A family of hierarchically self-assembling linear-dendritic hybrid polymers for highly efficient targeted gene delivery.
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Animals↗
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Results for “Gene Targeting”
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Chromatin reorganization accompanying cellular dedifferentiation is associated with modifications of histone H3, redistribution of HP1, and activation of E2F-target genes.
The remarkable regeneration capacity of plant cells is based on their capability to dedifferentiate. We recently reported that cellular dedifferentiation proceeds through two distinct phases, each accompanied by chromatin decondensation: acquisition of competence for fate switch followed by a signal-dependent reentry into S phase. The purpose of this study was to (1) characterize changes in chromatin factors associated with chromatin decondensation, and (2) study the relationship between chromatin decondensation and transcriptional activation of pRb/E2F-regulated genes. We show that plant cells competent for fate switch display a disruption of nucleolar domain appearance associated with condensation of 18S ribosomal DNA, as well as modifications of histone H3 and redistribution of heterochromatin protein 1 (HP1). We further show that the pRb/E2F-target genes RNR2 and PCNA are condensed and silent in differentiated leaf cells but become decondensed, although not yet activated, as cells acquire competence for fate switch; transcriptional activation becomes evident during progression into S phase, concomitantly with pRb phosphorylation. We propose that chromatin reorganization is central for reversion of the differentiation process leading to resetting of the gene expression program and activation of silent genes.
Cell Cycle Proteins↗
Conditional gene targeting of the Scnn1a (alphaENaC) gene locus.
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Animals↗
Comparative analysis of the human and mouse Hey1 promoter: Hey genes are new Notch target genes.
Hey genes (Hey1, Hey2 and HeyL) encode a new group of basic helix-loop-helix transcription factors that are related to the hairy/Enhancer of split genes. In the present study, we cloned and characterized the promoter region of the human and mouse Hey1 gene. The transcription initiation site was located 138 nucleotides upstream of the start codon. There is a minimal sequence element (nt -30 to -247) that is essential and important for basal transcription in three different cell types. Further upstream, a highly conserved sequence block (nt -324 to -646; approximately 90% human/mouse similarity) could be identified that contains several putative binding sites for transcription factors and likely represents an important regulatory region for this gene. Cotransfection experiments demonstrated that the mHey1 promoter activity is up-regulated by the activated form of all four mammalian Notch receptors via two functional RBP-Jkappa binding sites. The other members of the Hey gene family, Hey2 and HeyL, also possess RBP-Jkappa binding sites and they are similarly responsive to Notch signaling. Thus, our data clearly demonstrate that Hey genes form a new class of Notch signal transducers that should prove to be relevant in various developmental processes.
Animals↗
Targeted gene expression in dopamine and serotonin neurons of the mouse brain.
We used a knock-in strategy to generate two lines of mice expressing Cre recombinase under the transcriptional control of the dopamine transporter promoter (DAT-cre mice) or the serotonin transporter promoter (SERT-cre mice). In DAT-cre mice, immunocytochemical staining of adult brains for the dopamine-synthetic enzyme tyrosine hydroxylase and for Cre recombinase revealed that virtually all dopaminergic neurons in the ventral midbrain expressed Cre. Crossing DAT-cre mice with ROSA26-stop-lacZ or ROSA26-stop-YFP reporter mice revealed a near perfect correlation between staining for tyrosine hydroxylase and beta-galactosidase or YFP. YFP-labeled fluorescent dopaminergic neurons could be readily identified in live slices. Crossing SERT-cre mice with the ROSA26-stop-lacZ or ROSA26-stop-YFP reporter mice similarly revealed a near perfect correlation between staining for serotonin-synthetic enzyme tryptophan hydroxylase and beta-galactosidase or YFP. Additional Cre expression in the thalamus and cortex was observed, reflecting the known pattern of transient SERT expression during early postnatal development. These findings suggest a general strategy of using neurotransmitter transporter promoters to drive selective Cre expression and thus control mutations in specific neurotransmitter systems. Crossed with fluorescent-gene reporters, this strategy tags neurons by neurotransmitter status, providing new tools for electrophysiology and imaging.
Animals↗
Targeted gene disruption: applications in neurobiology.
