Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Gene Targeting”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,009 records · Page 56Linked to original sources
PubMed2002

Peroxisome proliferator-activated receptor alpha (PPARalpha ) turnover by the ubiquitin-proteasome system controls the ligand-induced expression level of its target genes.

Peroxisome proliferator activated-receptor alpha (PPARalpha) is a ligand-activated transcription factor belonging to the nuclear receptor family. PPARalpha is implicated in the regulation of lipid and glucose metabolism and in the control of inflammatory response. Recently, it has been demonstrated that a number of nuclear receptors are degraded by the ubiquitin-proteasome pathway. Since PPARalpha exhibits a circadian expression rhythm and since PPARalpha is rapidly regulated under certain pathophysiological conditions such as the acute phase inflammatory response, we hypothesized that PPARalpha protein levels must be under tight control. Here, we studied the mechanisms controlling PPARalpha protein levels and their consequences on the transcriptional control of PPARalpha target genes. Using pulse-chase experiments, it is shown that PPARalpha is a short-lived protein and that addition of its ligands stabilizes this nuclear receptor. By transient cotransfection experiments using expression vectors for PPARalpha and hemagglutinin-tagged ubiquitin, it is demonstrated that PPARalpha protein is ubiquitinated and that its ligands decrease the ubiquitination of this nuclear receptor, thus providing a mechanism for the ligand-dependent stabilization observed in pulse-chase experiments. In addition, treatment with MG132, a selective proteasome inhibitor, increases the level of ubiquitinated PPARalpha and inhibits its degradation in transfected cells. Furthermore, MG132 treatment enhances the level of endogenous PPARalpha in HepG2 cells. Finally, transient transfection and quantitative reverse transcription-PCR show that inhibition of PPARalpha degradation increases its transcriptional activation and expression of target genes such as apoA-II and fatty acid transport protein (FATP). Taken together, these data demonstrate that PPARalpha is degraded by the ubiquitin-proteasome system in a ligand-dependent manner. Regulation of its degradation provides a novel regulatory mechanism of transcriptional activity of this nuclear receptor.

Animals↗
PubMed2001

Maf and Jun nuclear oncoproteins share downstream target genes for inducing cell transformation.

The Maf oncoprotein is a basic leucine zipper (bZip)-bearing transcriptional activator that recognizes the Maf recognition element (MARE) DNA sequence. In this study, we investigated the role of Maf's transactivation function in cell transformation. Replacement of the conserved amino terminus transactivator domain of Maf by a heterologous and stronger transactivator domain (the acidic transactivator domain of VP16) resulted in enhanced transformation of chicken embryo fibroblast cells. In contrast, the fusing of a transcriptional repressor domain (Sin3 interaction domain of Mxi1) with the whole Maf protein masked the transactivator function of Maf, which in turn inhibited its transforming activity. Furthermore, the leucine zipper domain of Maf, which defines its dimer-forming specificity, was exchangeable with that of GCN4 yeast protein in terms of its transactivating and cell transforming activities. Thus, heterodimer formation with other bZip factors is not required for Maf's ability to transform. These results together suggest that transactivation through MARE is necessary for Maf-induced transformation and that there exist downstream target gene(s) for transformation. Since the MARE sequence overlaps with the recognition element of another bZip oncoprotein Jun, we assessed whether Jun and Maf induce cell transformation through activating the same genes. We thus constructed a mutated version of Jun that has a GCN4 leucine zipper and lacks the transactivator domain. This mutant repressed the cell transformation not only by Jun but also by Maf. Thus, Maf and Jun share downstream target gene(s) that are involved in cell transformation.

Animals↗
PubMed2005

Applications of gene targeting technology to mental retardation and developmental disability research.

The human and mouse genome projects elucidated the sequence and position map of innumerous genes expressed in the central nervous system (CNS), advancing our ability to manipulate these sequences and create models to investigate regulation of gene expression and function. In this article, we reviewed gene targeting methodologies with emphasis on applications to CNS development and neurodevelopmental disorders.

