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Results for “Targeted expression”

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PubMed2003

Targeted gene expression using the GAL4/UAS system in the silkworm Bombyx mori.

The silkworm Bombyx mori is one of the most well-studied insects in terms of both genetics and physiology and is recognized as the model lepidopteran insect. To develop an efficient system for analyzing gene function in the silkworm, we investigated the feasibility of using the GAL4/UAS system in conjunction with piggyBac vector-mediated germ-line transformation for targeted gene expression. To drive the GAL4 gene, we used two endogenous promoters that originated from the B. mori actin A3 (BmA3) and fibroin light-chain (FiL) genes and the artificial promoter 3xP3. GFP was used as the reporter. In initial tests of the function of the GAL4/UAS system, we generated transgenic animals that carried the UAS-GFP construct plus either BmA3-GAL4 or 3xP3-GAL4. GFP fluorescence was observed in the tissues of GFP-positive animals, in which both promoters drove GAL4 gene expression. Animals that possessed only the GAL4 gene or UAS-GFP construct did not show GFP fluorescence. In addition, as a further test of the ability of the GAL4/UAS system to drive tissue-specific expression we constructed FiL-GAL4 lines with 3xP3-CFP as the transformation marker. FiL-GAL4 x UAS-GFP crosses showed GFP expression in the posterior silk gland, in which the endogenous FiL gene is normally expressed. These results show that the GAL4/UAS system is applicable to B. mori and emphasize the potential of this system for controlled analyses of B. mori gene function.

Animals↗

PubMed1998

Functional role of CD95 ligand in concanavalin A-induced intestinal intraepithelial lymphocyte cytotoxicity.

Freshly isolated murine intestinal intraepithelial lymphocytes (IEL) express CD95 ligand (CD95L), as shown by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorter (FACS) analysis. Between 15 and 25% of IEL could be stained with an antibody to CD95L. Therefore it was investigated whether the CD95L/CD95 pathway was effective in IEL cytotoxicity. Stimulation of IEL in vitro with concanavalin A (Con A) induced a strong cytotoxic response, which was much higher when using CD95-expressing target cells. This effect was most evident when comparing the specific lysis of CD95-transfected target cells of the leukaemia cell line L1210 with that of the untransfected parental cell line. In addition, an antibody to CD95 was able to dramatically reduce the specific lysis of CD95-expressing target cells. After stimulation with Con A, which is able to bind to CD95L, the effects were more obvious compared with the triggering of the T-cell receptor (TCR)-alphabeta or gamma delta. On the other hand, EGTA reduced the Con A-induced cytotoxicity. Together these findings support a role of the CD95L/CD95 pathway in IEL cytotoxicity, even though the reaction was Ca2+ sensitive. As a function, CD95L-expressing IEL should be able to contribute to the elimination of CD95-expressing target cells in the intestine.

Animals↗

PubMed2007

Targeting SPARC expression decreases glioma cellular survival and invasion associated with reduced activities of FAK and ILK kinases.

Secreted protein acidic and rich in cysteine (SPARC) is an extracellular glycoprotein expressed in several solid cancers, including malignant gliomas, upon adoption of metastatic or invasive behaviors. SPARC expression in glioma cells promotes invasion and survival under stress, the latter process dependent on SPARC activation of AKT. Here we demonstrate that downregulation of SPARC expression with short interfering RNA (siRNA) in glioma cells decreased tumor cell survival and invasion. SPARC siRNA reduced the activating phosphorylation of AKT and two cytoplasmic kinases, focal adhesion kinase (FAK) and integrin-linked kinase (ILK). We determined the contributions of FAK and ILK to SPARC effects using SPARC protein and cell lines engineered to overexpress SPARC. SPARC activated FAK and ILK in glioma cells previously characterized as responsive to SPARC. Downregulation of either FAK or ILK expression inhibited SPARC-mediated AKT phosphorylation, and targeting both FAK and ILK attenuated AKT activation more potently than targeting either FAK or ILK alone. Decreased SPARC-mediated AKT activation correlated with a reduction in SPARC-dependent invasion and survival upon the downregulation of FAK and/or ILK expression. These data further demonstrate the role of SPARC in glioma tumor progression through the activation of intracellular kinases that may provide novel therapeutic targets for advanced cancers.