In classical gene inactivation approaches by homologous recombination in embryonic stem cells, the resulting knockout mice are genotypically homogeneous. The inactivation of a gene in the complete organism may sometimes lead to early embryonic lethality. The observation that bacterial recombinases can drive site-specific recombination in mammalian cells has allowed for spatiotemporally controlled genetic modifications. Thus, conditional gene inactivation can be achieved in a specific subset of cells, leaving the rest of the organism genotypically unchanged. Another application of bacterial recombinases is the generation of exon-specific knockout mice, allowing for the analysis of the role of tissue-specific splice variants. A combination of the above-mentioned bacterial recombinase technique with inducible promoter systems permits the investigator to choose precisely the onset of recombination. An extension of the above-mentioned techniques is the combination of the bacterial recombinase technique with adenovirus-based technology, which would open vast possibilities of tissue-specific genetic modifications in a controlled time frame.
Animals↗
Single gene target bacterial identification. groEL gene sequencing for discriminating clinical isolates of Burkholderia pseudomallei and Burkholderia thailandensis.
Proper identification of Burkholderia pseudomallei and Burkholderia thailandensis is crucial in guiding clinical management of patients with suspected melioidosis, as more than 99% of cases of melioidosis are caused by B. pseudomallei, whereas B. thailandensis is only responsible for causing less than 1% of the cases. However, the difference between the 16S ribosomal RNA gene sequences of B. pseudomallei and that of B. thailandensis is only 1%, and is therefore not discriminative enough for distinguishing the 2 species confidently. In this study, we amplified and sequenced the groEL genes of 7 strains of B. thailandensis and 6 strains of B. pseudomallei, and compared the sequences with 7 other groEL gene sequences of Burkholderia species. BLAST analysis revealed that the putative protein encoded by the groEL gene of B. thailandensis has 99.6%, 99.5%, 98.4%, 98.5%, and 96.5% amino acid identity with the groEL of B. pseudomallei, B. mallei, B. cepacia, B. vietnamiensis, and B. fungorum respectively. The amino acid sequences of GroEL of the strains of B. thailandensis and B. pseudomallei all showed >99.5% amino acid identity with each other. The nucleotide sequence of the groEL gene of any of the strains of B. thailandensis showed >99.8% nucleotide identity with that of any of the other strains of B. thailandensis, and the nucleotide sequence of the groEL gene of any of the strains of B. pseudomallei showed >99.5% nucleotide identity with that of any of the other strains of B. pseudomallei. However, the nucleotide sequence of any of the strains of B. thailandensis showed <97.6% nucleotide identity with any of the strains of B. pseudomallei. The amino acid sequences of GroEL of the 20 strains of Burkholderia species all showed >96% amino acid identity with each other. Furthermore, the nucleotide sequence of the groEL genes of the 2 strains of B. cepacia showed >99.5% nucleotide identity with each other, and the nucleotide sequence of the groEL gene of B. mallei showed >99.5% nucleotide identity with any of the strains of B. pseudomallei. The groEL gene sequence is therefore good for distinguishing between B. thailandensis and B. pseudomallei, and the GroEL amino acid and groEL nucleotide sequences of this single gene locus may potentially be useful for a 2-tier hierarchical identification of medically important Burkholderia at the genus and species levels respectively.
Base Sequence↗
Investigating the role of a Verticillium fungicola beta-1,6-glucanase during infection of Agaricus bisporus using targeted gene disruption.
Studies on the mycopathogen Verticillium fungicola have shown the up-regulation of beta-1,6-glucanases when grown in the presence of host cell walls and host cell wall components including chitin. These cell-wall-degrading enzymes are hypothesized to contribute to the pathogenic ability of mycopathogens. A beta-1,6-glucanase gene, VfGlu1, showing high similarity to beta-1,6-glucanase genes from Hypocrea virens, Neotyphodium sp., and Trichoderma harzianum, was isolated using degenerate PCR from V. fungicola, a serious mycopathogen of the cultivated mushroom Agaricus bisporus. Agrobacterium-mediated transformation of V. fungicola using homologous DNA from VfGlu1 resulted in homologous integration at the VfGlu1 locus in 75% of transformants, generating mutants disrupted in the VfGlu1 gene. VfGlu1 mutants displayed reduced virulence and diminished ability to utilize chitin as a carbon source, implicating VfGlu1 in the disease process. Agrobacterium-mediated transformation affords an efficient technique for the disruption of genes associated with disease symptom development in the complex V. fungicola-A. bisporus interaction.
