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Results for “gene knockout”

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At least 307 records · Page 17Linked to original sources
PubMed2001

Gene knockout demonstrates that vip3A contributes to the pathogenesis of Bacillus thuringiensis toward Agrotis ipsilon and Spodoptera exigua.

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. To determine the importance of Vip3A for the insect pathogenicity of B. thuringiensis the vip3A gene was deleted from strain HD1, yielding strain HD1Deltavip3A. Compared with HD1, strain HD1Deltavip3A was one-fourth as toxic to Agrotis ipsilon larvae and less than one-tenth as toxic to Spodoptera exigua larvae. When streptomycin was included in the S. exigua diet the toxicity of HD1Deltavip3A was approximately half that of HD1. Addition of HD1 spores increased the toxicity of purified Cry1 protein more than 600-fold against S. exigua, whereas addition of HD1Deltavip3A spores increased toxicity of Cry1 protein approximately 10-fold. These results demonstrate that an important component of B. thuringiensis insecticidal activity against S. exigua is the synthesis of Vip3A protein by B. thuringiensis cells after ingestion of spores and crystal proteins by insect larvae.

Animals↗
PubMed2005

Essential role of chicken ovalbumin upstream promoter-transcription factor II in insulin secretion and insulin sensitivity revealed by conditional gene knockout.

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) has been implicated in the control of blood glucose by its potent effect on expression and signaling of various nuclear receptors. To understand the role of COUP-TFII in glucose homeostasis, conditional COUP-TFII-deficient mice were generated and crossed with mice expressing Cre under the control of rat insulin II gene promoter, resulting in deletion of COUP-TFII in pancreatic beta-cells. Homozygous mutants died before birth for yet undetermined reasons. Heterozygous mice appeared healthy at birth and showed normal growth and fertility. When challenged intraperitoneally, the animals had glucose intolerance associated with reduced glucose-stimulated insulin secretion. Moreover, these heterozygous mice presented a mild increase in fasting and random-fed circulating insulin levels. In accordance, islets isolated from these animals exhibited higher insulin secretion in low glucose conditions and markedly decreased glucose-stimulated insulin secretion. Their pancreata presented normal microscopic architecture and insulin content up to 16 weeks of study. Altered insulin secretion was associated with peripheral insulin resistance in whole animals. It can be concluded that COUP-TFII is a new, important regulator of glucose homeostasis and insulin sensitivity.

Animals↗
PubMed2004

Gene knockout of amyloid precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in primary mouse cortical neurons and embryonic fibroblasts.

Alzheimer's disease is characterised by the accumulation of amyloid-beta peptide, which is cleaved from the copper-binding amyloid-beta precursor protein. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for amyloid-beta precursor protein and amyloid-beta in copper homeostasis. Amyloid-beta precursor protein is a member of a multigene family, including amyloid precursor-like proteins-1 and -2. The copper-binding domain is similar among amyloid-beta precursor protein family members, suggesting an overall conservation in its function or activity. Here, we demonstrate that double knockout of amyloid-beta precursor protein and amyloid precursor-like protein-2 expression results in significant increases in copper accumulation in mouse primary cortical neurons and embryonic fibroblasts. In contrast, over-expression of amyloid-beta precursor protein in transgenic mice results in significantly reduced copper levels in primary cortical neurons. These findings provide cellular neuronal evidence for the role of amyloid-beta precursor protein in copper homeostasis and support the existing hypothesis that amyloid-beta precursor protein and amyloid precursor-like protein-2 are copper-binding proteins with functionally interchangeable roles in copper homeostasis.

Amyloid beta-Protein Precursor↗
PubMed2005

Gene knockout analysis of two gamma-tubulin isoforms in mice.

Gamma-tubulin regulates the nucleation of microtubules, but knowledge of its functions in vivo is still fragmentary. Here, we report the identification of two closely related gamma-tubulin isoforms, TUBG1 and TUBG2, in mice, and the generation of TUBG1- and TUBG2-deficient mice. TUBG1 was expressed ubiquitously, whereas TUBG2 was primarily detected in the brain. The development of TUBG1-deficient (Tubg1-/-) embryos stopped at the morula/blastocyst stages due to a characteristic mitotic arrest: the mitotic spindle was highly disorganized, and disorganized spindles showed one or two pole-like foci of bundled MTs that were surrounded by condensed chromosomes. TUBG2 was expressed in blastocysts, but could not rescue the TUBG1 deficiency. By contrast, TUBG2-deficient (Tubg2-/-) mice were born, grew, and intercrossed normally. In the brain of wild-type mice, TUBG2 was expressed in approximately the same amount as TUBG1, but no histological abnormalities were found in the Tubg2-/- brain. These findings indicated that TUBG1 and TUBG2 are not functionally equivalent in vivo, that TUBG1 corresponds to conventional gamma-tubulin, and that TUBG2 may have some unidentified function in the brain.

