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Results for “Gene Targeting”

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At least 19 recordsLinked to original sources
PubMed2002

Identification and characterization of transcription factor target genes using gene-targeted mice.

Critical to understanding biological roles for transcription factors is an appreciation of the target genes regulated by the transcription factor. The identification of target genes can often expand the understanding of known biological roles for a transcription factor and may reveal unappreciated and unexpected functions. This article focuses on the identification and characterization of transcription factor target genes using mouse molecular genetics. The use of genetically engineered (knockout) mice and global gene expression analysis to identify transcription factor target genes is reviewed, with emphasis on important technical considerations. Detailed protocols for the application of real-time reverse transcription polymerase chain reaction and immunohistochemistry in target gene expression analysis are described. The article closes with a discussion of the use of mouse molecular genetics for the characterization of target genes as downstream effectors of transcription factors.

Algorithms↗
PubMed1997

[Modification of gene targeting method for functional analysis of the target gene in vivo].

Gene targeting in ES cells is a powerful tool to generate mice bearing predesigned mutations in the germ line. However, these mice carrying such constitutional mutations are often lethal and, therefore, we cannot study other functional aspects of the gene at later stages or in particular tissues. To inactivate the target gene in particular tissues or at particular stages of development, conditional gene inactivation based on the Cre-loxP recombination system of bacteriophage P1 is considered one of the applicable techniques. To express the Cre gene in a cell-type or developmental stage specific manner, either transgenic or adenoviral technology is most considerable technique in vivo.

Animals↗
PubMed2004

Differential target gene activation by TBX2 and TBX2VP16: evidence for activation domain-dependent modulation of gene target specificity.

The determinants of in vivo target site selectivity by transcription factors are poorly understood. To find targets for the developmentally regulated transcription factor TBX2, we generated stable transfectants of human embryonic kidney cells (293) that express a TBX2-ecdysone receptor (EcR) chimeric protein. While constitutive expression of TBX2 is toxic to 293 cells, clones expressing TBX2EcR are viable in the absence of an EcR ligand. Using cDNA arrays and quantitative PCR, we discovered nine genes whose expression was increased, but no genes whose expression was reduced, following 24 h of induction with Ponasterone A (PonA), a ligand for EcR. Since TBX2 was reported previously to be a transcriptional repressor, we also generated cell lines expressing a TBX2VP16EcR protein which we showed was a potent conditional transcriptional activator in transient transfection assays. Treatment of these cells with PonA induced the expression of five genes, none of which were affected in TBX2EcR-expressing cells. This discordance between TBX2- and TBX2VP16-regulated genes strongly suggests that specific transactivation domains can be a major determinant of gene target site selectivity by transcription factors that possess the same DNA-binding domain.

Binding Sites↗
PubMed1998

Antibodies for targeted gene therapy: extracellular gene targeting and intracellular expression.

Antibody genes of human origin and human antibodies directed against human proteins have become widely available in recent years. These are valuable reagents for gene therapy applications, in which the use of human proteins and genes allows for increased therapeutic benefit. Engineered human antibodies can be used in gene therapy both as a component of a gene delivery system and as a therapeutic gene. As the targeting moiety of a gene delivery system, the antibody should meet certain criteria that have been previously determined from other clinical applications of antibodies. These include bioavailability, specificity for the target cell, and rapid clearance. In addition, if repeat delivery of therapeutic genes is going to be needed, then gene delivery vectors should be non-immunogenic to allow repeated administration. The use of human antibodies in this application should therefore be superior to approaches which use rodent-derived antibodies. Another application of antibodies in gene therapy is the use of antibodies expressed inside the cell (intrabodies) as therapeutic agents. The power of the immune system to rearrange a limited set of genes to create recognition sites for any known molecule is well documented. The ability to harness this information and use these highly specific binding molecules as medicines to inhibit an unwanted cellular function is a promising advance in the field of molecular medicine, and in particular, in the field of intracellular immunization.

Journal Article↗
PubMed1998

Therapeutic gene targeting.

Gene targeting is the use of homologous recombination to make defined alterations to the genome. One of the possible outcomes of gene targeting is the accurate correction of genetic defects, and this would make it the ideal method of gene therapy for single gene disorder. While gene targeting has been achieved both in human cell lines and in nontransformed, primary human cells, its low efficiency has been a major limitation to its therapeutic potential. Gene therapy in vivo gene targeting is there for impractical without dramatic improvements in targeting efficiency. Ex vivo approaches might more realistically be considered, but would benefit from progress in the isolation and growth of somatic stem cells and improvements in targeting efficiency. We provide here a brief review of the challenges of gene therapy by gene targeting. This is followed by a critical overview of recent developments in gene targeting techniques, and in our understanding of the underlying processes of homologous and nonhomologous recombination.

Adenoviridae↗
PubMed2002

[Progress on gene targeting].