Agaricus↗
Targeted gene correction: a new strategy for molecular medicine.
Advances, over the past 20 years, in the genetic manipulation of mammalian cells form the scientific basis of gene therapy. A number of strategies are presently being used to replace or augment a dysfunctional gene with a correct copy of itself. Now, a novel approach to correct the dysfunctional gene in the chromosome is being developed. Data obtained from biochemical, cell-based and animal studies suggest that the era of gene repair is dawning. It is now conceivable that inherited and non-inherited disorders might be treated with a small molecular tool designed to fix the mutation directly. Here, the conceptualization of the technique and its barriers to success are discussed.
Animals↗
In vivo targeted gene transfer into liver cells mediated by a novel galactosyl-D-lysine/D-serine copolymer.
A novel synthetic polypeptide designed as a DNA binding-molecule for liver-specific, receptor-mediated, gene transfer was used to selectively introduce reporter genes into liver cells in the form of plasmid DNA-ligand complexes. The polypeptide was a D-lysine/D-serine copolymer (Lys/Ser = 33/36 or 53/60) modified with a polyethylene glycol 5000 at the carboxyl-terminus (PLSP). In addition, the lysine epsilon-amino groups were covalently bound to galactose (galactosyl-PLSP), a ligand for the asialoglycoprotein receptor on hepatocytes. Solutions of DNA-ligand complex were prepared by adding solutions of DNA and galactosyl-PLSP in a mixing ratio of DNA:galactosyl-PLSP = 1:3 (w/w). Following injection of the DNA-ligand complex into mice via the tail vein, high levels of luciferase and enhanced green fluorescent protein, which were encoded by the reporter genes, were observed in liver. In contrast, luciferase activity in kidney, spleen, lung and heart was negligible. The high levels of gene expression obtained with DNA/galactosyl-PLSP complexes were achieved without partial hepatectomy or administration of lysosomotrophic agents. Thus, the synthetic Lys/Ser copolymer used in the present study appears to be a promising new tool which enhances the efficacy of receptor-mediated gene transfer into hepatocytes and which may provide another step toward the clinical practice of organ-specific gene therapy.
Animals↗
Gene therapy progress and prospects: targeted gene repair.
The capacity to correct a mutant gene within the context of the chromosome holds great promise as a therapy for inherited disorders but fulfilling this promise has proven to be challenging. However, steady progress is being made and the development of gene repair as a viable and robust approach is underway. Here, we present some of the recent advances that are helping to shape our thinking about the feasibility and the limitations of this technique. For the most part, these advances center on understanding the regulation of the reaction and validating its application in animal models.
DNA Repair↗
Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates.
Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by active drug efflux out of the cells. In clinical C. albicans strains, fluconazole resistance frequently correlates with constitutive activation of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not expressed detectably in fluconazole-susceptible isolates. However, the molecular changes causing MDR1 activation have not yet been elucidated, and direct proof for MDR1 expression being the cause of drug resistance in clinical C. albicans strains is lacking as a result of difficulties in the genetic manipulation of C. albicans wild-type strains. We have developed a new strategy for sequential gene disruption in C. albicans wild-type strains that is based on the repeated use of a dominant selection marker conferring resistance against mycophenolic acid upon transformants and its subsequent excision from the genome by FLP-mediated, site-specific recombination (MPAR-flipping). This mutagenesis strategy was used to generate homozygous mdr1/mdr1 mutants from two fluconazole-resistant clinical C. albicans isolates in which drug resistance correlated with stable, constitutive MDR1 activation. In both cases, disruption of the MDR1 gene resulted in enhanced susceptibility of the mutants against fluconazole, providing the first direct genetic proof that MDR1 mediates fluconazole resistance in clinical C. albicans strains. The new gene disruption strategy allows the generation of specific knock-out mutations in any C. albicans wild-type strain and therefore opens completely novel approaches for studying this most important human pathogenic fungus at the molecular level.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Magnetofection: enhancing and targeting gene delivery with superparamagnetic nanoparticles and magnetic fields.
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Adenoviridae↗
The ecdysone-inducible Broad-complex and E74 early genes interact to regulate target gene transcription and Drosophila metamorphosis.