Amino Acid Sequence↗
PubMed2004

Gene knockout of glycine transporter 1: characterization of the behavioral phenotype.

N-methyl-d-aspartate receptor (NMDAR) activation requires both the binding of glutamate to its recognition site and occupancy of the strychnine insensitive glycine modulatory site (GMS). Pharmacological studies suggest that the glycine transporter, GlyT1, maintains subsaturating concentrations of glycine at synaptic NMDARs. To characterize further the role of GlyT1, we generated mice in which the gene encoding GlyT1 was inactivated by homologous recombination through insertion of a PGK-Neo cassette in place of exons 2 and 3. Real-time quantitative PCR revealed no transcripts in newborn homozygous [GlyT1(-/-)] mice and a 50% reduction in heterozygous (HZ) [GlyT1(+/-)] mice as compared with WT littermates. The activity of Na(+)-dependent glycine transport in forebrain homogenates was similarly affected. Homozygous mice died within 12 h of birth. In acute hippocampal slices, exogenous glycine or d-serine (10 microM) enhanced NMDAR currents with Schaffer collateral stimulation in WT mice but not HZ mice, suggesting that the GMS was more occupied in the latter. The NMDAR/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ratio of the excitatory postsynaptic currents was significantly increased in the HZ mice. In the water maze, the HZ mice exhibited better spatial retention. Furthermore, HZ mice were less sensitive to an amphetamine disruption of prepulse inhibition than WT mice but were more sensitive to the effects of MK-801. Thus, reduced expression of GlyT1 enhances hippocampal NMDAR function and memory retention and protects against an amphetamine disruption of sensory gating, suggesting that drugs which inhibit GlyT1 might have both cognitive enhancing and antipsychotic effects.

Amino Acid Transport Systems, Neutral↗
PubMed1997

Development and regenerative ability of bladder in the transgenic epidermal growth factor receptor gene knockout mouse.

PURPOSE: During embryogenesis we have previously shown that urothelium is essential for normal bladder growth and development. Urothelial growth may be mediated by peptides of the epidermal growth factor family, since the epidermal growth factor receptor is expressed in bladder urothelium and epidermal growth factor has been shown to induce deoxyribonucleic acid synthesis and migration of urothelial cells in vitro. Bladders from transgenic mice in which the epidermal growth factor receptor gene has been knocked out were used to examine the possible role of epidermal growth factor in bladder growth and development, detrusor neoformation and bladder regeneration. MATERIALS AND METHODS: Whole bladders from transgenic knockout mice 0 to 10 days old were surgically implanted into the "subdetrusor" space of adult athymic nude rat hosts. After 10 days the dome of the host rat bladder was resected with the distal half of the transplanted knockout mouse bladder. Augmentation cystoplasty was then performed on the host rat bladder using acellular tissue matrix with a portion of the acellular matrix sutured directly to the transplanted knockout mouse bladder. The animals were sacrificed 2 or 3 weeks postoperatively. To test the ability of knockout bladder tissue to regenerate into the transplanted matrix species specific Hoechst dye was used to determine whether the cells within the acellular matrix were of host (rat) or transplant (knockout mouse) origin. Immunocytochemical analysis was used to assess muscle neoformation. Controls consisted of wild-type mouse bladders from the same litter. Since epidermal growth factor receptor knockout mice usually die in the neonatal period, the role of the epidermal growth factor receptor signaling pathway in long-term muscle development was evaluated by transplanting knockout and wild-type control bladders under the renal capsule of athymic nude mouse hosts. These mice were sacrificed 30 days later and muscle development was assessed using immunocytochemical analysis. RESULTS: Histologically the transplanted acellular tissue matrix in the experimental and control animals appeared the same, containing well differentiated urothelial and smooth muscle cells that had migrated into the transplanted matrix. Staining with species specific Hoechst dye revealed that urothelial and smooth muscle cells transplanted from the knockout and wild-type mouse bladders invaded and regenerated in the transplanted matrix. There was no apparent difference in the amount of knockout or control mouse tissue in the transplanted matrix. Also, the long-term renal capsule transplants revealed no difference in the amount of smooth muscle in the epidermal growth factor receptor knockout and wild-type bladders. CONCLUSIONS: Signaling through the epidermal growth factor receptor pathway is not necessary for normal bladder development or bladder regeneration after injury.

