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Results for “Gene knockout”

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At least 793 records · Page 44Linked to original sources
PubMed2002

Effect of monoamine oxidase A knockout on resistance to long-term exposure to ethanol.

It was shown that transgenic Tg8 mice with monoamine oxidase A (MAO A) gene knockout demonstrate higher resistance to acute ethanol exposure compared to wild type C3H mice. This difference was observed at the early age (28-30 days). Long-term ethanol treatment changed the resistance to hypnotic, but not hypothermic action of this agent. Seven-day exposure increased the resistance to ethanol-induced narcotic sleep in Tg8 and C3H mice. After 30-day ethanol treatment the duration of narcotic sleep sharply decreased in C3H mice and increased in Tg8 mice, which attested to their decreased tolerance to ethanol.

Aging↗
PubMed2001

Lack of mu-opioid receptor leads to an increase in the NMDA receptor subunit mRNA expression and NMDA-induced convulsion.

The present study investigated in situ hybridization of N-methyl-D-aspartate (NMDA) receptor (NR) subunit mRNA and convulsion induced by intracerebroventricular injection of NMDA, in order to examine changes in NMDA receptor function in mu-opioid receptor gene knockout mice. Levels of NR1 and NR2A subunit mRNA were significantly increased in the parietal cortex (8.4 and 10.6%, respectively) and hypothalamus (8.7 and 15.2%, respectively) in mu-opioid receptor knockout mice. Levels of NR2B subunit mRNA were noted to be increased in the parietal cortex (9.1%), thalamus (7.7%), and hypothalamus (10.4%) in mu-opioid receptor knockout mice. The ED(50) for NMDA-induced convulsion in wild-type mice was 0.20 microg/10 microl/mouse. The ED(50) in mu-opioid receptor knockout mice was 0.14 microg/10 microl/mouse. There is a significant difference in the potency ratio of wild-type mice versus knockout mice (potency ratio: 1.44, P < 0.05). These results indicate that mu-opioid receptor knockout mice are more sensitive to NMDA-induced convulsion. Therefore, these results suggest that absence of mu-opioid receptor gene is accompanied by changes in the NMDA receptor system which can modulate the synaptic excitability in the process such as convulsion or epilepsy.

Animals↗
PubMedDate not supplied

Retinoic acid and retinoic acid receptors in development.

The vitamin A derivative retinoic acid (RA) and related compounds (retinoids) are utilized as signaling molecules in a diverse array of developmental and physiological regulatory processes, including many important in the developing and mature nervous system. Retinoids function by interaction with high affinity receptors of the nuclear receptor family, which also mediate the effects of steroid and thyroid hormones and which act in the nucleus as transcription factors. This review summarizes current knowledge of the molecular mechanisms of retinoid action, the complex role of retinoid receptors in a variety of hormonal signaling processes, and illustrates current efforts to more fully understand the biological functions of retinoid receptors through analysis of downstream gene regulatory networks and studies of mouse gene knockout systems.

Animals↗
PubMed2000

Tissue-specific actions of thyroid hormone: insights from animal models.

The major developmental targets for thyroid hormone are the brain, small intestine, and bone. Clear defects in gene regulation and tissue function as a consequence of TR gene inactivation can additionally be shown in the pituitary, hypothalamus, heart, and liver. TR gene knockout models show a clear distinction between thyroid hormone requirements for development and those that are required for functions in the adult animal. T3-mediated gene repression appears especially important in a number of tissues including brain, pituitary, and the heart. Preliminary evaluation of the combined TR knockout models suggests that hypothyroidism is associated with more significant abnormalities than receptor deficiency, indicating that the repressive action of the unliganded receptor may have physiological relevance. These various animal models should be very useful to design and test thyroid hormone analogues to selectively stimulate desired thyroid hormone actions.

Animals↗
PubMed2001

Nramp1 gene affects selective early steps in macrophage-mediated anti-cryptococcal defense.

Cryptococcus neoformans is an opportunistic fungus responsible for severe and often recurrent meningoencephalitis in immunodepressed patients. Initial evidence suggests that C. neoformans is a facultative intracellular pathogen; however, the strategies by which C. neoformans undergoes survival and eventually proliferation have not been elucidated. We investigated the role of Nrampl gene in macrophage-mediated anti-cryptococcal defense. Using cell lines expressing the functional, mutated or knockout gene, it was established that Nramp1 (1) is not involved in the phagocytic event, (2) influences anti-cryptococcal activity in the early steps but not at later times, and (3) is unrelated to the biomolecular pathways through which C. neoformans impairs macrophage secretory response. Although the functional role of Nramp1 is still far from being elucidated, the present data add insight into its involvement in macrophage-mediated antimicrobial defense, particularly in the initial steps allowing C. neoformans growth inhibition.

