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Results for “targeted integration”

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At least 127 records · Page 7Linked to original sources
PubMed2003

Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep(+) hybrid amplicon vector.

Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep(+) hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep(+), rep(-) (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep(+) hybrid amplicon vector than with rep(-) or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep(+) hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep(-) or standard amplicon vectors resulted in random integrations of the entire rep(-) hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.

Animals↗
PubMed2006

Fragile sites are preferential targets for integrations of MLV vectors in gene therapy.

Following gene therapy of SCID-X1 using murine leukemia virus (MLV) derived vector, two patients developed leukemia owing to an activating vector integration near the LMO2 gene. We found that these integrations reside within FRA11E, a common fragile site known to correlate with chromosomal breakpoints in tumors. Further analysis showed that fragile sites attract a nonrandom number of MLV integrations, shedding light on its integration mechanism and risk-to-benefit ratio in gene therapy.

Cells, Cultured↗
PubMed2006

A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element (LME) constructs.

Alternaria brassicicola causes black spot disease of cultivated Brassicas and has been used consistently as a necrotrophic fungal pathogen for studies with Arabidopsis. In A. brassicicola, mutant generation has been the most rate-limiting step for the functional analysis of individual genes due to low efficiency of both transformation and targeted integration. To improve the targeted gene disruption efficiency as well as to expedite gene disruption construct production, we used a short linear construct with minimal elements, an antibiotic resistance selectable marker gene, and a 250- to 600-bp-long partial target gene. The linear minimal element (LME) constructs consistently produced stable transformants for diverse categories of genes. Typically, 100% of the transformants were targeted gene disruption mutants when using the LME constructs, compared with inconsistent transformation and usually less than 10% targeted gene disruption with circular plasmid disruption constructs. Each mutant displayed a unique molecular signature thought to originate from endogenous exonuclease activities in fungal cells. Our data suggests that a DNA double-stranded break repair mechanism (DSBR) functions to increase targeting efficiency. This method is advantageous for high throughput gene disruption, overexpression, and reporter gene introduction within target genes, especially for asexual filamentous fungi where genetic approaches are unfavorable.

Acetylglucosaminidase↗
PubMed2002

Characterization of the Hansenula polymorpha PUR7 gene and its use as selectable marker for targeted chromosomal integration.

The Hansenula polymorpha genes encoding the putative functional homologs of the enzymes involved in the seventh and eighth step in purine biosynthesis, HpPUR7 and HpPUR8, were cloned and sequenced. An overexpression vector designated pHIPA4 was constructed, which contains the HpPUR7 gene as selectable marker and allows expression of genes of interest via the strong, inducible alcohol oxidase promoter. An ade11 auxotrophic mutant that is affected in the activity of the HpPUR7 gene product was used to construct strain NCYC495 ade11.1 leu1.1 ura3. This strain grew on methanol at wild-type rates (doubling time of approximately 4 h) and is suitable for independent introduction of four expression cassettes, each using one of the markers for selection, in addition to the zeocin resistance marker. It was subsequently used as a host for overproduction of two endogenous peroxisomal matrix proteins, amine oxidase and catalase. Efficient site-specific integration of pHIPA4 and overproduction of amine oxidase and catalase is demonstrated. The expression cassette appeared to be pre-eminently suited to mediate moderate protein production levels. The advantages of pHIPA4 and the new triple auxotrophic strain in relation to the use of H. polymorpha as a versatile cell factory or as a model organism for fundamental studies on the principles of peroxisome homeostasis is discussed.

Alcohol Oxidoreductases↗
PubMed2005

Inactivation of retroviruses with preservation of structural integrity by targeting the hydrophobic domain of the viral envelope.

We describe a new approach for the preparation of inactivated retroviruses for vaccine application. The lipid domain of the viral envelope was selectively targeted to inactivate proteins and lipids therein and block fusion of the virus with the target cell membrane. In this way, complete elimination of the infectivity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) could be achieved with preservation of antigenic determinants on the surface of the viral envelope. Inactivation was accomplished by modification of proteins and lipids in the viral envelope using the hydrophobic photoinduced alkylating probe 1,5 iodonaphthylazide (INA). Treatment of HIV and SIV isolates with INA plus light completely blocked fusion of the viral envelope and abolished infectivity. The inactivated virus remained structurally unchanged, with no detectable loss of viral proteins. Modifications to envelope and nucleocapsid proteins were detected by changes in their elution pattern on reverse-phase high-performance liquid chromatography. These modifications had no effect on primary and secondary structure epitopes as determined by monoclonal antibodies. Likewise, the inactivated HIV reacted as well as the live virus with the conformation-sensitive and broadly neutralizing anti-HIV type 1 monoclonal antibodies 2G12, b12, and 4E10. Targeting the lipid domain of biological membranes with hydrophobic alkylating compounds could be used as a general approach for inactivation of enveloped viruses and other pathogenic microorganisms for vaccine application.