Animals↗
PubMed2006

The effect of pyruvate decarboxylase gene knockout in Saccharomyces cerevisiae on L-lactic acid production.

A plant- and crop-based renewable plastic, poly-lactic acid (PLA), is receiving attention as a new material for a sustainable society in place of petroleum-based plastics. We constructed a metabolically engineered Saccharomyces cerevisiae that has both pyruvate decarboxylase genes (PDC1 and PDC5) disrupted in the genetic background to express two copies of the bovine L-lactate dehydrogenase (LDH) gene. With this recombinant, the yield of lactate was 82.3 g/liter, up to 81.5% of the glucose being transformed into lactic acid on neutralizing cultivation, although pdc1 pdc5 double disruption led to ineffective decreases in cell growth and fermentation speed. This strain showed lactate productivity improvement as much as 1.5 times higher than the previous strain. This production yield is the highest value for a lactic acid-producing yeast yet reported.

Bioreactors↗
PubMed2000

Cutaneous overexpression of neurotrophin-3 (NT3) selectively restores sensory innervation in NT3 gene knockout mice.

Neurotrophin-3 (NT3) is essential for development of sensory innervation to the skin. NT3 supports the postnatal survival of primary sensory neurons that mediate mechanoreception and their Merkel cell containing touch dome end organs (Airaksinen et al., 1996). In this study we determined whether NT3 overexpressed in the skin could restore innervation lost when endogenous NT3 levels were reduced. Hybrid mice that overexpress NT3 in basal keratinocytes but lack one endogenous NT3 allele (K14-NT3/NT3(+/-)) were compared to NT3 overexpresser (K14-NT3) mice, heterozygous knockout (NT3(+/-)) mice, and littermate control mice. In line with previous analyses, NT3(+/-) mice lost 63% of the Merkel cells associated with touch domes, 67% of touch dome units and the associated SAI innervation. All of these parameters were restored to overexpresser levels in K14-NT3/NT3(+/-) mice. Knockout NT3(+/-) mice also had a 31% reduction of L4/L5 dorsal root ganglion cells and a 24% reduction of myelinated axons in the saphenous cutaneous nerve. These losses were also restored in hybrid K14-NT3/NT3(+/-) mice, though only to control mouse values. These results indicate that overexpression of NT3 in skin of NT3(+/-) knockout mice rescued most cutaneous neurons lost in NT3(+/-) mice, but was unable to rescue NT3-dependent neurons that project to noncutaneous sensory targets.

Animals↗
PubMed2005

Why do Nogo/Nogo-66 receptor gene knockouts result in inferior regeneration compared to treatment with neutralizing agents?

IN-1, the monoclonal antibody against the exon 3-encoded N-terminal domain of Nogo-A, and the Nogo-66 receptor (NgR) antagonist NEP1-40 have both shown efficacy in promoting regeneration in animal spinal cord injury models, the latter even when administered subcutaneously 1 week after injury. These results are supportive of the hypothesis that the Nogo-NgR axis is a major path for inhibition of spinal cord axonal regeneration and uphold the promises of these neutralizing agents in clinical applications. However, mice with targeted disruption of Nogo and NgR have, surprisingly, only modest regenerative capacity (if any) compared with treatment with IN-1 or NEP1-40. Disruption of the Nogo gene by various groups yielded results ranging from significant regenerative improvement in young mice to no improvement. Likewise, knockout of NgR produced some improvement in raphespinal and rubrospinal axonal regeneration, but not that of corticospinal neurons. Other than invoking possible differences in genetic background, we suggest here some possible and testable explanations for the above phenomena. These possibilities include effects of IN-1 and NEP1-40 on the CNS beyond neutralization of Nogo and NgR functions, and the latter's possible role in the CNS beyond that of neuronal growth inhibition.

Animals↗
PubMed2003

Increased neurogenesis and brain-derived neurotrophic factor in neurokinin-1 receptor gene knockout mice.

