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Results for “Targeted expression”

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PubMed2000

Target-specific expression of pre- and postsynaptic mechanisms.

Target-specific expression of pre- and postsynaptic mechanisms of synaptic transmission has been shown in a variety of central neurons by a number of laboratories. These data have demonstrated that synaptic transmission between single axons diverging onto distinct target neurons can behave independently, differentially influencing activity in the target neuron. Similarly, single neurons are capable of manufacturing molecularly distinct ligand-gated receptors and targeting them to synapses innervated by distinct converging afferent projections. A picture is emerging consistent with a role for both pre- and postsynaptic mechanisms in influencing the target-specific nature of transmission at numerous diverse synapses throughout the mammalian CNS. This target specificity adds another level of complexity in unravelling the roles played by individual neurons within a computational network. To begin to understand the coordinated activity of large ensembles of neurons it is becoming clear that the nature of transmission between individual pre- and postsynaptic elements within a circuit must first be understood for each and every neural element involved.

Animals↗

PubMed2003

Evidence for lack of enhanced hedgehog target gene expression in common extracutaneous tumors.

Abnormal hedgehog signaling, most commonly caused by loss of PTCH1 inhibitor activity,drives tumorigenesis of basal cell carcinomas (BCCs). To assess whether other tumors also have abnormal hedgehog signaling, we have assayed RNA from common cancers at nine different sites for levels of expression of hedgehog target genes that are up-regulated uniformly in BCCs. We report here that such dysregulation appears not to be common in the types of non-BCC cancers studied, indicating that the molecular pathogenesis of BCCs, like their frequency and behavior, differs markedly from that of most other cancers.

Adolescent↗

PubMed2001

Targeting transgene expression for cystic fibrosis gene therapy.

We have developed an expression cassette for cystic fibrosis (CF) gene therapy using control elements from the human cytokeratin 18 gene (KRT18, also known as K18). KRT18 is naturally expressed in a spatial pattern similar to that of CFTR, the gene mutated in CF. We delivered a KRT18-driven lacZ plasmid complexed with cationic liposomes intravenously to mice and examined expression in various tissues. We found expression in nasal and bronchial epithelium, airway submucosal glands, gall bladder, and kidneys. Expression was low in pancreas and gut, and absent from liver and alveolar lung. This is consistent with the expression pattern reported for a K18lacZ transgenic mouse. Following delivery of a cytomegalovirus (CMV) major immediate-early promoter/enhancer-driven lacZ plasmid, we found expression in bronchi, submucosal glands, alveolar cells, liver, and kidney. We did not detect expression in nose, pancreas, gall bladder, or gut. Using fluorescently labeled plasmid delivered by means of liposomes, we identified the liver, alveolar lung, and kidneys as the major plasmid deposition sites. Our data demonstrate that a KRT18-driven expression vector delivered systemically can target gene expression to CF-affected tissues, despite an uneven distribution of plasmid DNA. A KRT18-based vector may be a useful alternative to viral promoter-based vectors in clinical gene therapy trials to treat CF.

Animals↗

PubMed2002

Mks1 in concert with TOR signaling negatively regulates RTG target gene expression in S. cerevisiae.

The target of rapamycin (TOR) signaling pathway allows eukaryotic cells to regulate their growth in response to nutritional cues. In S. cerevisiae, TOR controls the expression of genes involved in several nutrient-responsive biosynthetic pathways. In particular, we have demonstrated that TOR negatively regulates a concise cluster of genes (termed RTG target genes) that encode mitochondrial and peroxisomal enzymes required for de novo amino acid biosynthesis. TOR acts in part by regulating the subcellular localization of the Rtg1/Rtg3 transcription factor complex. Nuclear entry of this complex requires the cytoplasmic protein Rtg2, whose precise function has remained ill defined. Here we establish that the likely role of Rtg2 is to antagonize the activity of another protein, Mks1, which we demonstrate is itself a negative regulator of RTG target gene activation. Results of epistasis analyses suggest that Rtg2 and Mks1 act downstream of TOR and upstream of Rtg1 and Rtg3. Moreover, we find that Mks1 phosphorylation responds to TOR as well as to each of the Rtg1-Rtg3 proteins, indicative of complex regulation within this branch of TOR signaling. In addition to RTG target genes, microarray analysis reveals robust expression of lysine biosynthetic genes in mks1Delta cells, which depends on a functional RTG pathway. This latter result provides a molecular explanation for the previous identification of MKS1 as LYS80, a negative regulator of lysine biosynthesis [8].