Gene targeting is a rising technology in molecular biology,which is defined as the introduction of exogeneous DNA to specific site in genome by homologous recombination,and consequently change the hereditary character of the cell. This technology provides a new and powerful means for research in developmental biology,molecular genetics,immunology and medicine. Progresses have been made in exploring gene structure and function,gene expression and regulation,transgene and gene therapy with the application of gene targeting. But there are some problems in gene targeting,especially for the low efficiency. This article just provided a review of the principle and program of gene targeting,and discussed the possible approaches to increase the efficiency of gene targeting.

English Abstract↗
PubMed2003

[Homologous recombination and gene targeting].

Gene therapy and the production of mutated cell lines or model animals both require the development of efficient, controlled gene-targeting strategies. Classical approaches are based on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. The low frequency of homologous recombination in mammalian cells leads to inefficient targeting. Here, we review the limiting steps of classical approaches and the new strategies developed to improve the efficiency of homologous recombination in gene-targeting experiments.

Animals↗
PubMed1998

Analysis of antigen receptor signalling using mouse gene targeting.

Gene targeting in mice has enabled the study of antigen receptor signalling in primary lymphocytes. Furthermore, it has provided the tools to directly assess the function of individual signalling proteins by mutation of the genes that code for them. Some of the results that gene targeting has produced have confirmed previous views of the function of particular proteins. Others have given surprising results and overturned accepted viewpoints.

Animals↗
PubMed1989

The new mouse genetics: altering the genome by gene targeting.

Gene targeting (homologous recombination between DNA sequences residing in the chromosome and newly introduced DNA sequences) in pluripotent, mouse embryo-derived stem (ES) cells promises to provide the means to generate mice of any desired genotype. This review describes some of the background and current advances of gene targeting in mouse ES cells.

Animals↗
PubMed1995

Gene targeting and gene transfer studies of the plasminogen/plasmin system: implications in thrombosis, hemostasis, neointima formation, and atherosclerosis.

The plasminogen/plasmin or fibrinolytic system with its physiological triggers, tissue-type plasminogen activator, and urokinase-type plasminogen activator has been presumed to participate in normal and pathological processes of the vessel wall such as blood clot dissolution (thrombolysis), hemostasis, aneurysm formation, neovascularization, restenosis, and atherosclerosis. The implied role of the fibrinolytic system in vivo is, however, deduced from correlations between fibrinolytic activity and (patho)physiological phenomena, which does not allow establishing a cause/consequence relationship. Gene targeting and gene transfer technologies allow establishment of the in vivo role of gene products more conclusively. This article reviews briefly the findings of such studies on thrombolysis/thrombosis, hemostasis, neointima formation, atherosclerosis, and associated effects on survival.

Arteriosclerosis↗
PubMed1999

Gene targeting for gene therapy: prospects.

Ideally, gene therapy involves the correction of genetic defects through the natural means of gene targeting. This therapy possesses a number of conceptual advantages. However, a major obstacle to successful gene therapy is the relative inefficiency of the targeting process in mammalian cells. Gene targeting may be accomplished by two different mechanisms: the homologous recombination and the mismatch correction of DNA heteroduplexes. Based on the model of homologous recombination for the well-studied prokaryotic and the less studied eukaryotic systems, three approaches have been employed to improve the efficiency and accuracy of homologous recombination events. These are: (1) artificial double-strand breaks in both the exogenous and the chromosomal DNA, (2) a contiguous long homology between the exogenous and chromosomal DNA, and (3) a transient overproduction of an active recombinase, the bacterial RecA or mammalian RecA-like proteins, in mammalian cell nuclei. Combining these approaches can result in more effective gene targeting protocols. The second mechanism has been improved based on recent observations of recombinogenic activity of oligonucleotides and, especially, specifically designed chimeric RNA/DNA oligonucleotides. The use of RecA-like proteins to stimulate searching for homology and forming stable DNA heteroduplexes between oligonucleotides and chromosomal DNA remains an attractive idea for additional improvement of gene targeting events.

Animals↗
PubMed1997

Introduction of precise alterations into the mouse genome with high efficiency by stable tag-exchange gene targeting: implications for gene targeting in ES cells.

The efficiency of tag-and-exchange gene targeting approaches for the introduction of precise genomic modifications is compromised by high levels of non-homologous recombinants which survive selection due to loss of tag gene expression, often by physical loss of the tag gene. We describe a modified approach, termed stable tag-exchange, which incorporates an additional positive selection (stability) cassette to circumvent this limitation. HPRT (tag) and neo (stability) cassettes, separated by 4.9 kb of homologous DNA, were introduced efficiently into the LIF locus of ES cells. The tag cassette was substituted for abeta-galactosidase gene in exchange step targeting. Direct comparison of the tag-and-exchange and stable tag-exchange approaches indicated respective targeting efficiencies of 21% and 88%. The increased stable tag-exchange targeting efficiency resulted from elimination of >75% of background lines which survived tag-and-exchange selection due to physical loss of the tag gene. These resulted from reversion of the tagged allele to wild-type which is therefore a major contributor to tag-and-exchange targeting background. Our results extend the application of gene targeting by demonstrating a rationale for single-step integration of multiple regions of extended non-homology, and providing an efficient system for the repeated introduction of precise alterations into the mammalian genome.

