The most 5' fragment includes the natural translation initiation site and the sequence encoding the signal peptide (SP). After subcloning, the nucleotide sequence was determined and compared with the published human sequence, resulting in the detection of 30 nucleotide variations. For validation purposes, all variations were screened in 6-12 normal human alleles. Twenty-one were present in all screened alleles and have to be revised in the published nucleotide sequence. Since one variation concerns a triplet insertion, the coding sequence of the mature human thyroglobulin is 8307 nucleotides encoding 2750 amino acids. The TG cDNA constructs were transiently transfected in HTori 3 and COS-1 cells and protein expression was detected using a polyclonal anti-human-TG on fixed cells and after SDS-PAGE. In both cell-lines all four TG protein fragments were expressed. The mannose structures detected on the proteins by lectins and localization after expression in the cells suggest that only the N-terminal TG fragment (containing the SP) is directed to the endoplasmatic reticulum but is unable to reach the Golgi complex. The described expression system in human thyrocytes will be a helpful tool in studying the structure-function relationship of human TG in thyroid hormonogenesis.

Alleles↗

PubMed1994

Transient expression of a Drosophila melanogaster hsp70 promoter/lacZ construct injected into larvae of two species of predatory mites (Acari: Phytoseiidae).

The Drosophila melanogaster heat shock 70 promoter (hsp70) was used to regulate expression of the Escherichia coli beta-galactosidase gene (lacZ) in transiently-transformed predatory mite larvae. A construct containing the hsp70 promoter upstream of the D. melanogaster alcohol dehydrogenase (adh) translational start site and Escherichia coli lacZ gene fusion (adh/lacZ) was injected into larvae of Metaseiulus occidentalis and Amblyseius finlandicus. LacZ expression was compared to expression of a similar construct lacking any upstream regulatory sequence. Expression from the hsp70 promoter was strong and heat shock-dependent in both species. The Drosophila hsp70 promoter therefore appears useful for regulating expression of exogenous DNA in both phytoseiid species and may be broadly applicable in the Phytoseiidae. Furthermore, the lacZ gene is a useful gene for analysis of expression in both species. Larval microinjection provides a method of assessing transient expression and of examining native regulatory sequences in these two phytoseiids and will likely be useful in other phytoseiid mites with only minor modifications.

Animals↗

PubMed1998

Evidence for NOS-containing renal neuronal somata transiently expressing a catecholaminergic phenotype during development in the rat.

Transiently catecholaminergic cells (TC-cells) expressing tyrosine hydroxylase (TH) have been shown in a variety of tissues during embryonic life. To investigate the possible relationship of nitric oxide synthase (NOS)-containing renal neuronal somata (RNS) and the TC-cells, we examined serial 100 microm slices of whole kidneys for TH-immunofluorescence and NADPH-d histochemistry during prenatal and postnatal development. The number of TH-cells increased during the prenatal period, peaked at birth and were very rare by PD21. A subpopulation of TH-immunoreactive RNS displayed NADPH-d activity. By PD21 the TH-positive RNS had practically disappeared while the number of NADPH-d positive RNS was markedly increased. These results suggest that kidneys possess transient catecholaminergic cells which display NOS-activity and that NOS expression may be the end-point in the differentiation of the RNS.

Animals↗

PubMed2001

The transient expression of C-C chemokine receptor 8 in thymus identifies a thymocyte subset committed to become CD4+ single-positive T cells.

Developing T cells journey through the different thymic microenvironments while receiving signals that eventually will allow some of them to become mature naive T cells exported to the periphery. This maturation can be visualized by the phenotype of the developing cells. CCR8 is a ss-chemokine receptor preferentially expressed in the thymus. We have developed 8F4, an anti-mouse CCR8 mAb that is able to neutralize the ligand-induced activation of CCR8, and used it to characterize the CCR8 protein expression in the different thymocyte subsets. Taking into account the intrathymic lineage relationships, our data showed that CCR8 expression in thymus followed two transient waves along T cell maturation. The first one took place in CD4(-) CD8(-) double-negative thymocytes, which showed a low CCR8 expression, and the second wave occurred after TCR activation by the Ag-dependent positive selection in CD4(+) CD8(+) double-positive cells. From that maturation stage, CCR8 expression gradually increased as the CD4(+) cell differentiation proceeded, reaching a maximum at the CD4(+) CD8(-) single-positive stage. These CD4(+) cells expressing CCR8 were also CD69(high) CD62L(low) thymocytes, suggesting that they still needed to undergo some differentiation step before becoming functionally competent naive T cells ready to be exported from the thymus. Interestingly, no significant amounts of CCR8 protein were detectable in CD4(-) CD8(+) thymocytes. Our data showing a clear regulation of the CCR8 protein in thymus suggest a relevant role for CCR8 in this lymphoid organ, and identify CCR8 as a possible marker of thymocyte subsets recently committed to the CD4(+) lineage.