Animals↗
PubMed2004

OptiRNAi, an RNAi design tool.

RNA interference (RNAi), a recently developed reverse genetics tool, has many advantages compared to traditional gene knockout methods. Appropriate selection of double stranded RNAs identical to a specific region(s) of the target gene is critical for the successful implementation of this technology. Recently, Elbashir et al. [Methods 26 (2002) 199] has established empirical criteria for siRNA sequence selection that significantly improved the success rate for RNAi attempts. We have developed OptiRNAi, a computational tool, which uses the Elbashir et al. criteria to predict appropriate target sequences for siRNA production. Specificity of these siRNAs for the target of interest can then be assessed by the investigator using the embedded Blast search engine optimized for RNAi design. Thus, OptiRNAi is an efficient and user friendly tool for RNAi design based on criteria that are more stringent than other available tools.

Animals↗
PubMed2002

Mouse cone arrestin gene characterization: promoter targets expression to cone photoreceptors.

Cone arrestin (CAR) is a novel member of the arrestin superfamily expressed in retinal cone photoreceptors and the pineal gland. To understand the regulatory mechanisms controlling its cone- and pineal-specific expression, and to facilitate further functional studies using gene knockout approaches, we characterized the genomic organization and the 5'-flanking region of the mouse CAR (mCAR) gene. The mCAR gene is comprised of 17 exons and 16 introns, encoding five alternatively spliced transcripts. A 215-bp proximal promoter fragment containing a TATA box, an Sp1 site and four cone-rod homeobox-binding sites is sufficient to direct expression in cultured retinoblastoma cells and in cone photoreceptors and the pineal gland in transgenic Xenopus laevis.

Alternative Splicing↗
PubMed2006

Wound healing is defective in mice lacking tetraspanin CD151.

The tetraspanin CD151 forms complexes in epithelial cell membranes with laminin-binding integrins alpha6beta4, alpha3beta1, and alpha6beta1, and modifies integrin-mediated cell migration in vitro. We demonstrate in this study that CD151 expression is upregulated in a distinct temporal and spatial pattern during wound healing, particularly in the migrating epidermal tongue at the wound edge, suggesting a role for CD151 in keratinocyte migration. We show that healing is significantly impaired in CD151-null mice, with wounds gaping wider at 7 days post-injury. The rate of re-epithelialization of the CD151-null wounds is adversely affected, with significantly less wound area being covered by migrating epidermal cells. Our studies reveal that although laminin levels are similar in wild-type and CD151-null wounds, the organization of the laminin in the basement membrane is impaired. Furthermore, upregulation of alpha6 and beta4 integrin expression is adversely affected in CD151-null mice wounds. In contrast, we find no significant effect of CD151 gene knockout on alpha3 and beta1 integrin expression in wound repair. We suggest that mice lacking the CD151 gene are defective in wound healing, primarily owing to impairment of the re-epithelialization process. This may be due to defective basement membrane formation and epithelial cell adhesion and migration.

Animals↗
PubMed2005

DNA microarray-based identification of genes associated with glycopeptide resistance in Staphylococcus aureus.