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed1993

Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.

Animals↗

PubMed1989

Rejection of skin allografts by CD4+ T cells is antigen-specific and requires expression of target alloantigen on Ia- epidermal cells.

The effector mechanism of skin allograft rejection has been characterized as Ag specific, rejecting cells that express the target alloantigen but sparing those that do not. However, the rejection of MHC class II disparate skin grafts, in which very few cells (Langerhans cells) actually express the target Ia Ag could conceivably proceed by either one of two distinct rejection mechanisms. One possibility is that Ia- cells are destroyed by a sequence of events in which CD4+ T cells, activated by Ia+ LC, elaborate soluble factors that are either directly cytolytic or that recruit and activate non-specific effector cells. The alternative possibility is that activated CD4+ T cells elaborate soluble factors which induce Ia expression on Ia- cell populations, and that these Ia+ cells are subsequently destroyed by effector cells specific for the induced Ia alloantigens. We found that rejection of Ia+ LC was not of itself sufficient to cause rejection of skin grafts, indicating that skin allograft rejection is contingent on the destruction not only of LC but of other graft cell populations as well. We then investigated whether CD4+ T cells rejected allogeneic skin grafts in an antigen specific fashion. To do so, we engrafted immunoincompetent H-2b nude mice with trunk skin grafts from B6----A/J allophenic mice because such skin is composed of mutually exclusive cell populations expressing either H-2a or H-2b histocompatibility Ag, but not both. The engrafted mice were subsequently reconstituted with H-2b CD4+ T cells. The CD4+ T cells destroyed keratinocytes of A/J origin but spared keratinocytes of B6 origin, even though neither cell population constitutively expresses target IAk alloantigen. The targeted rejection of A/J keratinocytes but not of B6 keratinocytes indicates that the target Ia alloantigen must have been induced on Ia- A/J keratinocytes, rendering them susceptible to destruction by anti-Iak-specific CD4+ effector cells. These data demonstrate that CD4+ T cell rejection of skin allografts is mediated by Ag-specific CD4+ cytolytic T cells and hence, requires the induction of target Ia alloantigens on epidermal cells within the graft.

Animals↗

PubMed1997

Effect of multiple, repeated doses of radioimmunotherapy on target antigen expression (breast MUC-1 mucin) in breast carcinomas.

The effect of radioimmunotherapy (RIT) on target antigen expression was studied in breast carcinomas transplanted in immunodeficient mice. In nine separate experiments, a single dose of 1500 microCi of 131I-labeled monoclonal antibody (MAb) Mc5 was given to groups of mice carrying well-established, vascularized, transplantable breast tumors (MX-1). Mc5 recognizes an epitope on the tandem repeat of the breast epithelial MUC-1 mucin. This dose suppressed tumor growth for at least 20 days, after which the tumors began to regrow. At various times thereafter, tumors were removed and analyzed for target antigen expression by flow cytometry and immunohistochemistry. In no case was there any significant decrease in antigen content/cell in the tumors of treated mice compared to tumors in control untreated mice. Similar results were obtained with four other breast carcinomas (MCF-7, MDA-MB-331, MDA-MB-435, and MX-2A). To assess the effect of repeated RIT doses on target antigen expression, groups of mice with MX-1 tumors were given 2, 3, and 4 consecutive doses of 1200 microCi of 131I-labeled Mc5. One mouse each at 2, 3, and 4 doses (3 of 18) was cured of its tumor. Control mice were sacrificed after 50 days due to the excessive size of their tumors. Tumors from four mice from each group (2, 3, and 4 doses), after they began to regrow, were excised and analyzed for mucin content and compared to tumors from untreated mice with similar-size tumors transplanted at later dates. In none of the treated groups was there any decrease in mucin content. These results demonstrate that RIT with an anti-breast mucin MAb does not result in the appearance of antigen-negative tumor cells, thus indicating that repeated fractionated doses, which will most likely be necessary for an eventual cure of breast cancer with MAb therapy, are possible.