Alleles↗
PubMed1996

Gene targeting in rat embryo fibroblasts promoted by the polyomavirus large T antigen.

We used the recombination-promoting activity of the polyomavirus large T antigen (T-ag) to increase the frequency of gene targeting in rat fibroblasts. We constructed a cell line carrying a functional polyomavirus replication origin and a transformation-defective middle T-ag oncogene. The structure of the locus was such that homologous recombination with the targeting DNA reconstituted a functional transforming gene and converted the cells from the normal to the transformed state. Introduction of the large T-ag with the targeting DNA promoted recombinational events that corrected the mutation in either the target locus or the targeting DNA. The frequency of recombination was not substantially influenced by the extent of homology between the recombining sequences. However, it was reduced when the replication origin was inactivated in the targeting DNA, and was reduced further when the origin was inactivated in the target locus.

Animals↗
PubMed2006

Induction of progesterone target genes requires activation of Erk and Msk kinases and phosphorylation of histone H3.

How genes are regulated in the context of chromatin is a central question of biology. Steroid hormones control gene expression via interaction of their receptors with target sequences on DNA but can also activate cytoplasmic signaling cascades. Here we report that rapid Erk activation by progestins participates in induction of target genes by preparing the chromatin for transcription. Five minutes after hormone treatment, Erk activation leads to phosphorylation of the progesterone receptor (PR), activation of Msk1, and recruitment of a complex of the three proteins to a nucleosome on the MMTV promoter. Msk1 phosphorylates histone H3, leading to displacement of HP1gamma and recruitment of Brg1 and RNA polymerase II. Cell-free experiments show a direct interaction between PR, Erk, and Msk1 and support the importance of H3 phosphorylation for nucleosome remodeling. Inhibition of Msk1 activation blocks recruitment of the kinase complex, H3 phosphorylation, and HP1gamma displacement, thus precluding remodeling and induction of the promoter.

Animals↗
PubMed2005

Targeting gene expression selectively in cancer cells by using the progression-elevated gene-3 promoter.

One impediment to effective cancer-specific gene therapy is the rarity of regulatory sequences targeting gene expression selectively in tumor cells. Although many tissue-specific promoters are recognized, few cancer-selective gene promoters are available. Progression-elevated gene-3 (PEG-3) is a rodent gene identified by subtraction hybridization that displays elevated expression as a function of transformation by diversely acting oncogenes, DNA damage, and cancer cell progression. The promoter of PEG-3, PEG-Prom, displays robust expression in a broad spectrum of human cancer cell lines with marginal expression in normal cellular counterparts. Whereas GFP expression, when under the control of a CMV promoter, is detected in both normal and cancer cells, when GFP is expressed under the control of the PEG-Prom, cancer-selective expression is evident. Mutational analysis identifies the AP-1 and PEA-3 transcription factors as primary mediators of selective, cancer-specific expression of the PEG-Prom. Synthesis of apoptosis-inducing genes, under the control of the CMV promoter, inhibits the growth of both normal and cancer cells, whereas PEG-Prom-mediated expression of these genes kills only cancer cells and spares normal cells. The efficacy of the PEG-Prom as part of a cancer gene therapeutic regimen is further documented by in vivo experiments in which PEG-Prom-controlled expression of an apoptosis-inducing gene completely inhibited prostate cancer xenograft growth in nude mice. These compelling observations indicate that the PEG-Prom, with its cancer-specific expression, provides a means of selectively delivering genes to cancer cells, thereby providing a crucial component in developing effective cancer gene therapies.

Animals↗
PubMed2005

Global analysis of IL-2 target genes: identification of chromosomal clusters of expressed genes.