Pulses of the steroid hormone ecdysone initiate Drosophila metamorphosis by inducing widespread changes in gene expression. The Broad-Complex (BR-C) and E74 are induced directly by ecdysone and encode families of transcription factors that regulate ecdysone primary- and secondary-response genes. Genetic analyses have revealed that mutations in the BR-C and E74 are lethal during metamorphosis and that these mutations cause some similar lethal phenotypes and alterations in secondary-response gene transcription. To examine whether the BR-C and E74 function together during development, we have combined representative alleles from each BR-C and E74 complementation group. Analysis of the morphological and molecular phenotypes of the double-mutant animals reveals that BR-C and E74 alleles act together to produce both novel and synergistic effects. We find that the BR-C and E74 share functions in puparium formation, pupation and early gene induction. In addition, our evidence suggests that the BR-C and E74 transcription factors may directly interact to regulate the expression of salivary gland glue and late genes. This data is consistent with current models which propose that combinations of ecdysone primary-response genes regulate common morphogenetic pathways during insect metamorphosis.
Alleles↗
Targeted gene transfer increases contractility and decreases oxygen cost of contractility in normal rat hearts.
The aim of this study was to examine how global cardiac gene transfer of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) can influence left ventricular (LV) mechanical and energetic function, especially in terms of O(2) cost of LV contractility, in normal rats. Normal rats were randomized to receive an adenovirus carrying the SERCA2a (SERCA) or beta-galactosidase (beta-Gal) gene or saline by a catheter-based technique. LV mechanical and energetic function was measured in cross-circulated heart preparations 2-3 days after the infection. The end-systolic pressure-volume relation was shifted upward, end-systolic pressure at 0.1 ml of intraballoon water volume was higher, and equivalent maximal elastance, i.e., enhanced LV contractility, was higher in the SERCA group than in the normal, beta-Gal, and saline groups. Moreover, the LV relaxation rate was faster in the SERCA group. There was no significant difference in myocardial O(2) consumption per beat-systolic pressure-volume area relation among the groups. Finally, O(2) cost of LV contractility was decreased to subnormal levels in the SERCA group but remained unchanged in the beta-Gal and saline groups. This lowered O(2) cost of LV contractility in SERCA hearts indicates energy saving in Ca(2+) handling during excitation-contraction coupling. Thus overexpression of SERCA2a transformed the normal energy utilization to a more efficient state in Ca(2+) handling and superinduced the supranormal contraction/relaxation due to enhanced Ca(2+) handling.
Animals↗
Gene expression profiling of target genes in ventilator-induced lung injury.
In the lungs, high-pressure mechanical ventilation induces an inflammatory response similar to that observed in acute respiratory distress syndrome. To further characterize these responses and to compare them with classical inflammatory pathways, we performed gene expression profiling analysis of 20,000 mouse genes in isolated blood-free (to exclude genes from sequestered leukocytes) perfused mouse lungs exposed to low-pressure ventilation (10 cmH2O), high-pressure ventilation (25 cmH2O, overventilation), and LPS treatment. A large number of inflammatory and apoptotic genes were increased by both overventilation and LPS. However, certain growth factor-related genes, as well as genes related to development, cellular communication, and the cytoskeleton, were only regulated by overventilation. We validated and confirmed increased mRNA expression pattern of five genes (amphiregulin, gravin, Nur77, Cyr61, interleukin-11) by real-time PCR; furthermore, we confirmed increased protein expression of amphiregulin by immunohistochemistry and immunoblotting assays. These genes represent novel candidate genes in ventilator-induced lung injury.
A Kinase Anchor Proteins↗
Gene targeting and in vivo gene transfer.
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Animals↗
Efficiency of targeted gene delivery of ligand-poly-L-lysine hybrids with different crosslinks.
Hybrids of a ligand protein crosslinked to a DNA binding protein have been developed as gene delivery vehicles mediated by receptors. To identify the effect of the crosslinks between the ligand and DNA binding protein on gene expression caused by an internalized hybrid-DNA complex, we prepared two kinds of transferrin-poly-L-lysine (TF-PL) hybrids: one was crosslinked by probably cleavable disulfide bonds (TF-ss-PL) and the other was linked by a probably uncleavable Schiff's base (TF-Schiff-PL). The binding affinity of the hybrids to HeLa cells was not different. However, the expression of a reporter gene (for luciferase) bound to these hybrids in HeLa cells transfected with TF-Schiff-PL was greater than that of TF-ss-PL.
Asialoglycoproteins↗