Animals↗

PubMed1998

[The target--gene expression of interleukin--12 in HepG2 cells].

EB virus vector pEBAF, which contains human alpha-fetoprotein gene promoter and enhancer was used as expression vector. Two recombinant expression vectors, pEBAF/P35 and pEBAF/P40, were constructed and co-transfected into 4 cell lines. RT-PCR suggested that both P35 and P40 mRNA are expressed in HepG2 cell secreting alpha-fetoprotein, in contrast, neither P35 mRNA nor P40m RNA was expressed in non-secreting alpha-fetoprotein cell lines. This proved that the pEBAF may lead to target--expression of IL-12 in hepatoma and has tissue specificity and exclusiveness for hepatoma. The cotransfected Hyg resistant clone of HepG2 cell was selected. Northern blot indicated that the HepG2 cell clone had transcription of P35 mRNA and P40 mRNA. Expression of IL-12 in the supernatant of HepG2 cell culture had been testified by ELISA and Western blot. IL-12 biological activity test indicated that the expressed IL-12 had activity of inducing IFN-gamma production. This research laid the foundation to develop an in vivo gene therapy strategy pointing to the treatment of hepatocellular carcinoma, with selective killing of the tumor cells and no effect on the normal hepatic cells.

Carcinoma, Hepatocellular↗

PubMed1998

Targeting gene expression to the head: the Drosophila orthodenticle gene is a direct target of the Bicoid morphogen.

The Bicoid (Bcd) morphogen establishes the head and thorax of the Drosophila embryo. Bcd activates the transcription of identified target genes in the thoracic segments, but its mechanism of action in the head remains poorly understood. It has been proposed that Bcd directly activates the cephalic gap genes, which are the first zygotic genes to be expressed in the head primordium. It has also been suggested that the affinity of Bcd-binding sites in the promoters of Bcd target genes determines the posterior extent of their expression (the Gene X model). However, both these hypotheses remain untested. Here, we show that a small regulatory region upstream of the cephalic gap gene orthodenticle (otd) is sufficient to recapitulate early otd expression in the head primordium. This region contains two control elements, each capable of driving otd-like expression. The first element has consensus Bcd target sites that bind Bcd in vitro and are necessary for head-specific expression. As predicted by the Gene X model, this element has a relatively low affinity for Bcd. Surprisingly, the second regulatory element has no Bcd sites. Instead, it contains a repeated sequence motif similar to a regulatory element found in the promoters of otd-related genes in vertebrates. Our study is the first demonstration that a cephalic gap gene is directly regulated by Bcd. However, it also shows that zygotic gene expression can be targeted to the head primordium without direct Bcd regulation.

Animals↗

PubMed1998

Studying development of disease through temporally controlled gene expression in the salivary gland.