T lymphocytes play a central role in controlling adaptive immune responses. IL-2 critically regulates both T cell growth and death and is involved in maintaining peripheral tolerance, but the molecules involved in these and other IL-2 actions are only partially known. We now provide a comprehensive compendium of the genes expressed in T cells and of those regulated by IL-2 based on a combination of DNA microarrays and serial analysis of gene expression (SAGE). The newly identified IL-2 target genes include many genes previously linked to apoptosis in other cellular systems that may contribute to IL-2-dependent survival functions. We also studied the mRNA expression of known regulators of signaling pathways for their induction in response to IL-2 in order to identify potential novel positive and/or negative feedback regulators of IL-2 signaling. We show that IL-2 regulates only a limited number of these genes. These include suppressors of cytokine signaling (SOCS) 1, SOCS2, dual-specificity phosphatase (DUSP) 5, DUSP6 and non-receptor type phosphatase-7 (PTPN7). Additionally, we provide evidence that many genes expressed in T cells locate in chromosomal clusters, and that select IL-2-regulated genes are located in at least two clusters, one at 5q31, a known cytokine gene cluster, and the other at 6p21.3, a region that contains genes encoding the tumor necrosis factor (TNF) superfamily members TNF, LT-alpha and LT-beta.

Animals↗
PubMed2003

Gene-targeted mice reveal a critical role for inducible nitric oxide synthase in vascular dysfunction during diabetes.

BACKGROUND AND PURPOSE: Inducible nitric oxide synthase (iNOS) is a mediator of vascular dysfunction during inflammation. The purpose of this study was to test the hypothesis that vascular dysfunction during diabetes is dependent on expression of iNOS. METHODS: Diabetes was produced in mice with streptozotocin (150 mg/kg IP). After 4 to 6 months of diabetes, vasomotor function was examined in vitro in carotid arteries from mice with targeted disruption of the gene for iNOS (iNOS-deficient mice) and from normal, wild-type (WT) mice. RESULTS: Contractile responses of carotid arteries to U46619, a thromboxane A2 analogue, were not altered by diabetes in WT mice. Responses to U46619 were increased in arteries from diabetic iNOS-deficient mice compared with diabetic WT and nondiabetic mice (iNOS-deficient and WT mice). These results indicate that expression of iNOS inhibits an increased vasoconstrictor response during diabetes. Arteries from nondiabetic WT mice relaxed 83+/-2% (mean+/-SE) in response to acetylcholine (1 micromol/L) compared with 58+/-6% in arteries from diabetic WT mice (P<0.05 versus nondiabetic mice). In contrast, relaxation of carotid arteries to acetylcholine was similar (81+/-4% versus 76+/-6%; P>0.05) in iNOS-deficient mice under nondiabetic and diabetic conditions, respectively. Thus, diabetes produced impairment of endothelium-dependent relaxation in arteries from WT but not iNOS-deficient mice. Endothelium-independent relaxation in response to nitroprusside was similar in arteries from all mice. CONCLUSIONS: These results provide the first direct evidence that impairment of endothelium-dependent relaxation during diabetes is dependent on expression of iNOS.

Animals↗
PubMed2006

Schistosoma mansoni TGF-beta receptor II: role in host ligand-induced regulation of a schistosome target gene.

Members of transforming growth factor-beta (TGF-beta) superfamily play pivotal roles in development in multicellular organisms. We report the functional characterization of the Schistosoma mansoni type II receptor (SmTbetaRII). Mining of the S. mansoni expressed sequence tag (EST) database identified an EST clone that shows homology to the kinase domain of type II receptors from different species. The amplified EST sequence was used as a probe to isolate a cDNA clone spanning the entire coding region of a type II serine/threonine kinase receptor. The interaction of SmTbetaRII with SmTbetaRI was elucidated and shown to be dependent on TGF-beta ligand binding. Furthermore, in the presence of human TGF-beta1, SmTbetaRII was able to activate SmTbetaRI, which in turn activated SmSmad2 and promoted its interaction with SmSmad4, proving the transfer of the signal from the receptor complex to the Smad proteins. Gynaecophoral canal protein (GCP), whose expression in male worms is limited to the gynaecophoric canal, was identified as a potential TGF-beta target gene in schistosomes. Knocking down the expression of SmTbetaRII using short interfering RNA molecules (siRNA) resulted in a concomitant reduction in the expression of GCP. These data provide evidence for the direct involvement of SmTbetaRII in mediating TGF-beta-induced activation of the TGF-beta target gene, SmGCP, within schistosome parasites. The results also provide additional evidence for a role for the TGF-beta signaling pathway in male-induced female reproductive development.