Antibodies, Monoclonal↗
PubMed2003

Integration of targeted therapies in gemcitabine chemotherapy regimens.

Lung cancer is one of the most common malignancies in the United States and the most lethal. Non-small-cell lung cancer (NSCLC), which accounts for over 80% of all lung cancer cases, results in a particularly poor prognosis and a high mortality rate. The 5-year survival rate is still only 15%, and most patients ultimately die from this disease. Unfortunately, the therapeutic improvements resulting from the new generation of cytotoxic agents seems to have reached a plateau. Targeted therapeutics, or agents aimed at specific biologic pathways, represent a potential for great improvements in survival from this disease. Because of their greater specificity, targeted therapeutics have the potential for decreased toxicity and increased efficacy. The main categories of targeted therapies applicable for NSCLC include receptor-targeted therapy, signal transduction or cell-cycle inhibition, angiogenesis inhibition, gene therapy, and vaccines. These new agents are, in theory, more target-specific, less toxic, easier to administer, and may lead to enhanced safety and survival for patients with advanced NSCLC. Targeted therapies are in different phases of clinical testing and have shown encouraging activity as single agents or in combination with chemotherapy as well as radiation therapy. Herein we explore targeted therapy in combination with gemcitabine.

Journal Article↗
PubMed1999

Inactive and active mutants of rab1b are not tightly integrated into target membranes.

The rab1b GTPase is localized on the ER and cis-Golgi membranes and involved in early exocytotic membrane traffic processes. To analyze the influence of rab1b conformation on specific membrane targeting processes three point mutants simulating permanently inactive or active rab1b proteins were constructed and expressed in BHK cells. To increase the expression of the growth inhibiting rab1b S22N and rab1b N121I mutants we co-expressed the Mss4 protein and observed an elevated expression of the inactive rab1b mutants. Surprisingly, only the rab1b wild-type protein shows the correct intracellular localization, and a tight membrane association. We conclude that the targeting process of rab1b depends predominantly on GDP/GTP exchange.

Amino Acid Substitution↗
PubMed1991

Increased ratio of targeted to random integration after transfection of chicken B cell lines.

Constructs of four different genetic loci were transfected into the avian leukosis virus-induced chicken B cell line DT40, which continues diversification of its rearranged light chain immunoglobulin gene by gene conversion. Analysis of stable transfectants revealed an unexpectedly high frequency of targeted integration into the homologous gene loci of DT40. Transcriptional activity of the target gene locus is not required, since a construct of the untranscribed ovalbumin gene also integrated predominantly by homologous recombination. A construct derived from the beta-actin locus was transfected into other chicken cell lines to determine the cell type specificity of the phenomenon. Targeted integration still occurred at high frequency in two other B cell lines that do not have the gene conversion activity. However, the ratios of targeted to random integration were reduced by at least one order of magnitude in three non-B cell lines.

Actins↗
PubMed1988

Highly preferred targets for retrovirus integration.

A central feature of retrovirus replication is integration of the provirus into host cell DNA, but the specificity of this step for cell target sequences has not been clarified. To investigate this issue, we developed a method for screening and comparing large numbers of unselected integration events. Using a replication-competent Rous sarcoma virus containing a bacterial suppressor tRNA gene as a selectable marker, we obtained collections of clones comprising integrated provirus together with host flanking sequences. Hybridization and sequence analysis of the flanking sequence reveals the presence of a number of strongly preferred integration targets. Within these targets, independent integration events occur at sites identical to the base.

Animals↗
PubMed2026

Molecular biomarker profiling in noninfectious uveitis: a chronological review of discovery.