Multistep tumorigenesis proceeds through activation of oncogenes and inactivation of tumor suppressor genes. Initiating oncoproteins induce secondary changes that maintain transformation in the absence of original stimuli. Time-dependent reversal of SV40 T antigen (TAg)-induced hyperplasia was studied using temporally controlled gene expression. Targeting TAg expression to the submandibular salivary gland of transgenic mice produces focal hyperplasias at age two weeks, which extend through large areas of the gland by four months. At twelve months, fibrosis and tumor foci accompany hyperplasia. Hyperplasia reverses when TAg expression in discontinued at four months but not at seven months. Secondary changes that maintain transformation appear to be time dependent. The system can be used to identify genetic events resulting in phenotypic reversal at four months and to expose factors preventing its occurrence at seven months. Expression of other proteins can be targeted to the salivary gland, and temporally controlled gene deletions can also be made using this system.

Animals↗

PubMedDate not supplied

Multicomponent DNA carrier with a vesicular stomatitis virus G-peptide greatly enhances liver-targeted gene expression in mice.

Genes can be targeted to hepatocytes in vitro and in vivo by the use of asialoorosomucoid-polylysine conjugates. After systemic application, this nonviral vector is recognized by highly selective asialoglycoprotein (AsGP) receptors on the sinusoidal liver cell membrane and is taken up via receptor-mediated endocytosis. As most of the DNA is rapidly transferred to lysosomes where it is degraded, transfection efficiency is low and gene expression transient. To address this problem, we incorporated a pH-dependent synthetic hemolytic peptide derived of the G-protein of Vesicular Stomatitis Virus (VSV) into the gene transfer system, to increase endosomal escape of internalized DNA. The multicomponent carrier binds DNA in a nondamaging way, is still recognized by the AsGP receptor, and is targeted to the liver in vivo. Injection of DNA complexes containing a luciferase marker gene resulted in luciferase expression of 29 000 pg/g liver which corresponded to an increase of a factor of 10(3) overexpression after injection of DNA complexes without endosomolytic peptide. Furthermore, the amount of intact transgene within isolated liver cell nuclei was increased by a factor of 10(1)-10(2) by the use of the multicomponent carriers. These results demonstrate that incorporation of a hemolytic peptide into a nonviral vector can greatly increase gene expression while retaining cell type targetability in vivo.

Animals↗

PubMed1991

Correlation between CD8 dependency and determinant density using peptide-induced, Ld-restricted cytotoxic T lymphocytes.

We have taken advantage of some unique properties of H-2Ld to investigate the determinant density requirements for cytotoxic T lymphocyte (CTL) priming versus effector function and to correlate the determinant density requirements with CD8 dependency. In a previous study (Lie, W.-R., N. B. Myers, J. Gorka, R. J. Rubocki, J. M. Connolly, and T. H. Hansen. 1990. Nature [Lond.]. 344:439), we demonstrated that culturing normal cells with peptides known to be restricted by H-2Ld led to a two- to fourfold increase in surface Ld expression. In the present study, we demonstrate the generation of Ld-restricted, peptide-specific in vitro primary CTL by culturing spleen cells with murine cytomegalovirus or tum- peptide at concentrations previously shown to result in maximum induction of Ld expression. Target cells can be sensitized for recognition by these CTL with lower dose of peptide than are required for the primary sensitization. This demonstrates differences in the determinant density requirements for priming versus effector function. The in vitro primary CTL generated with peptide can weakly lyse target cells that express the determinant endogenously, and CTL lines and clones capable of strong lysis of endogenous expressors are easily obtained. In both cases, target cells treated with exogenous peptide are lysed better than target cells expressing antigen endogenously. This suggested that there are differences in the determinant density of peptide-fed versus endogenous targets. This interpretation was substantiated when it was observed that the level of lysis of target cells expressing endogenous determinants correlated inversely with the amount of peptide required to sensitize targets for recognition by various tum- -specific CTL clones. Furthermore, simultaneous titration of both the peptide used to treat target cells and the antibody to CD8 revealed that the various CTL clones analyzed displayed widely disparate CD8 dependencies. In each case, the CD8 dependency correlated inversely with the determinant density requirement. Therefore, CD8 dependency of CTL is relative, but shows an absolute and quantitative correlation with their dependency on determinant density. These findings suggest that under physiologic conditions, where only low determinant densities are likely to be encountered, all CTL clones will show at least partial CD8 dependency.