Animals↗

PubMed1991

Simultaneous transient expression assays of the trypanosomatid parasite Leishmania using beta-galactosidase and beta-glucuronidase as reporter enzymes.

We describe a transient transfection protocol for cultured Leishmania major promastigotes, utilizing Escherichia coli genes encoding beta-galactosidase and beta-glucuronidase inserted into an expression vector derived from the dihydrofolate reductase-thymidylate synthase locus. Less than 0.1 pg of either reporter enzyme can be detected with a simple fluorimetric assay, and transfection of 10 micrograms of either reporter construct yields activities at least 100-fold over background. Simultaneous introduction of both constructs showed that the activity of each reporter gene was unaffected by the presence of the other, allowing one reporter construct to serve as a control for experimental variability in test gene constructs containing the second reporter gene. These results show that it is feasible to apply transient expression assays to the identification of cis-acting elements of genes encoding nonabundant mRNAs in the genus Leishmania.

Animals↗

PubMed1989

Transient expression of the angiotensin II receptor: a rapid and functional analysis of a calcium-mobilizing seven-transmembrane domain receptor in COS-7 cells.

The mas oncogene/angiotensin II receptor was subcloned into a mammalian expression vector pCDM8 and used to transiently transfect monkey kidney derived COS-7 cells. As a result, the mas transfected COS-7 cells expressed a functional angiotensin II receptor capable of transducing an increase in intracellular Ca2+ following stimulation with angiotensin II. The angiotensin II stimulated changes in Ca2+ could be measured 24 hours after transfection in both a fluorimeter and a fluorescence activated cell sorter. These results describe a rapid method for the functional analysis of the 7-transmembrane domain receptor genes.

Angiotensin II↗

PubMed1996

Capsid proteins of cowpea mosaic virus transiently expressed in protoplasts form virus-like particles.

The coding regions for cowpea mosaic virus (CPMV) capsid proteins VP37 and VP23 were introduced separately into a transient plant expression vector containing an enhanced CaMV 358 promoter. Significant expression of either capsid protein was observed only in protoplasts transfected simultaneously with both constructs. Immunosorbent electron microscopy revealed the presence of virus-like particles in extracts of these protoplasts. An extract of protoplasts transfected with both constructs together with RNA-1 was able to initiate a new infection, showing that the two capsid proteins of CPMV can form functional particles containing RNA-1 and that the 60-kDa capsid precursor is not essential for this process.

Capsid↗

PubMed2003

Neutral amino acid transporter ASCT1 is preferentially expressed in L-Ser-synthetic/storing glial cells in the mouse brain with transient expression in developing capillaries.

Nonessential amino acid L-Ser plays an essential role in neuronal survival and differentiation, through preferential expression of the L-Ser biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH), in particular in glial cells but not in neurons. To seek the molecular candidates responsible for glia-borne L-Ser transport, we performed histochemical analyses on amino acid transporter ASCT1, which prefers small neutral amino acids, such as Ala, Ser, Cys, and Thr, and mediates their obligatory exchange. At early developmental stages, neuroepithelial cells constituting the ventricular zone expressed ASCT1 mRNA and protein ubiquitously. Thereafter, ASCT1 expression was gradually downregulated in neuronal populations during the late embryonic and neonatal periods, whereas its high expression was transmitted to radial glial cells and then to astrocytes. High levels of ASCT1 were also detected in the olfactory ensheathing glia. The preferential glial expression of ASCT1 was consistent with that of 3PGDH, and their extensive colocalization was demonstrated at the cellular level. Moreover, high cellular contents of L-Ser were revealed in these glial cells by using a specific antibody to L-Ser. These results strongly suggest that a large amount of L-Ser is synthesized and stored in these glial cells and is released through ASCT1 in exchange for other extracellular substrates. In addition, we observed prominent expression of ASCT1 in capillary endothelial cells of embryonic and neonatal brains. Therefore, ASCT1 appears to be regulated to meet metabolic demands by differentiating and mature neurons through the transport of glia- and blood-borne small neutral amino acids.