Animals↗
PubMed2006

A novel integrated strategy (full length gene targeting) for mRNA accessible site tagging combined with microarray hybridization/RNase H cleavage to screen effective antisense oligonucleotides.

PURPOSE: Down regulation of targeted gene by antisense oligonucleotides (ASOs) has been an effective approach for molecular therapy and the study of gene function. However, it is difficult to find optimal and effective ASOs. We describe a novel integrated strategy called full length gene targeting (FLGT), involving mRNA accessible site tagging combined with microarray hybridization/RNase H cleavage for screening effective ASOs in full length of target gene. METHODS: Initially, transcripts representing mRNA (cRNA) were hybridized with randomized oligonucleotides library, then oligonucleotides tags were sequenced, aligned to target mRNA, and found to be able to precisely define the accessible sites of the mRNA by TargetFinder softeware. Further, selected ASO probes were synthesized and used to construct microarrays. Target mRNA labeled alpha-(32)P-UTP was hybridized to the microarrays, and the substrate heteroduplexes were followed by RNase H catalytic reaction on microarrays. Those ASOs with strong signal and shorter T(1/2) (time of 50% heteroduplex cleavage by RNase H) were selected in the combinatorial assays. Survivin, an inhibitor of apoptosis, was chosen as a target to screen ASOs by the FLGT process. RESULTS: Using the integrated strategy, five ASOs against survivin were selected and showed significant down regulation of survivin expression and inhibition of tumor cells growth in vitro. Furthermore, one ASO was used to further investigate its antitumor activity on Human hepatocellular carcinoma (HCC) orthotopic transplant model in mice. CONCLUSIONS: This study demonstrated that FLGT is useful for screening effective ASOs. FLGT may become a useful tool for screening more effective ASOs in full length of target gene.

Animals↗
PubMed1995

Gene targeting and gene transfer studies of the biological role of the plasminogen/plasmin system.

A possible role of the plasminogen/plasmin or fibrinolytic system in several biological processes has been implied from correlations between fibrinolytic activity and (patho)physiological phenomena. However, such indirect evidence does not allow to definitively establish the biological relevance of this system. Two recently developed technologies, gene targeting and gene transfer, have allowed to more definitively characterize the in vivo role of gene products. The consequences of gain or loss of function of fibrinolytic system components on reproduction, development, health, survival and on hemostasis, thrombosis, neointima formation, tissue remodeling, brain function, malignancy and neovascularization is summarized below. In addition, the possible use of transgenic mice to study gene regulation or to generate monoclonal antibodies against conserved epitopes in the targeted proteins is discussed.

Animals↗
PubMed1996

Essential and non-redundant roles of p48 (ISGF3 gamma) and IRF-1 in both type I and type II interferon responses, as revealed by gene targeting studies.

BACKGROUND: Interferons (IFNs) are a class of cytokines which confer cellular resistance against viral infections. Type I (IFN-alpha and -beta) and type II (IFN-gamma) IFNs utilize distinct receptors, the stimulation of which results in the induction of downstream target genes. These target genes usually contain within their promoter region an IFN responsive element, termed ISRE (IFN stimulated response element) which binds a heterotrimeric transcription factor, ISGF3 (IFN-stimulated gene factor 3) consisting of p48 (ISGF3 gamma), Stat1 (Signal transducers and activators of transcription-1; alpha or beta), and Stat2. The ISRE sequence overlaps with that of IRF-E which binds another IFN-inducible factor, IRF-1 (IFN regulatory factor-1). RESULTS: We generated mice lacking p48 by gene targeting. We show that p48 plays an essential role in both type I and type II IFN responses; activation of IFN-inducible genes and establishment of the antiviral state by IFN-alpha or -gamma are both severely impaired, and ISRE-binding activities induced by both IFNs are absent in the p48-negative embryonic fibroblasts (EFs). Furthermore, we generated mice deficient for both p48 and IRF-1 and found that at least one IFN-inducible gene is dependent on both factors. CONCLUSIONS: p48 and IRF-1 do not perform redundant functions in the cell, but rather complement one another in both type I and II IFN responses.

Animals↗
PubMed2006

Targeted gene repair: the ups and downs of a promising gene therapy approach.