Brain Neoplasms↗

PubMed1996

A mammalian patched homolog is expressed in target tissues of sonic hedgehog and maps to a region associated with developmental abnormalities.

Drosophila patched is a segment polarity gene required for the correct patterning of larval segments and imaginal discs during fly development and has a close functional relationship with hedgehog. We have isolated a complete human PATCHED cDNA sequence, which encodes a putative protein of 1296 amino acids, and displays 39% identity and 60% similarity to the Drosophila PATCHED protein. Hydropathy analysis suggests that human PATCHED is an integral membrane protein with a pattern of hydrophobic and hydrophilic stretches nearly identical to that of Drosophila patched. In the developing mouse embryo, patched is initially detected within the ventral neural tube and later in the somites and limb buds. Expression in the limb buds is restricted to the posterior ectoderm surrounding the zone of polarizing activity. The results show that patched is expressed in target tissues of sonic hedgehog, a murine homolog of Drosophila hedgehog suggesting that patched/hedgehog interactions have been conserved during evolution. Human PATCHED maps to human chromosome 9q22.3, the candidate region for the nevoid basal cell carcinoma syndrome. Patched expression is compatible with the congenital defects observed in the nevoid basal cell carcinoma syndrome.

Amino Acid Sequence↗

PubMed2002

Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression.

The TCL1 gene at 14q32.1 is involved in chromosomal translocations and inversions in mature T cell leukemias. These leukemias are classified either as T prolymphocytic leukemias, which occur very late in life, or as T chronic lymphocytic leukemias, which often arise in patients with ataxia telangiectasia (AT) at a young age. In transgenic animals, the deregulated expression of TCL1 leads to mature T cell leukemia, demonstrating the role of TCL1 in the initiation of malignant transformation in T cell neoplasia. Expression of high levels of Tcl1 have also been found in a variety of human tumor-derived B cell lines ranging from pre-B cell to mature B cell. Here we describe the phenotype of transgenic mice, E mu-TCL1, established with TCL1 under the control of a V(H) promoter-Ig(H)-E mu enhancer to target TCL1 expression to immature and mature B cells. Flow cytometric analysis reveals a markedly expanded CD5(+) population in the peritoneal cavity of E mu-TCL1 mice starting at 2 mo of age that becomes evident in the spleen by 3-5 mo and in the bone marrow by 5-8 mo. Analysis of Ig gene rearrangements indicates monoclonality or oligoclonality in these populations, suggesting a preneoplastic expansion of CD5(+) B cell clones, with the elder mice eventually developing a chronic lymphocytic leukemia (CLL)-like disorder resembling human B-CLL. Our findings provide an animal model for CLL, the most common human leukemia, and demonstrate that deregulation of the Tcl1 pathway plays a crucial role in CLL pathogenesis.

Aging↗

PubMed2003

Cyclic nucleotide phosphodiesterase activity, expression, and targeting in cells of the cardiovascular system.

Cyclic AMP (cAMP) and cGMP regulate a myriad of cellular functions, such as metabolism, contractility, motility, and transcription in virtually all cell types, including those of the cardiovascular system. Considerable effort over the last 20 years has allowed identification of the cellular components involved in the synthesis of cyclic nucleotides, as well as effectors of cyclic nucleotide-mediated signaling. More recently, a central role for cyclic nucleotide phosphodiesterase (PDE) has also been elaborated in many cell types, including those involved in regulating the activities of the cardiovascular system. In this review, we introduce the PDE families whose members are expressed in cells of the cardiovascular system including cardiomyocytes, vascular smooth muscle cells, and vascular endothelial cells. Because cell behavior is a dynamic process influenced by numerous factors, we will attempt to emphasize how changes in the activity, expression, and targeting of PDE influence cyclic nucleotide-mediated regulation of the behavior of these cells.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed2001

Epidermal Langerhans cell-targeted gene expression by a dectin-2 promoter.