Amino Acid Sequence↗

PubMed1994

Multiple natural killer cell-activating signals are inhibited by major histocompatibility complex class I expression in target cells.

Several lines of evidence indicate that major histocompatibility complex class I molecules expressed by target cells can prevent natural killer cell (NK) lysis, possibly by engaging inhibitory receptors expressed by NK cells. On the other hand it is likely that NK cells must be activated to lysis by the recognition of unidentified NK target structures on target cells. To investigate the relationship between positive activation of NK cells by NK target structures versus inhibition by target cell class I molecules, we have examined various NK/target cell interactions for which the expression of inhibitory class I molecules by the target cells is known. The results suggests that specific properties of the target cell other than the absence of class I expression are necessary to activate NK-mediated lysis. Furthermore, different effector cell populations, i.e. freshly isolated versus interleukin-2 activated NK cells, differ in their capacity to kill class I-deficient lymphoblast target cells. In general, class I-deficient target cells that are resistant to direct lysis by a given NK population can be lysed by the NK cells when the reaction is mediated by antibody-dependent cellular cytotoxicity (ADCC). Most significantly, all types of NK-mediated lysis of lymphoblasts, of tumor cells and of almost any target by ADCC can be inhibited by appropriate class I gene expression in the target cell. These results suggest a model in which lysis by NK cells must be triggered by any one of a set of distinct target cell ligands, but that all of these signals can be overruled by class I-mediated inhibition.

Animals↗

PubMed2002

Investigating the site of human saccadic adaptation with express and targeting saccades.

To focus on various objects of interest within the visual environment, primates employ rapid eye movements called saccades. When the accuracy of these movements becomes impaired, the brain can adjust their amplitude by a process known as saccadic adaptation. To investigate the locus of this plasticity in the human brain, we behaviorally adapted two types of saccade thought to be generated through different neuronal pathways. Targeting saccades, which are made to sequentially illuminated targets and have long latencies, are thought to involve higher cortical processing whereas express saccades, which have very short latencies, apparently do not. If adaptation transfers between these two types of saccade, one may conclude that the plasticity must exist at a locus common to the two pathways generating these saccades. We directly reduced the gain of either targeting or express saccades by intrasaccadically moving the target one-third of its amplitude back toward the initial fixation location and then examined whether the gain was also reduced in the other type of saccade. When targeting saccades were adapted directly, all subjects showed significant reductions in the gain of these saccades. In 75% of the 32 experimental target conditions across all subjects, there were also significant reductions in the gain of express saccades, thus providing evidence of adaptation transfer. In 71% of these conditions (i.e., 53% of all target conditions) there was no significant difference between the reductions in gain of the two types of saccade, suggesting that adaptation transfer was complete (100%). Similar results were obtained when express saccades were adapted directly: significant reductions in gain occurred in 91% of express saccades and in 100% of targeting saccades. In 86% of the target conditions, across subjects, in which both express and targeting saccades showed significant reductions in gain, the two types of saccade did not differ significantly in the amount of gain reduction. This suggests that adaptation transfer was complete for 78% of all target conditions. Therefore, we conclude that saccadic adaptation transfers robustly between targeting and express saccades. These results suggest that adaptation in humans occurs after the pathways generating these two types of saccade converge, probably at or downstream from the superior colliculus.

Adaptation, Physiological↗

PubMed1982

Expression of target antigen for Epstein-Barr virus-specific cytotoxic T cells on BJAB cells freshly infected with EBV.