Six pairs of transcription profiles between glycopeptide-intermediate S. aureus (GISA [or vancomycin-intermediate S. aureus; VISA]) and glycopeptide-susceptible S. aureus (vancomycin-susceptible S. aureus [VSSA], including glycopeptide-susceptible isogenic mutants from VISA) strains were compared using a microarray. Ninety-two open reading frames which were or tended to be increased in transcription in VISA in at least five out of six array combination pairs were evaluated for their effects on glycopeptide susceptibility by introducing these genes one by one into VSSA strain N315 to construct an overexpression library. By screening the library, 17 genes including 8 novel genes were identified as associated with glycopeptide resistance since their experimental overexpression reduced vancomycin and/or teicoplanin susceptibility of N315. The raised MICs of vancomycin and teicoplanin were 1.25 to 3.0 and 1.5 to 6.0 mg/liter, respectively, as compared to 1.0 mg/liter of N315. Three of these genes, namely graF, msrA2, and mgrA, also raised the oxacillin MIC from 8.0 mg/liter for N315 to 64 to approximately 128 mg/liter when they were overexpressed in N315. Their contribution to vancomycin and beta-lactam resistance was further supported by gene knockout and trans-complementation assay. By using a plasmid-based promoter-green fluorescent protein gene (gfp) transcriptional fusion system, graF promoter-activated cells were purified, and subsequent susceptibility tests and Northern blot analysis demonstrated that the cells with up-regulated activity of graF promoter showed reduced susceptibility to vancomycin, teicoplanin, and oxacillin. In addition, cell morphology studies showed that graF and msrA2 overexpression increased cell wall thickness of N315 by factors of 23.91 and 22.27%, respectively, accompanied by glycopeptide MIC increments of 3- to 6-fold, when they were overexpressed in N315. Moreover, extended experiments and analyses indicate that many of the genes identified above are related to the cell wall biosynthetic pathway, including active nutrient transport systems. We propose that the genes which raise glycopeptide resistance in S. aureus function toward altering the cell wall metabolic pathway.

Anti-Bacterial Agents↗
PubMed2004

Deciphering the renal code: advances in conditional gene targeting.

Several powerful new techniques can examine gene function in mammals. Recombinase systems and kidney-specific promoters enable gene knockout and overexpression. Genetic systems induced on administration or removal of antibiotics or hormones permit control of gene expression. Gene silencing using short interfering RNA expression systems should accelerate loss-of-function studies. Thorough characterization of animals that have undergone conditional gene targeting has already provided insights into renal development and diseases. Here we discuss the advantages and pitfalls of currently available gene-targeting systems.

Animals↗
PubMed1996

Models of experimental hypertension in mice.

Experimental models of hypertension in various animals are useful in the research of vasoactive mechanisms. Recombinant DNA technology has produced genetically engineered animals, mostly mice, useful in hypertension research. However, the development of hypertensive models in mice is fraught with technical difficulties. We describe here the successful development in mice of two common types of experimental hypertension: the renovascular two-kidney, one clip and mineralocorticoid deoxycorticosterone-salt models. By adapting technology previously used in rats, we succeeded in developing hypertension (defined as systolic pressures higher than 140 mm Hg) in more than 50% of mice so treated. We also adapted the methodology for indirect tail-cuff blood pressure measurements as well as for direct intra-arterial monitoring of blood pressure in conscious, freely moving mice. Application of these techniques in transgenic or gene knockout mice with altered vasoactive hormones or receptors should allow elucidation of the role of the target gene products in various types of hypertension.

Animals↗
PubMed2006

TLR4-dependent NF-kappaB activation and mitogen- and stress-activated protein kinase 1-triggered phosphorylation events are central to Helicobacter pylori peptidyl prolyl cis-, trans-isomerase (HP0175)-mediated induction of IL-6 release from macrophages.

Helicobacter pylori infection is associated with the local production of chemokines and cytokines, of which IL-6 is overexpressed at the margin of gastric ulcer in H. pylori-positive gastritis. Cells of the monocytic lineage are the major sources of IL-6, and mononuclear cell infiltration in the lamina propria is characteristic of H. pylori-induced chronic infection. Our study shows for the first time that a secreted peptidyl prolyl cis-, trans-isomerase, HP0175 elicits IL-6 gene expression and IL-6 release from macrophages. An isogenic strain inactivated in the HP0175 gene (knockout) was attenuated in its IL-6-inducing ability, which was restored after complementation with the HP0175 gene. The specificity of the HP0175-induced effect was confirmed by the fact that rHP0175 purified from HEK293 cells could also induce IL-6 release, ruling out the possibility that the observed effect was due to bacterial contaminants. HP0175 was capable of interacting directly with the extracellular domain of TLR4. HP0175-induced IL-6 gene expression was critically dependent on TLR4-dependent NF-kappaB and MAPK activation. TLR4/PI3K-dependent ERK1/2 and p38 MAPK signaling converged upon activation of mitogen- and stress-activated protein kinase 1 (MSK1). The central role of MSK1 was borne out by the fact that silencing of MSK1 expression abrogated HP0175-mediated NF-kappaB-dependent IL-6 gene transcription. MSK1 regulated the recruitment of p65 and phopho-Ser(10)-histone H3 to the IL-6 promoter. HP0175 therefore regulated IL-6 gene transcription through chromatin modification at the IL-6 promoter.