Despite their critical function as APCs for primary immune responses, dendritic cells (DC) and Langerhans cells (LC) have been rarely used as targets of gene-based manipulation because well-defined regulatory elements controlling LC/DC-specific expression have not been identified. Previously, we identified dectin-2, a C-type lectin receptor expressed selectively by LC-like XS cell lines and by LC within mouse epidermis. Because these characteristics raised the possibility that dectin-2 promoter may direct LC/DC-specific gene expression, we isolated a 3.2-kb nucleotide fragment from the 5'-flanking region of the dectin-2 gene (Dec2FR) and characterized its regulatory elements and the transcriptional activity using a luciferase (Luc) reporter system. The Dec2FR contains a putative TATA box and cis-acting elements, such as the IFN-stimulated response element, that drive gene expression specifically in XS cells. Dec2FR comprises repressor, enhancer, and promoter regions, and the latter two regions coregulate XS cell-specific gene expression. In transgenic mice bearing a Dec2FR-regulated Luc gene, the skin was the predominant site of Luc activity and LC were the exclusive source of such activity within epidermis. By contrast, other APCs (DC, macrophages, and B cells) and T cells expressed Luc activity close to background levels. We conclude that epidermal LC are targeted selectively for high-level constitutive gene expression by Dec2FR in vitro and in vivo. Our findings lay the foundation for use of the dectin-2 promoter in LC-targeted gene expression systems that may enhance vaccination efficacy and regulate immune responses.

5' Flanking Region↗

PubMed2005

Peroxisome proliferator-activated receptor gamma and its ligands in controlling interleukin-1beta target gene expression: a confusing story.

Antiinflammatory effects by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists have been previously reported. However, PPARgamma dependency and the molecular mechanism involved in these effects require more investigation to clearly demonstrate whether PPARgamma is a key modulator of the antiinflammatory process. This would permit the design of more specific agonists or antagonists able to address the gamma subtype without cross reactions with other transcription factors, thus preventing undesirable side effects. However, several hurdles need to be taken into consideration, such as the coexpression of several PPAR isotypes in the same cell type. As PPARgamma and -alpha seem to play equal antiinflammatory roles, determining the subset of specific PPAR subtype target genes appears to be crucial. The work described here is our current understanding of the modulations of interleukin-1 target gene expression by PPARgamma and its ligands.

Animals↗

PubMed1997

Ligand-inducible and liver-specific target gene expression in transgenic mice.

Transgenic mice have been used as models for tissue-specific gene regulation and to examine the molecular and cellular effects of altered expression of specific gene in disease processes such as tumorigenesis. Because of the deleterious effects of constitutive expression of transgenes, which frequently result in prenatal or postnatal death, only a limited number of disease models have been established in transgenic mice. We report an inducible binary transactivation system that permits the control of transgene expression in a tissue-specific and inducible fashion in mice. In this system, transcription of the target transgene is kept silent until turned on by the administration of an exogenous compound. We also demonstrate that expression level of the target gene can be induced three to four orders of magnitude and can be controlled by the administrated compound in a dose-dependent manner.

Animals↗

PubMed2004

Genome-wide analysis reveals inositol, not choline, as the major effector of Ino2p-Ino4p and unfolded protein response target gene expression in yeast.