PURPOSE OR REVIEW: Noninfectious uveitis (NIU) encompasses a heterogeneous group of immune-mediated intraocular inflammatory diseases whose complexity has driven systematic molecular biomarker discovery. This review presents NIU molecular biomarkers organized by biological category; autoantigens, human leukocyte antigens (HLA) and genetic markers, cellular immune subsets, cytokines, chemokines, and multiomics platforms including proteomics, microbiome metagenomics, metabolomics, and single-cell transcriptomics with each category presented in strict chronological order of landmark discovery. RECENT FINDINGS: We present a review organized along two nested timelines. Categories are presented in the order they historically emerged in the field, and within each category, landmark discoveries appear in chronological sequence. This allows the reader to trace how each biomarker category evolved: from foundational autoantigen identification in experimental uveitis models, through the genomic revolution of HLA association studies, into cellular immunophenotyping, cytokine profiling of aqueous humor, chemokine mapping of intraocular trafficking, and finally the emerging omics platforms that may potentially anchor precision medicine in NIU. Each biomarker is paired in line with its linked targeted therapeutic. SUMMARY: Biomarker research has transformed the understanding of NIU from a clinically defined syndrome into a group of molecularly distinct immune disorders. Advances spanning autoantigens, genetics, immune-cell profiling, cytokines, chemokines, and multiomics have revealed novel pathogenic mechanisms and therapeutic targets. Integration of these biomarkers with targeted therapies may accelerate the transition toward precision medicine in uveitis care.

cytokines↗
PubMed2003

Patterns of Hermes transposition in Drosophila melanogaster.

Transposable elements are being developed as tools for genomics and for the manipulation of insect genotypes for the purposes of biological control. An understanding of their transposition behavior will facilitate the use of these elements. The behavior of an autonomous Hermes transposable element from Musca domestica in the soma and germ-line of Drosophila melanogaster was investigated using the method of transposon display. In the germ-line, Hermes transposed at a rate of approximately 0.03 jumps per element per generation. Within the soma Hermes exhibited markedly non-random patterns of integration. Certain regions of the genome were distinctly preferred over others as integration targets, while other regions were underrepresented among the integration sites used. One particular site accounted for 4.4% of the transpositions recovered in this experiment, all of which were located within a 2.5-kb region of the actin5C promoter. This region was also present within the Hermes element itself, suggesting that this clustering is an example of transposable element "homing". Clusters of integration sites were also observed near the original donor sites; these represent examples of local hopping. The information content (sequence specificity) of the 8-bp target site was low, and the consensus target site resembles that determined from plasmid-based integration assays.

Animals↗
PubMed2025

Genome-wide profiling the integration patterns with T7-PCR.

Integration of exogenous gene fragments into the host genomes is a widely used and powerful method for studying gene functions, advancing molecular breeding, and conducting gene therapy. Accurately identifying the integration sites is essential for ensuring both the safety and efficacy of genome engineering efforts. However, current mapping techniques are constrained by high costs and a low signal-to-noise ratio. In this study, we developed an innovative tool for mapping integration sites, leveraging T7 polymerase-mediated in vitro transcription (T7-IVT) to capture the junction fragments surrounding integration loci. This approach converts genomic flanking sequences into RNA, enabling the simultaneous enrichment of junction fragments and the elimination of background genomic DNA, thereby significantly enhancing the signal-to-noise ratio. We have validated the efficiency of this method, named T7-PCR, across yeast, plant, and human cells under diverse integration scenarios. T7-PCR outperforms current next-generation sequencing (NGS)-based mapping strategies in terms of efficiency and accuracy, with minimal positional effects. This method is highly applicable for high-throughput transgene screening and also supports the development of next-generation tools for targeted integration of large fragments.

Humans↗
PubMed1994

The frequency of gene targeting in yeast depends on the number of target copies.

We have compared the efficiency of transformation by linear DNA fragments in yeast strains carrying different numbers of homologous targets for recombination. In strains carrying dispersed copies of a target and in strains carrying tandem arrays, the frequency of transformation is proportional to the number of targets. This result is in contrast to previous studies of transformation in mammalian cells, where targeted integration was insensitive to the number of targets. We conclude that, in yeast, the search for a homologous partner is a rate-limiting step in the successful recombination of linearized DNA fragments. Furthermore, the fact that we obtain the same results with both dispersed and clustered targets argues against models of homology searching in which DNA becomes nonspecifically associated with a chromosome and then slides along the DNA until homology is encountered.

Chromosome Mapping↗