Activin Receptors, Type I↗
PubMed2006

Human eosinophils have an intact Smad signaling pathway leading to a major transforming growth factor-beta target gene expression.

BACKGROUND: There is a paradoxical finding that eosinophils are frequently accumulated at the sites of allergic inflammation where transforming growth factor (TGF)-beta, a negative regulator of eosinophil survival, is upregulated; however, eosinophil accumulation is persistent. We thus hypothesized that eosinophils might have aberrant TGF-beta signaling and be unresponsive to TGF-beta. To test the hypothesis, we examined the expression and function of Smad proteins, which are central mediators for TGF-beta signaling, in human eosinophils. METHODS: Eosinophils were isolated from the peripheral blood of normal donors, and the expression and activation of endogenous Smad proteins were examined by reverse transcription polymerase chain reaction and Western blotting. The Smad function in the transcription of the major TGF-beta target gene Smad7 was investigated using a dominant negative form of Smad3. The effect of TGF-beta on eosinophil survival was then evaluated by a cell viability assay using normal and asthmatic eosinophils. RESULTS: Human eosinophils expressed mRNAs and proteins of TGF-beta typeI and type II receptors, Smad2, Smad3 and Smad4. TGF-beta induced the phosphorylation of Smad2 in eosinophils, which was blocked by SB431542, an inhibitor of TGF-beta type I receptor kinase. A dominant negative Smad3 protein suppressed TGF-beta-induced Smad7 mRNA expression in eosinophils. Finally, TGF-beta prevented granulocyte macrophage colony-stimulating factor- or interferon-gamma-mediated survival of eosinophils obtained from asthmatic patients as well as normal subjects. CONCLUSION: Human eosinophils have an intact Smad signaling pathway leading to a major TGF-beta target gene expression. Thus, eosinophils might become resistant to TGF-beta only in in vivo circumstances.

Activin Receptors, Type I↗
PubMed2003

Tumor-targeted gene therapy: strategies for the preparation of ligand-polyethylene glycol-polyethylenimine/DNA complexes.

Surface-shielded DNA delivery systems have been synthesized with virus-like characteristics that target gene expression into distant tumor tissues. Polyethylenimine (PEI)/DNA complexes ('polyplexes') conjugated with the cell-binding ligand transferrin (Tf) or epidermal growth factor (EGF) were used to achieve receptor-mediated endocytosis. The surface charge of the complexes was masked by covalently linking PEI to polyethylene glycol (PEG). Three alternatives for generating these surface-shielded formulations were utilized, attaching ligand and PEG molecules to PEI either before or after DNA complex formation. The stabilized formulations could be ultra-concentrated, stored frozen, and applied systemically after thawing. Intravenous injection of Tf-PEG-coated polyplexes resulted in gene transfer to subcutaneous Neuro2a neuroblastoma tumors of syngeneic A/J mice; EGF-PEG-coated polyplexes were intravenously applied for targeting human hepatocellular carcinoma xenografts in SCID mice. In these models, luciferase marker gene expression levels in tumor tissues were 10- to 100-fold higher than in other organ tissues. Repeated systemic application of Tf-PEG-PEI/DNA complexes encoding tumor necrosis factor alpha (TNF-alpha) into tumor-bearing mice induced tumor necrosis and inhibition of tumor growth in three murine tumor models of different tissue origin (Neuro2a, M-3 or B16 melanoma).