In the yeast Saccharomyces cerevisiae, the transcription of many genes encoding enzymes of phospholipid biosynthesis are repressed in cells grown in the presence of the phospholipid precursors inositol and choline. A genome-wide approach using cDNA microarray technology was used to profile the changes in the expression of all genes in yeast that respond to the exogenous presence of inositol and choline. We report that the global response to inositol is completely distinct from the effect of choline. Whereas the effect of inositol on gene expression was primarily repressing, the effect of choline on gene expression was activating. Moreover, the combination of inositol and choline increased the number of repressed genes compared with inositol alone and enhanced the repression levels of a subset of genes that responded to inositol. In all, 110 genes were repressed in the presence of inositol and choline. Two distinct sets of genes exhibited differential expression in response to inositol or the combination of inositol and choline in wild-type cells. One set of genes contained the UASINO sequence and were bound by Ino2p and Ino4p. Many of these genes were also negatively regulated by OPI1, suggesting a common regulatory mechanism for Ino2p, Ino4p, and Opi1p. Another nonoverlapping set of genes was coregulated by the unfolded protein response pathway, an ER-localized stress response pathway, but was not dependent on OPI1 and did not show further repression when choline was present together with inositol. These results suggest that inositol is the major effector of target gene expression, whereas choline plays a minor role.

Basic Helix-Loop-Helix Proteins↗

PubMed1994

Incorporation of adenovirus into a ligand-based DNA carrier system results in retention of original receptor specificity and enhances targeted gene expression.

Adenovirus type 5 was modified by coupling an asialoglycoprotein-polylysine conjugate to the virus by reactions that activate carbohydrate residues. Wild-type virus modified in this manner had greatly decreased infectivity toward normally susceptible HeLa S3 (asialoglycoprotein receptor (-)) and SK Hep1 (asialoglycoprotein receptor (-)) cells leaving 91 and 86% viable, respectively, after 48 h. However, with Huh 7 (asialoglycoprotein receptor (+)) cells, modified virus retained its infectivity leaving only 19% of cells viable under identical conditions. Modified virus was complexed to DNA in the form of a plasmid, pSVHBV surf, containing the gene for hepatitis B surface antigen as a marker of gene expression. Huh 7, receptor (+), cells treated with modified wild type, and modified replication-defective d1312 virus complexed to DNA raised antigen levels by approximately 13- and 30-fold, respectively, compared with asialoglycoprotein-polylysine DNA complex alone. Competition with a large excess of an asialoglycoprotein blocked the enhancement by more than 95%. Using a beta-galactosidase marker gene, the number of cells transfected by modified virus was found to be 200-fold higher than complex alone. Yet, specificity was retained exclusively for asialoglycoprotein receptor-bearing cells. These data indicate that adenovirus can be chemically modified by coupling ligands resulting in targeted gene expression dictated specifically by receptor recognition of the attached ligand.

Adenoviruses, Human↗

PubMed2000

Plasmid DNA adsorbed onto cationic microparticles mediates target gene expression and antigen presentation by dendritic cells.

Dendritic cells (DC) play a key role in antigen presentation and activation of specific immunity. Much current research focuses on harnessing the potency of DC for vaccines, gene therapy, and cancer immunotherapy applications. However, DC are not readily transfected in vitro by traditional nonviral techniques. A novel DNA vaccine formulation was used to determine if DC are transfected in vitro. The formulation consists of plasmid DNA adsorbed on to cationic microparticles composed of the biodegradable polymer polylactide-co-glycolide (PLG) and the cationic surfactant, cetyltrimethylammonium bromide (CTAB). Using preparations of fluorescent-labeled plasmid DNA formulated on PLG-CTAB microparticles to study internalization by macrophages and dendritic cells in vitro and in vivo, we found that most, but not all, of the fluorescence was concentrated in endosomal compartments. Furthermore, uptake of plasmid DNA encoding HIV p55 gag adsorbed to PLG-CTAB microparticles by murine bone marrow-derived dendritic cells resulted in target gene expression, as detected by RT-PCR. The antigen was subsequently processed and presented, resulting in stimulation of an H-2kd-restricted, gag-specific T cell hybridoma. Activation of the hybridoma, detected by IL-2 production, was dose-dependent in the range of 0.1-20 microg DNA (10-2000 microg PLG) and was sustained up to 5 days after transfection. Thus, adsorption of plasmid DNA on PLG-CTAB microparticles provides a potentially useful nonviral approach for in vitro transfection of dendritic cells. Gene Therapy (2000) 7, 2105-2112.