The target antigen for Epstein-Barr virus (EBV)-specific cytotoxic T cells (Tc) was expressed on BJAB cells exposed to the B95-8 strain of EBV for at least one hour. Ultraviolet-light (UV)-irradiated noninfectious B95-8 virus also induced the target antigen on BJAB cells. Cold target competition tests suggested that the target antigen expressed on EBV-infected BJAB cells was distinct from the lymphocyte-detected membrane antigen (LYDMA) which was also recognized by the EBV-specific Tc and expressed on autologous EBV-Transformed lymphoblastoid cell line (LCL) cells. Neither of these target antigens for EBV- specific Tc was detected on the surface of EBV-genome positive BJAB cells which had been kept in a long term culture after EBV-infection. Thus, the virion antigen, especially the EBV-membrane antigen (MA), is a possible candidate for the target antigen expressed on EBV-infected BJAB cells. Lysis of EBV-infected BJAB cells was inhibited by target cell treatment with anti-beta 2 microglobulin (anti-beta 2M) antibody and induction of the effector Tc was dependent on the donor individual. These results suggested the possibility that the Tc recognizing EBV-infected BJAB cells are restricted by the major histocompatibility complex (MHC).

Animals↗

PubMed1997

Targeting gene expression to hypoxic tumor cells.

Solid tumors with areas of low oxygen tension (hypoxia) have a poor prognosis, as cells in this environment often survive radiation and chemotherapy. In this report we describe how this hypoxic environment can be used to activate heterologous gene expression driven by a hypoxia-responsive element (HRE), which interacts with the transcriptional complex hypoxia-inducible factor-1 (HIF-1). Our results demonstrate that the HIF-1/HRE system of gene regulation is active in hypoxic tumor cells and show the potential of exploiting tumor-specific conditions for the targeted expression of diagnostic or therapeutic genes in cancer therapy.

Animals↗

PubMed1997

HIV-1 induces cytotoxic T lymphocytes in the cervix of infected women.

Although T lymphocytes are present in the genital mucosa, their function in sexually transmitted diseases is unproven. To determine if cervical T cells mediate HIV-specific cytolysis, mononuclear cells in cytobrush specimens from HIV-1-infected women were stimulated in vitro with antigen. Resultant cell lines lysed autologous targets expressing HIV-1 proteins in 12/19 (63%) subjects, and these responses were detected intermittently on repeated visits. All 8 subjects with blood CD4+ counts > or =500 cells/microl had HIV-1-specific cervical CTL, whereas only 4/11 with counts <500 cells/microl had detectable responses (P = 0.008). Class II MHC-restricted CD4+ CTL clones lysed targets expressing Env gp41 or infected with HIV-1. Class I MHC-restricted CD8+ clones recognized HIV-1 Gag- or Pol-expressing targets, and the epitopes were mapped to within 9-20 amino acids. Comparisons of intra-individual cervical and blood CTL specificities indicate that epitopes recognized by CTL in the cervix were commonly recognized in the blood. These studies provide the first definitive evidence for an MHC-restricted effector function in human cervical lymphocytes.

Cell Line, Transformed↗

PubMed1991

Binary system for regulating transgene expression in mice: targeting int-2 gene expression with yeast GAL4/UAS control elements.

We have developed a binary transgenic system that activates an otherwise silent transgene in the progeny of a simple genetic cross. The system consists of two types of transgenic mouse strains, targets and transactivators. A target strain bears a transgene controlled by yeast regulatory sequences (UAS) that respond only to the yeast transcriptional activator GAL4. A transactivator strain expresses an active GAL4 gene that can be driven by any selected promoter. The current paradigm uses the murine growth factor int-2 cDNA as the target gene and the GAL4 gene driven by the mouse mammary tumor virus long terminal repeat as the transactivator. Both target and transactivator strains are phenotypically normal. By contrast, the bigenic offspring of these two strains express high levels of the target int-2 gene in each organ expressing the GAL4 transactivator. They also display a characteristic dominant int-2 phenotype that consists of epithelial hyperplasia in mammary and salivary glands, as well as prostatic and epididymal hypertrophy, which results in male sterility.

Animals↗