As a novel form of molecular medicine based on direct actions over the genes, targeted gene repair has raised consideration recently above classical gene therapy strategies based on genetic augmentation or complementation. Targeted gene repair relies on the local induction of the cell's endogenous DNA repair mechanisms to attain a therapeutic gene conversion event within the genome of the diseased cell. Successful repair has been achieved both in vitro and in vivo with a variety of corrective molecules ranging from oligonucleotides (chimeraplasts, modified single-stranded oligonucleotides, triplex-forming oligonucleotides), to small DNA fragments (small fragment homologous replacement (SFHR)), and even viral vectors (AAV-based). However, controversy on the consistency and lack of reproducibility of early experiments regarding frequencies and persistence of targeted gene repair, particularly for chimeraplasty, has flecked the field. Nevertheless, several hurdles such as inefficient nuclear uptake of the corrective molecules, and misleading assessment of targeted repair frequencies have been identified and are being addressed. One of the key bottlenecks for exploiting the overall potential of the different targeted gene repair modalities is the lack of a detailed knowledge of their mechanisms of action at the molecular level. Several studies are now focusing on the assessment of the specific repair pathway(s) involved (homologous recombination, mismatch repair, etc.), devising additional strategies to increase their activity (using chemotherapeutic drugs, chimeric nucleases, etc.), and assessing the influence of the cell cycle in the regulation of the repair process. Until therapeutic correction frequencies for single gene disorders are reached both in cellular and animal models, precision and undesired side effects of this promising gene therapy approach will not be thoroughly evaluated.

Animals↗
PubMed1997

Inhibitory role of plasminogen activator inhibitor-1 in arterial wound healing and neointima formation: a gene targeting and gene transfer study in mice.

BACKGROUND: Plasminogen-deficient mice display impaired vascular wound healing and reduced arterial neointima formation after arterial injury, suggesting that inhibition of plasmin generation might reduce arterial neointima formation. Therefore, we studied the consequences of plasminogen activator inhibitor-1 (PAI-1) gene inactivation and adenoviral PAI-1 gene transfer on arterial neointima formation. METHODS AND RESULTS: Neointima formation was evaluated in PAI-1-deficient (PAI-1(-/-)) mice with perivascular electric or transluminal mechanical injury. PAI-1 deficiency improved vascular wound healing in both models: the cross-sectional neointimal area was 0.001+/-0.001 mm2 in PAI-1(+/+) and 0.016+/-0.008 mm2 in PAI-1(-/-) mice within 1 week after electric injury (P<.02) and 0.055+/-0.008 mm2 in PAI-1(+/+) and 0.126+/-0.006 mm2 in PAI-1(-/-) mice within 3 weeks after mechanical injury (P<.001). Proliferation of smooth muscle cells was not affected by PAI-1 deficiency. Topographic analysis of arterial wound healing after electric injury revealed that PAI-1(-/-) smooth muscle cells, originating from the uninjured borders, more rapidly migrated into the necrotic center of the arterial wound than wild-type smooth muscle cells. On the basis of immunostaining, PAI-1 expression was markedly upregulated during vascular wound healing. There were no genotypic differences in reendothelialization of the vascular wound. When PAI-1(-/-) mice were intravenously injected with replication-defective adenovirus expressing human PAI-1 (AdCMVPAI-1), plasma PAI-1 antigen levels increased in a dose-dependent fashion up to to 61+/-8 microg/mL with 2x10(9) plaque-forming units (pfu) virus. Luminal stenosis was 35+/-13% in control AdRR5-treated (2x10(9) pfu) and suppressed to 5+/-5% in AdCMVPAI-1-treated (6x10(8) pfu) PAI-1(-/-) mice (P<.002). CONCLUSIONS: By affecting cellular migration, PAI-1 plays an inhibitory role in vascular wound healing and arterial neointima formation after injury, and adenoviral PAI-1 gene transfer reduces arterial neointima formation in mice.

Adenoviridae↗
PubMed2000

Gene targeting and gene transfer to unravel the molecular basis of the formation and disorders of blood vessels.

Novel gene technologies have allowed to manipulate the genetic balance of candidate molecules in mice in a controllable manner. Homologous or site-specific recombination in embryonic stem cells allows to study the consequences of deficiencies, mutations, conditional or tissue specific expression of gene products in transgenic mice. These technological breakthroughs have significantly advanced biomedical research and our understanding of the pathophysiological role of candidate disease genes. In addition, gene transfer allows to test the possible therapeutic use of gene products for gene therapy. With the advent of genome sequencing programs, these gene technologies provide means to study gene function in a conclusive manner. Furthermore, disease models can be generated which can be used as testmodels for (gene) therapy or for discovery of novel genes using differential gene profiling techniques. The present review will focus on the use of these technologies to study the molecular basis of how blood vessels form (angiogenesis) and how vessels can become affected by disorders. A selected number of molecules that have been studied in our laboratory will be reviewed in particular.

Adult↗