Adsorption↗

PubMed1999

A novel gene delivery system targeting cells expressing VEGF receptors.

Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors. GV1, GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex. Using pSV2-beta-galactosidase as a reporter gene, it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) and human malignant melanoma cell lines (A375) in vitro. In vivo experiments, exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO, human malignant melanoma A375 and human hepatoma graft in nude mice. This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice. These results are correlated with the relevant receptors (flt-1, flk-1/KDR) expression on the targeted cells and tissues.

Amino Acid Sequence↗

PubMed2003

Antiviral immune responses in gene-targeted mice expressing the immunoglobulin heavy chain of virus-neutralizing antibodies.

Two gene-targeted immunoglobulin heavy chain transgenic mouse strains, TgH(KL25) and TgH(VI10), expressing neutralizing specificities for lymphocytic choriomeningitis virus and vesicular stomatitis virus, respectively, have been generated. Three days after lymphocytic choriomeningitis virus infection, TgH(KL25) mice showed a thymus-independent neutralizing IgM response followed by thymus-dependent (TD) IgG. In contrast, WT mice mounted only a TD IgG response around day 80. These observations indicated that not only structural properties of the virus but also immunological parameters such as the frequency of B cells were indicative for the induction of thymus-independent versus TD Ig responses. Naïve vesicular stomatitis virusspecific Ig heavy chain transgenic mice displayed greatly elevated natural antibody titers. However, despite these high naïve titers, de novo activation of naïve CD4+ T and B cells was not blocked. Therefore, B cells giving rise to natural antibodies do not participate in virus-induced antibody responses.

Animals↗

PubMed1999

Interleukin-10 reduces natural killer (NK) sensitivity of tumor cells by downregulating NK target structure expression.

We examined the effect of exogenous IL-10 on the sensitivity of rat W14 and W31 tumor cells to natural killer (NK) cell-mediated cytotoxicity in relation to previously identified NK target structure (NKTS) expressed on these cells. We also examined the effect of endogenous interleukin-10 (IL-10) on rat IL-10 cDNA-introduced W31 cells (W31T-H, a high-IL-10-producer clone; W31T-L, a low-IL-10-producer clone). Both exogenous and endogenous IL-10 had no effect on the proliferative activity of these cells, but incubation of cells with recombinant human (rh) IL-10 resulted in a dose-dependent decrease in the expression of NKTS recognized by mAb 109. The expression level of NKTS on W31T-H cells was dramatically decreased compared with that on W31T-L cells and parental W31 cells. In addition, treatment of W31 cells with the culture supernatants of W31T-H cells could downregulate the expression of the NKTS. Moreover, NK sensitivity of W31T-H was suppressed down to a level equivalent to that of W31 cells pretreated with exogenous rhIL-10, and cytolysis could no longer be inhibited by mAb 109. We previously demonstrated that IL-10 downregulated MHC class I expression in this model. Nevertheless, NK susceptibility was also decreased. Taken together, these results suggest that the IL-10-mediated decrease in NKTS expression has a larger effect than decreased MHC class I expression on NK sensitivity. Thus, our data reveal a novel mechanism for an IL-10-mediated escape of tumor cells from host immune surveillance by downregulation of NKTS expression.

Animals↗

PubMed2004

Defining strategies to extend duration of gene expression from targeted compacted DNA vectors.