Blotting, Western↗
PubMed2004

Functional genomic approaches using the nematode Caenorhabditis elegans as a model system.

Since the completion of the genome project of the nematode C. elegans in 1998, functional genomic approaches have been applied to elucidate the gene and protein networks in this model organism. The recent completion of the whole genome of C. briggsae, a close sister species of C. elegans, now makes it possible to employ the comparative genomic approaches for identifying regulatory mechanisms that are conserved in these species and to make more precise annotation of the predicted genes. RNA interference (RNAi) screenings in C. elegans have been performed to screen the whole genome for the genes whose mutations give rise to specific phenotypes of interest. RNAi screens can also be used to identify genes that act genetically together with a gene of interest. Microarray experiments have been very useful in identifying genes that exhibit co-regulated expression profiles in given genetic or environmental conditions. Proteomic approaches also can be applied to the nematode, just as in other species whose genomes are known. With all these functional genomic tools, genetics will still remain an important tool for gene function studies in the post genome era. New breakthroughs in C. elegans biology, such as establishing a feasible gene knockout method, immortalized cell lines, or identifying viruses that can be used as vectors for introducing exogenous gene constructs into the worms, will augment the usage of this small organism for genome-wide biology.

Animals↗
PubMed2001

Studying TGF-beta superfamily signaling by knockouts and knockins.

The transforming growth factor beta (TGF-beta) superfamily has profound effects on many aspects of animal development. In the last decade, our laboratory and others have performed in vivo functional studies on multiple components of the TGF-beta superfamily signal transduction pathway, including upstream ligands, transmembrane receptors, receptor-associated proteins and downstream Smad proteins. We have taken gene knockout approaches to generate null alleles of the genes of interest, as well as a gene knockin approach to replace the mature region of one TGF-beta superfamily ligand with another. We found that activin betaB, expressed in the spatiotemporal pattern of activin betaA, can function as a hypomorphic allele of activin betaA and rescue the craniofacial defects and neonatal lethal phenotype of activin betaA-deficient mice. With the knockout approach, we have shown that the expression pattern of a component in the TGF-beta superfamily signal transduction cascade does not necessarily predict its in vivo function. Two liver-specific activins, activin betaC and activin betaE are dispensable for liver development, regeneration and function, whereas ubiquitously expressed Smad5 has specific roles in the development of multiple embryonic and extraembryonic tissues.

Activins↗
PubMed1998

Cytochrome P450 knockout mice: new toxicological models.

1. Despite the frenetic pace of cytochrome P450 (CYP) research, important questions remain about the possible role of xenobiotic-metabolizing CYP in development and cellular homeostasis. 2. The recent experimental development of CYP null mouse lines has provided valuable new models for addressing these issues, as well as providing novel in vivo models to study the precise role of CYP in chemical-mediated toxicity. 3. The present review summarizes results from the three Cyp gene knockout mouse lines generated so far and looks at likely future directions of this research field. 4. Also discussed are null mouse lines with targeted mutations in genes encoding receptors having primary roles in Cyp gene regulation.

Animals↗
PubMed2002

Conditional disruption of the peroxisome proliferator-activated receptor gamma gene in mice results in lowered expression of ABCA1, ABCG1, and apoE in macrophages and reduced cholesterol efflux.

Disruption of the peroxisome proliferator-activated receptor gamma (PPAR gamma) gene causes embryonic lethality due to placental dysfunction. To circumvent this, a PPAR gamma conditional gene knockout mouse was produced by using the Cre-loxP system. The targeted allele, containing loxP sites flanking exon 2 of the PPAR gamma gene, was crossed into a transgenic mouse line expressing Cre recombinase under the control of the alpha/beta interferon-inducible (MX) promoter. Induction of the MX promoter by pIpC resulted in nearly complete deletion of the targeted exon, a corresponding loss of full-length PPAR gamma mRNA transcript and protein, and marked reductions in basal and troglitazone-stimulated expression of the genes encoding lipoprotein lipase, CD36, LXR alpha, and ABCG1 in thioglycolate-elicited peritoneal macrophages. Reductions in the basal levels of apolipoprotein E (apoE) mRNA in macrophages and apoE protein in total plasma and high-density lipoprotein (HDL) were also observed in pIpC-treated PPAR gamma-MXCre(+) mice. Basal cholesterol efflux from cholesterol-loaded macrophages to HDL was significantly reduced after disruption of the PPAR gamma gene. Troglitazone selectively inhibited ABCA1 expression (while rosiglitazone, ciglitazone, and pioglitazone had little effect) and cholesterol efflux in both PPAR gamma-deficient and control macrophages, indicating that this drug can exert paradoxical effects on cholesterol homeostasis that are independent of PPAR gamma. Together, these data indicate that PPAR gamma plays a critical role in the regulation of cholesterol homeostasis by controlling the expression of a network of genes that mediate cholesterol efflux from cells and its transport in plasma.