Amino Acid Transport System ASC↗

PubMed2001

Electroporated intact BY-2 tobacco culture cells as a model of transient expression study.

Transfer of foreign genes into plant cells can be accomplished by several methods: agrobacterium-mediated, microinjection, biolistic particle bombardment and electroporation. The last one is frequently used for transfection of plant protoplasts for transient gene expression. Electroporation is a simple procedure and allows transfecting a large number of cells at one time. Square wave-modulated porators are the most efficient for introducing expression cassettes into plant protoplasts. Based on a protocol developed by Wu & Feng (Plant Cell Reports, 1999, 18, 381-386), we optimized conditions for transfection of intact Nicotiana tabacum BY-2 cells using square wave-modulated electroporator. To simplify screening for transfected gene expression we used constructs with a GFP marker gene.

Cell Line↗

PubMed2002

Transient expression of fluorescent tau proteins promotes process formation in PC12 cells: contributions of the tau C-terminus to this process.

The neuronal microtubule-associated protein tau promotes microtubule assembly and has been implicated in the development of axonal morphology. In this study, PC12 cells were transiently transfected with constructs coding fusion proteins of human tau with green fluorescent protein (GFP). Expression of tau constructs actively stabilized microtubules. Expression of the C-terminus of tau can mimic this effect in living cells, though to a lesser extent because of the absence of the tau N-terminus. However, tau colocalization with microtubules did not require the presence of the tau N-terminus. Transient expression of tau (including tau24, a four-repeat human tau isoform encoded in 383 residues, and tau23, human fetal tau isoform encoded in 352 residues) stimulated process formation in PC12 cells, and this occurred faster with tau24 than with tau23. The residues (residues 154-172 in tau23) that confer microtubule nucleation activity of tau in vitro are not required for tau-directed process formation. However, when tau induces the formation of cellular processes in response to cortical breakdown by cytochalasin B, residues 154-172 must be present. Thus, it appears that tau may serve to promote cellular process outgrowth in cultured neuronal cells and that C-terminus of tau is essential to this process.

Animals↗

PubMed1991

Transient expression of somatostatin messenger RNA and peptide in the hypoglossal nucleus of the neonatal rat.

The postnatal developmental expression of somatostatin mRNA and peptide in the rat hypoglossal nucleus was analyzed using immunocytochemical and in situ hybridization techniques. Both the neuropeptide and its cognate mRNA were found to be transiently present within a subpopulation of hypoglossal motoneurons during the neonatal period. At the day of birth, a large population of perikarya situated in caudal, ventral regions of the hypoglossal nucleus expressed somatostatin. By postnatal day 7, the number of hypoglossal somata which expressed somatostatin had diminished considerably, and by 2 weeks postnatal, only few such cell bodies were found. By 3-4 weeks postnatal, somatostatin peptide- and mRNA-containing hypoglossal motoneurons were rarely observed, and in the adult, they were never detected, despite the use of colchicine. A double-labeling co-localization technique was used to demonstrate that somatostatin, when present perinatally, always coexisted with calcitonin gene-related peptide in hypoglossal motoneurons. The latter peptide, in contrast to somatostatin, was expressed in large numbers of somata throughout the entire hypoglossal nucleus and persisted within the motoneurons throughout development into adulthood. These results demonstrate that somatostatin is transiently expressed in motoneurons of the caudal, ventral tier of the hypoglossal nucleus in the neonatal rat. The developmental disappearance of somatostatin is most likely not due to cell death; hypoglossal somata continue to express calcitonin gene-related peptide, with which somatostatin coexisted perinatally, a high levels throughout development. Thus, it appears that the regulation of somatostatin expression in hypoglossal neurons occurs at the level of gene transcription or mRNA stability/degradation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗

PubMed2001

Transient expression of a GUS reporter gene from cauliflower mosaic virus replacement vectors in the presence and absence of helper virus.