Gene transfer complexes containing poly-L-lysine (poly-K) and DNA with ligands directed at the serpin enzyme complex receptor (sec-R) deliver reporter genes to receptor-bearing cells in vivo. Expression lasts for about 30 days, when complexes containing long-chain poly-K are used. Extending the duration of expression would be desirable if correction of genetic defects is the goal. To test whether the mechanism by which expression is extinguished was due to an immune response to the transgene, or the loss of the transgene, we conducted two experiments. In the first, we injected sec-R-targeted lacZ complexes intravenously (i.v.) into mice genetically engineered to express this gene briefly during development. These mice, who should recognize the protein as 'self', also extinguished lacZ expression after 30 days. In a second experiment, we injected immunodeficient animals with sec-R-targeted human factor IX complexes. A similar temporal pattern of expression was observed in Rag-1 -/- mice, in whom expression also extinguished by 40 days. Moreover, factor IX plasmid DNA was detected in the lung and spleen 50 days after injection of complexes, suggesting that not all cells which had taken up the transgene had been destroyed. Thus, the host's immune response to the transgene may not account for the loss of reporter gene expression from these molecular conjugates. We further tested whether repeat administration of sec-R-targeted complexes will be limited by host immune responses. Mice were pre-dosed twice with sec-R-targeted complexes containing lacZ over a 40-day period. We then injected the animals i.v. with sec-R-targeted human factor IX complexes and measured gene expression and antibody production. Although 14 of 36 animals displayed low-titer antibodies to the ligand in targeted complex, expression levels were unaffected compared with virgin dosing. When the complexes were administered three times intranasally (n=10), no antibodies against the complex were detected in blood. Plasma from mice dosed with saline, nontargeted complex or naked DNA did not react with the ligand, ligand-poly K conjugate or targeted complex. All animals exhibiting human factor IX expression developed antibodies to that transgene by 21 days. Thus, at least three repeat administrations of sec-R-directed molecular conjugates are possible, provided that immune responses to the transgene itself are not limiting.

Animals↗

PubMed2003

Conditionally replicative adenovirus expressing a targeting adapter molecule exhibits enhanced oncolytic potency on CAR-deficient tumors.

Conditionally replicative adenoviruses (CRAds) are potentially useful agents for anticancer virotherapy approaches. However, lack of coxsackievirus and adenovirus receptor (CAR) expression on many primary tumor cells limits the oncolytic potency of CRAds. This makes the concept of targeting, that is, redirecting infection via CAR-independent entry pathways, relevant for CRAd development. Bispecific adapter molecules constitute highly versatile means for adenovirus targeting. Here, we constructed a CRAd with the Delta24 E1A mutation that produces a bispecific single-chain antibody directed towards the adenovirus fiber knob and the epidermal growth factor receptor (EGFR). This EGFR-targeted CRAd exhibited increased infection efficiency and oncolytic replication on CAR-deficient cancer cells and augmented lateral spread in CAR-deficient 3-D tumor spheroids in vitro. When compared to its parent control with native tropism, the new CRAd exhibited similar cytotoxicity on CAR-positive cancer cells, but up to 1000-fold enhanced oncolytic potency on CAR-deficient, EGFR-positive cancer cells. In addition, EGFR-targeted CRAd killed primary human CAR-deficient brain tumor specimens that were refractory to the parent control virus. We conclude, therefore, that CRAds expressing bispecific targeting adapter molecules are promising agents for cancer treatment. Their use is likely to result in enhanced oncolytic replication in cancerous tissues and thus in more effective tumor regression.

Adenovirus E1A Proteins↗

PubMed1996

Targeting gene expression to the wool follicle in transgenic sheep.

To establish the feasibility of overexpressing foreign genes in the wool follicle, transgenic sheep were produced by pronuclear microinjection of a DNA construct consisting of a mouse ultrahigh-sulfur keratin promoter linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Four of 31 lambs born were transgenic. The overall efficiency of transgenesis was 1.1% of zygotes injected and transferred. Two transgenic rams were mated to nontransgenic ewes, and both transmitted the gene to their offspring in Mendelian fashion. CAT expression was found in the skin of one G0 ram and in 9 out of 26 transgenic G1 progeny. Two G1 lambs were sacrificed to study tissue specificity. Both had high levels of expression in skin but One had high expression in spleen and kidney with lower levels of expression in lung; the other had low expression in spleen, lung, and muscle. In situ hybridization demonstrated that transgene expression in the skin was confined to the keratogenous zone of the wool follicle cortex. Expression of CAT activity in skin was correlated with diet-induced or seasonal changes in the rate of wool growth. This keratin promoter appears useful for overexpressing factors in the wool follicle that might influence wool production or properties.

Animals↗