Animals↗
PubMed1994

Targeted gene walking by low stringency polymerase chain reaction: assignment of a putative human brain sodium channel gene (SCN3A) to chromosome 2q24-31.

We have developed a low stringency polymerase chain reaction (LSPCR) to isolate the unknown neighboring region around a known DNA sequence, thus allowing efficient targeted gene walking. The method involves the polymerase chain reaction (PCR) with a single primer under conditions of low stringency for primer annealing (40 degrees C) for the first few cycles followed by more cycles at high stringency (55 degrees C). This enables the amplification of a targeted DNA fragment along with other nontargeted fragments. High stringency (55 degrees C) nested PCRs with end-labeled primers are then used to generate a ladder of radioactive bands, which accurately identifies the targeted fragment(s). We performed LSPCR on human placental DNA using a highly conserved sodium channel-specific primer for 5 cycles at 40 degrees C followed by 27 cycles at 55 degrees C for primer annealing. Subsequently, using higher stringency (55 degrees C) PCR with radiolabeled nested primers for 8 cycles, we have isolated a 0.66-kb fragment of a putative human sodium channel gene. Partial sequence (325 bp) of this fragment revealed a 270-bp region (exon) with homology to the rat brain sodium channel III alpha (RBIII) gene at the nucleotide (87%) and amino acid (92%) levels. Therefore, we putatively assign this sequence as a part of a gene coding the alpha-subunit of a human brain type III sodium channel (SCN3A). Using PCR on two human/rodent somatic cell hybrid panels with primers specific to this putative SCN3A gene, we have localized this gene to chromosome 2. Fluorescence in situ hybridization to human metaphase chromosomes was used to sublocalize the SCN3A gene to chromosome at 2q24-31. In conclusion, LSPCR is an efficient and sensitive method for targeted gene walking and is also useful for the isolation of homologous genes in related species.

Animals↗
PubMed2000

Enhancing the efficiency of introducing precise mutations into the mouse genome by hit and run gene targeting.

The creation of precise clinical mutations by targeting is important in elucidating disease pathogenesis using mouse models. 'Hit and run' gene targeting is an elegant method to achieve this goal. This uses first a positive selection to introduce the targeting vector carrying the required mutation and then a negative selection to identify clones which have removed vector and wild-type sequences by intrachromosomal recombination. However, this approach has only been successfully used in a handful of cases. We used this procedure to introduce precise clinical mutations into the exon 10 region of the cystic fibrosis transmembrane conductance regulator (Cftr) gene. Using a CMV promoter driven hygromycin/thymidine kinase (hyg/tk) fusion gene as both our dominant and negative selectable marker, we targeted the Cftr locus very efficiently but only identified false runs after the negative selection step. This defect in thymidine kinase induced toxicity to gancyclovir correlated with methylation of the transgene. Consequently we devised a stringent screening procedure to select only true 'run' clones. Unfortunately these 'run' clones had lost the mutation so we altered the vector design to bias the run step to retain the mutation and used a different tk selection cassette with a HSVtk promoter sequence. This new vector design allowed both efficient 'hit and run' for two cystic fibrosis (CF) mutations with no false positives and successful germline transmission of the novel G480C missense mutation.

Animals↗
PubMed2006

Characterization of target genes at the 2p15-16 amplicon in diffuse large B-cell lymphoma.

Amplification of 2p has been observed as a recurrent alteration in diffuse large B-cell lymphoma (DLBCL). Whereas two candidate oncogenes, REL and BCL11A, have been investigated as targets for 2p amplification, the question remains as to whether the true target gene in the amplicon is REL, BCL11A or both. We previously identified frequent genomic gains of chromosomal 2p in 25 out of 99 DLBCL cases by means of genome-wide array comparative genomic hybridization (CGH). All of these 25 cases included recurrent copy number gain at 2p15-16. In the study presented here, cases were analyzed in greater detail by means of contig bacterial artificial chromosome (BAC) array CGH for the 4.5-Mb region at 2p15-16, which contained 33 BAC clones. We confined the minimal common region to 500-kb in length, where only the candidate oncogene REL, and not BCL11A, is located. Real-time quantitative PCR was carried out to investigate the correlation between genomic gain and expression. It showed a significant correlation for both genes, indicating that these two genes are common targets for the 2p15-16 amplicon. However, given the fact that REL is more frequently amplified than BCL11A, the REL gene may play a more important role than BCL11A in the pathogenesis of DLBCL.