It has previously been shown that chronic treatment with antidepressant drugs increases neurogenesis and levels of brain-derived neurotrophic factor in the hippocampus. These changes have been correlated with changes in learning and long-term potentiation and may contribute to the therapeutic efficacy of antidepressant drug treatment. Recently, antagonists at the neurokinin-1 receptor, the preferred receptor for the neuropeptide substance P, have been shown to have antidepressant activity. Mice with disruption of the neurokinin-1 receptor gene are remarkably similar both behaviourally and neurochemically to mice maintained chronically on antidepressant drugs. We demonstrate here that there is a significant elevation of neurogenesis but not cell survival in the hippocampus of neurokinin-1 receptor knockout mice. Neurogenesis can be increased in wild-type but not neurokinin-1 receptor knockout mice by chronic treatment with antidepressant drugs which preferentially target noradrenergic and serotonergic pathways. Hippocampal levels of brain-derived neurotrophic factor are also two-fold higher in neurokinin-1 receptor knockout mice, whereas cortical levels are similar. Finally, we examined hippocampus-dependent learning and memory but found no clear enhancement in neurokinin-1 receptor knockout mice. These data argue against a simple correlation between increased levels of neurogenesis or brain-derived neurotrophic factor and mnemonic processes in the absence of increased cell survival. They support the hypothesis that increased neurogenesis, perhaps accompanied by higher levels of brain-derived neurotrophic factor, may contribute to the efficacy of antidepressant drug therapy.

Animals↗
PubMed2005

Effects of HSP70.1/3 gene knockout on acute respiratory distress syndrome and the inflammatory response following sepsis.

Heat shock response has been implicated in attenuating NF-kappaB activation and inflammation following sepsis. Studies utilizing sublethal heat stress or chemical enhancers to induce in vivo HSP70 expression have demonstrated survival benefit after experimental sepsis. However, it is likely these methods of manipulating HSP70 expression have effects on other stress proteins. The aim of this study was to evaluate the role of specific deletion of HSP70.1/3 gene expression on ARDS, NF-kappaB activation, inflammatory cytokine expression, and survival following sepsis. To address this question, we induced sepsis in HSP70.1/3 KO and HSP70.1/3 WT mice via cecal ligation and puncture (CLP). We evaluated lung tissue NF-kappaB activation and TNF-alpha protein expression at 1 and 2 h, IL-6 protein expression at 1, 2, and 6, and lung histopathology 24 h after sepsis initiation. Survival was assessed for 5 days post-CLP. NF-kappaB activation in lung tissue was increased in HSP70.1/3((-/-)) mice at all time points after sepsis initiation. Deletion of HSP70.1/3 prolonged NF-kappaB binding/activation in lung tissue. Peak expression of lung TNF-alpha at 1 and 2 h was also significantly increased in HSP70.1/3((-/-)) mice. Expression of IL-6 was significantly increased at 2 and 6 h, and histopathology revealed a significant increase in lung injury in HSP70.1/3((-/-)) mice. Last, deletion of the HSP70 gene led to increased mortality 5 days after sepsis initiation. These data reveal that absence of HSP70 alone can significantly increase ARDS, activation of NF-kappaB, and inflammatory cytokine response. The specific absence of HSP70 gene expression also leads to increased mortality after septic insult.

Animals↗
PubMed2000

A gene knockout corroborates the integral function of cellular retinol-binding protein in retinoid metabolism.

Continually expanding evidence has moved inexorably toward establishing key functions for cellular retinol-binding protein (CRBP) in retinoid metabolism. These experimental data integrate into a model of CRBP as a chaperone that protects retinol from the cellular milieu and interacts with certain retinoid-metabolizing enzymes. Mutant mice with an inactivated CRBP gene show decreased liver retinyl ester storage, a shorter elimination half-life of liver retinoids, and predisposition to vitamin A deficiency. No morphologic phenotype was observed until vitamin A was exhausted. Although the mechanisms underlying diminished vitamin A in the CRBP-null mice have not been elucidated, the observations support the model of CRBP as a chaperone of retinoid metabolism.

Amino Acids↗
PubMed1999

Normal development of mice and unimpaired cell adhesion/cell motility/actin-based cytoskeleton without compensatory up-regulation of ezrin or radixin in moesin gene knockout.