Vectors based upon the genome of cauliflower mosaic virus (CaMV) have only a limited capacity for replicating foreign DNA in plants. A helper virus system has been developed to complement CaMV constructs capable of carrying a large foreign gene (glucuronidase; GUS). GUS replaced part or all of the non-essential CaMV gene II and the essential genes III, IV and V. This construct was co-inoculated mechanically with wild-type CaMV helper virus onto Brassica rapa leaves to promote GUS vector complementation. After 1 week, blue foci of GUS activity were observed in the centres of the local lesions. Leaves inoculated with the GUS construct in the absence of helper virus showed randomly distributed foci of GUS activity that were generally smaller than the lesion-associated GUS foci. Inoculation with a simple non-replicating CaMV 35S promoter-GUS construct also produced small GUS foci. Co-inoculation of helper virus with CaMV gene replacement vectors in which replication was prevented by moving the primer-binding site or by deletion of an essential splice acceptor produced only small, randomly distributed GUS activity foci, demonstrating that the lesion-associated foci were produced by gene expression from replicating constructs. These experiments show that CaMV genes III-V can be complemented by wild-type virus and replacement gene vectors can be used for transient gene expression studies with CaMV constructs that distinguish gene expression associated with a replicating vector from that associated with a non-replicating vector.

Amino Acid Sequence↗

PubMed1995

Transient expression of luciferase in Entamoeba histolytica driven by the ferredoxin gene 5' and 3' regions.

We have successfully transfected Entamoeba histolytica trophozoites with constructs containing ferredoxin sequences fused to the reporter gene luciferase. We have determined the conditions and parameters necessary to maximise transient luciferase expression in our system. Our optimal construct gave values of 268 x 10(3) relative light units per second (RLU s-1) when assayed representing a stimulation of 18,000-fold over the control vector. Comparison of differing constructs allowed us to conclude that the 5' and 3' ferredoxin sequences are both necessary for optimal luciferase expression from our vectors. Transcriptional initiation occurs within the consensus sequence ATTCA in both construct and chromosomal ferredoxin promoters.

Animals↗

PubMed1999

Induction of the hair growth phase in postnatal mice by localized transient expression of Sonic hedgehog.

Hair follicles form in prenatal skin and mature in the postnatal period, establishing a growth cycle in 3 phases: telogen (resting), anagen (growth), and catagen (regression). Based on the knowledge that Sonic hedgehog (Shh) expression is necessary for the embryonic development of hair follicles, and that anagen in the postnatal cycling follicle has morphologic similarities to the epithelial invagination process in embryonic skin, we hypothesized that localized, but transient, enhanced expression of the Shh gene in postnatal skin would accelerate initiation of anagen in the hair follicle cycle, with concomitant accelerated hair growth. To assess this concept, an E1(-) adenovirus vector, AdShh, was used to transfer the murine Shh cDNA to skin of postnatal day 19 C57BL/6 mice. The treated skin showed increased mRNA expression of Shh, Patched (the Shh receptor), and Gli1 (a transcription factor in the Shh pathway). In mice receiving AdShh, but not in controls, acceleration into anagen was evident, since hair follicle size and melanogenesis increased and the hair-specific keratin ghHb-1 and the melanin synthesis-related tyrosinase mRNAs accumulated. Finally, C57BL/6 mice showed marked acceleration of the onset of new hair growth in the region of AdShh administration to skin 2 weeks after treatment, but not in control vector-treated or untreated areas. After 6 months, AdShh-treated skin showed normal hair and normal skin morphology. Together, these observations are consistent with the concept that upregulation of Shh activity in postnatal skin functions as a biologic switch that induces resting hair follicles to enter anagen with consequent hair growth.

Adenoviridae↗

PubMed1998

Dorsal root ganglion nerve cells transiently express increased immunoreactivity of the calcium-binding protein S-100beta after sciatic nerve transection.

Transiently increased immunoreactivity of the calcium binding protein S-100beta was demonstrated in spinal ganglion nerve cells after sciatic nerve transection. Neuropeptide Y (NPY), normally not seen in these nerve cells, appeared concomitantly. The transiently elevated co-expression of S-100beta and NPY is proposed to reflect an increased demand of neurotrophic and neuroprotective compounds in reactive neurons, tentatively regulating calcium ions.

Animals↗