Chromosomes, Artificial, Bacterial↗
PubMed1997

Gene targeting: things go better with Cre.

New technologies are changing the way in which gene targeting experiments are being designed. It is now becoming possible to analyze gene function in defined tissues at specific times during the life of a mouse.

Animals↗
PubMedDate not supplied

Polycation-based DNA complexes for tumor-targeted gene delivery in vivo.

BACKGROUND: Efficient and target-specific in vivo gene delivery is a major challenge in gene therapy. Compared to cell culture application, in vivo gene delivery faces a variety of additional obstacles such as anatomical size constraints, interactions with biological fluids and extracellular matrix, and binding to a broad variety of non-target cell types. METHODS: Polycation-based vectors, including adenovirus-enhanced transferrinfection (AVET) and transferrin-polyethylenimine (Tf-PEI), were tested for gene delivery into subcutaneously growing tumors after local and systemic application. DNA biodistribution and reporter gene expression was measured in the major organs and in the tumor. RESULTS: Gene transfer after intratumoral application was 10-100 fold more efficient with Tf-PEI/DNA or AVET complexes in comparison to naked DNA. Targeted gene delivery into subcutaneously growing tumors after systemic application was achieved using electroneutral AVET complexes and sterically stabilized PEGylated Tf-PEI/DNA complexes, whereas application of positively charged polycation/DNA complexes resulted in predominant gene expression in the lungs and was associated by considerable toxicity. CONCLUSION: For systemic application, the physical and colloidal parameters of the transfection complexes, such as particle size, stability, and surface charge, determine DNA biodistribution, toxicity, and transfection efficacy. By controlling these parameters, DNA biodistribution and gene expression can be targeted to different organs.

Adenoviridae↗
PubMed2001

Generation of bidirectional hypoxia/HIF-responsive expression vectors to target gene expression to hypoxic cells.

Hypoxia initiates an adaptive physiological response in all organisms and plays a role in the pathogenesis of several human diseases. The hypoxia/HIF-inducible factor-1 (HIF-1) transcription factor mediates transcriptional responses to hypoxia by binding to a cis-acting hypoxia-responsive element (HRE) present within target genes. The use of the HIF-1/HRE system of gene regulation can be utilized as a mechanism to target expression of therapeutic genes to hypoxic cells or cells that have a constitutively active HIF-1/HRE pathway due to cell transformation. Given the rapid resistance of tumors to single therapeutic strategies, new vector systems need to be developed that can deliver multimodal therapy. Here we show that HREs function as classical enhancer elements and function bidirectionally to co-regulate the expression of two genes. We designed a large series of novel bidirectional hypoxia/HIF-responsive expression vectors using HREs derived from the human vascular endothelial growth factor (VEGF) and erythropoietin (EPO) genes. We measured the ability of these constructs to express the luciferase and LacZ/beta-galactosidase (beta-gal) reporter genes bidirectionally under normoxic (21% O(2)) versus hypoxic (1, 3, 5, and 10% O(2)) conditions by transient transfection in three human glioma cell lines (LN229, U251MG and U138MG). Nine constructs were identified that exhibited moderate to high inducibility at 1% O(2) while maintaining tight regulation under normoxic conditions. Moreover, the level of activation was a function of O(2) concentration and was exponential at O(2) levels below 5%. These vectors will be valuable tools in a variety of gene therapy applications targeting pathological activation of the HIF-1/HRE pathway.

Cell Hypoxia↗