Ezrin/radixin/moesin (ERM) proteins are general cross-linkers between the plasma membrane and actin filaments. Because their expression is regulated in a tissue-specific manner, each ERM protein has been proposed to have unique functions. On the other hand, experiments at the cellular level and in vitro have suggested their functional redundancy. To assess the possible unique functions of ERM proteins in vivo, the moesin gene located on the X chromosome was disrupted by gene targeting in embryonic stem cells. Male mice hemizygous for the mutation as well as homozygous females were completely devoid of moesin but developed normally and were fertile, with no obvious histological abnormalities in any of the tissues examined. In the tissues of the mutant mice, moesin completely disappeared without affecting the expression levels or subcellular distribution of ezrin and radixin. Also, in platelets, fibroblasts, and mast cells isolated from moesin-deficient mice, targeted disruption of the moesin gene did not affect their ERM-dependent functions, i.e. platelet aggregation, stress fiber/focal contact formation of fibroblasts, and microvillar formation of mast cells, without compensatory up-regulation of ezrin or radixin. These findings favor the notion that ERM proteins are functionally redundant at the cellular as well as the whole body level.

Actins↗
PubMed2001

Conditional gene knockout using Cre recombinase.

Cre recombinase has become an important instrument for achieving precise genetic manipulation in mice. Many of these desired genetic manipulations rely on Cre's ability to direct spatially and temporally specified excision of a predesignated DNA sequence that has been flanked by directly repeated copies of the loxP recombination site. Success in achieving such conditional mutagenesis in mice depends both on the careful design of conditional alleles and on reliable detection of cre gene expression. These procedures include PCR, immunohistochemistry and the use of a recombination-proficient GFP-tagged Cre protein.

Animals↗
PubMed2003

Gene knockout or pharmacological inhibition of poly(ADP-ribose) polymerase-1 prevents lung inflammation in a murine model of asthma.

Airway inflammation is a central feature of asthma and chronic obstructive pulmonary disease. Reactive oxygen species (ROS) contribute to inflammation by damaging DNA, which, in turn, results in the activation of poly(ADP-ribose) polymerase-1 (PARP-1) and depletion of its substrate, nicotinamide adenine dinucleotide. Here we show that prevention of PARP-1 activation protects against both ROS-induced airway epithelial cell injury in vitro and airway inflammation in vivo. H(2)O(2) induced the generation of ROS, PARP-1 activation and concomitant nicotinamide adenine dinucleotide depletion, and release of lactate dehydrogenase in A549 human airway epithelial cells. These effects were blocked by the PARP-1 inhibitor 3-aminobenzamide (3-AB). Furthermore, 3-AB inhibited both activation of the proinflammatory transcription factor nuclear factor-kappaB and expression of the interleukin-8 gene induced by H(2)O(2) in these cells. In a murine model of allergen-induced asthma, 3-AB prevented airway inflammation elicited by ovalbumin. Moreover, PARP-1 knockout mice were resistant to such ovalbumin-induced inflammation. These protective effects were associated with an inhibition of expression of the inducible nitric oxide synthase. These results implicate PARP-1 activation in airway inflammation, and suggest this enzyme as a potential target for the development of new therapeutic strategies in the treatment of asthma as well as other respiratory disorders such as chronic obstructive pulmonary disease.

Allergens↗
PubMed2005

Single-gene knockout of a novel regulatory element confers ethionine resistance and elevates methionine production in Corynebacterium glutamicum.

Despite the availability of genome data and recent advances in methionine regulation in Corynebacterium glutamicum, sulfur metabolism and its underlying molecular mechanisms are still poorly characterized in this organism. Here, we describe the identification of an ORF coding for a putative regulatory protein that controls the expression of genes involved in sulfur reduction dependent on extracellular methionine levels. C. glutamicum was randomly mutagenized by transposon mutagenesis and 7,000 mutants were screened for rapid growth on agar plates containing the methionine antimetabolite D,L-ethionine. In all obtained mutants, the site of insertion was located in the ORF NCgl2640 of unknown function that has several homologues in other bacteria. All mutants exhibited similar ethionine resistance and this phenotype could be transferred to another strain by the defined deletion of the NCgl2640 gene. Moreover, inactivation of NCgl2640 resulted in significantly increased methionine production. Using promoter lacZ-fusions of genes involved in sulfur metabolism, we demonstrated the relief of L-methionine repression in the NCgl2640 mutant for cysteine synthase, o-acetylhomoserine sulfhydrolase (metY) and sulfite reductase. Complementation of the mutant strain with plasmid-borne NCgl2640 restored the wild-type phenotype for metY and sulfite reductase.

Carbon-Oxygen Lyases↗