ATP Binding Cassette Transporter 1↗
PubMedDate not supplied

The genes of systemic autoimmunity.

Autoimmune diseases include a wide spectrum of disorders, which have been divided into systemic and organ-specific disorders. Lupus, the prototypic systemic autoimmune disease, is characterized by female predominance, multiorgan pathology, and autoantibodies, primarily directed against nuclear antigens. The disease is heterogeneous, with variable organ involvement, serology, and clinical course. Susceptibility to lupus is inherited as a polygenic trait with added contributions from environmental and stochastic variance. Concerted efforts have recently been made by several laboratories to define the genetic basis of this disease in predisposed mice and humans. The identification of the Fas/FasL defects in lpr and gld lupus mice was the first example of spontaneous mutations of apoptosis-promoting genes being associated with systemic autoimmunity. This research was instrumental in clarifying the roles of these genes in tolerance and immunoregulation, and in extrapolating these results to other autoimmune diseases, as well as cancer and transplantation. To these findings have been added those from transgenic and gene knockout mouse studies that have helped to define the systemic autoimmunity-inducing or -modifying effects of specific genes in normal background and lupus-congenic mice. In addition, the findings from genome-wide searches have begun to identify predisposing loci (and ultimately genes) for the spontaneous lupus-like diseases in various mouse strains and in humans. The emerging picture is that multiple genetic contributions can independently lead to systemic autoimmunity in mice, which reinforces the view that human lupus may be similarly composed of diverse genotypes. This complexity underscores the importance of defining the predisposing alleles and mechanisms of action, an undertaking that is certainly feasible given current technologies and future advances in the definition of mammalian genomes.

Animals↗
PubMed1997

Development of a hammerhead ribozyme against bcl-2. I. Preliminary evaluation of a potential gene therapeutic agent for hormone-refractory human prostate cancer.

BACKGROUND: The bcl-2 oncoprotein suppresses apoptosis and, when overexpressed in prostate cancer cells, makes these cells resistant to a variety of therapeutic agents, including hormonal ablation. Therefore, bcl-2 provides a strategic target for the development of gene knockout therapies to treat human prostate cancers. Towards this end, we have synthesized an anti-bcl-2 gene therapeutic reagent based on ribozyme technology and have tested its effectiveness against bcl-2 mRNA in vitro and in vivo. METHODS: A divalent hammerhead ribozyme was constructed by recombining two catalytic RNA domains into an antisense segment of the coding region for human bcl-2 mRNA. A disabled ribozyme lacking catalytic activity was also constructed as a control reagent for our experiments. The ribozymes were tested for endonucleolytic activity against synthetic and natural bcl-2 mRNAs. Simple transfection procedures were then utilized to introduce the ribozymes into cultured prostate cancer cells (LNCaP derivatives). We measured the effects of the ribozymes on endogenous expression of bcl-2 mRNA and protein in these cells as well as their ability to induce apoptosis. RESULTS: The functional but not the disabled ribozyme was able to rapidly degrade bcl-2 mRNA in vitro, without the requirement for any other cellular protein or factor. When directly transfected into LNCaP cell variants, it significantly reduced bcl-2 mRNA and protein levels within 18 hr of treatment. This activity was sufficient to induce apoptosis in a low-bcl-2-expressing variant of LNCaP, but not in a high-bcl-2-expressing LNCaP line. For the high-bcl-2-expressing variant, however, it did restore the ability to genetically respond to a secondary apoptotic agent, phorbol ester, as evidenced by the renewed ability of phorbol ester to induce NGF1A mRNA in these cells. CONCLUSIONS: This study supports the potential utility of an anti-bcl-2 ribozyme reagent for reducing or eliminating bcl-2 expression from hormone-refractory prostate cancer cells and for killing prostate cancer cells. As such, it is the first step toward an effective gene therapy against hormone-refractory human prostate cancers.

Base Sequence↗