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Results for “Gene knockout”

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PubMed1999

B1 B cell numbers and antibodies against phosphorylcholine and LPS are increased in IL-6 gene knockout mice.

Peritoneal cavity cells were isolated from IL6-gene knockout (IL6(-/-)) and wild-type mice and stained for expression of IgM, CD5, and CD23. B1 cell (IgM(+)/CD23(-), CD5(+)/IgM(+)) numbers were increased twofold in IL6(-/-) mice compared to normals while IgM(+)/CD23(+) (B2) cell numbers were reduced threefold. Intestinal antibody levels were also determined for both total immunoglobulin and phosphorylcholine (PC)-specific and LPS-specific antibody following oral challenge with attenuated Salmonella typhimurium. Total immunoglobulin levels (IgM, IgG, and IgA) were reduced 60-80% in intestinal secretions of IL6(-/-) mice compared to wild-type controls; however, PC-specific antibody was significantly higher in IL6(-/-) mice. Anti-LPS antibodies were also three- to sevenfold higher in IL6(-/-) mice compared to controls following Salmonella challenge. These data suggest that in IL6(-/-) mice the development of mucosal B2 cells is impaired but that intestinal B1 cells responding to microbial antigens such as PC and LPS develop normally and are fully functional.

Animals↗

PubMed2002

Enhanced antinociception by intrathecally-administered morphine in histamine H1 receptor gene knockout mice.

We previously reported that histamine H(1) receptor gene knockout mice (H1KO) showed lower spontaneous nociceptive threshold to pain stimuli when compared to wild-type mice. The objective of the present study was to examine the antinociceptive effect of intrathecally-administered morphine in H1KO mice. The antinociceptive effects of morphine were examined using assays for thermal (tail-flick, hot-plate, paw-withdrawal), mechanical (tail-pressure) and chemical nociception (formalin and capsaicin tests) using H1KO and wild-type mice. In these nociceptive assays, intrathecally-administered morphine produced significant antinociceptive effects in wild-type mice. The antinociceptive effect produced by intrathecally administered morphine was enhanced in the knockout mice. We also examined the effect of an histamine H(1) receptor antagonist, an active (d-) isomer of chlorpheniramine, on morphine-induced antinociception in ICR mice. The intrathecal co-administration of d-chlorpheniramine enhanced the effect of morphine in all nociceptive assays examined. The pharmacological experiments using d-chlorpheniramine further substantiate the evidence for the histamine H(1) receptor-mediated suppression of morphine-induced antinociception. These results suggest that existing H(1) receptors play an inhibitory role in morphine-induced antinociception at the spinal cord level.

Analgesics↗

PubMed2002

Cyclooxygenase-1 gene knockout does not alter middle cerebral artery occlusion in a mouse stroke model.

Cyclooxygenase (COX) is crucial in inflammation; COX-1 is constitutional, and COX-2 is inducible. In this study, neurological function and infarct volume were evaluated at 24 h after permanent endovascular middle cerebral artery occlusion (MCAO) in both COX-1-gene knockout (homozygous or heterozygous) and wide-type mice. Similar infarct volumes and neurological deficits were seen among mice of different genotypes. There was no difference among the groups in arterial blood pressure and regional cerebral blood flow during the first 30 min of ischemia. Our results failed to confirm the harmful effect of losing COX-1 activity due to gene knockout in a permanent endovascular MCAO mouse stroke model.

Animals↗

PubMed1997

Beta3-adrenergic receptors on white and brown adipocytes mediate beta3-selective agonist-induced effects on energy expenditure, insulin secretion, and food intake. A study using transgenic and gene knockout mice.

beta3-Adrenergic receptors (beta3-ARs) are expressed predominantly on white and brown adipocytes, and acute treatment of mice with CL 316,243, a potent and highly selective beta3-AR agonist, produces a 2-fold increase in energy expenditure, a 50-100-fold increase in insulin levels, and a 40-50% reduction in food intake. Recently, we generated gene knockout mice lacking functional beta3-ARs and demonstrated that each of these responses were mediated exclusively by beta3-ARs. However, the tissue site responsible for producing these actions is unknown. In the present study, genetically engineered mice were created in which beta3-ARs are expressed exclusively in white and brown adipocytes (WAT+BAT-mice), or in brown adipocytes only (BAT-mice). This was accomplished by injecting tissue-specific beta3-AR transgenic constructs into mouse zygotes homozygous for the beta3-AR knockout allele. Control, knockout, WAT+BAT, and BAT-mice were then treated acutely with CL, and the effects on various parameters were assessed. As previously observed, all effects of CL were completely absent in gene knockout mice lacking beta3-ARs. The effects on O2 consumption, insulin secretion, and food intake were completely rescued with transgenic re-expression of beta3-ARs in white and brown adipocytes (WAT+BAT-mice), demonstrating that each of these responses is mediated exclusively by beta3-ARs in white and/or brown adipocytes, and that beta3-ARs in other tissue sites were not required. Importantly, transgenic re-expression of beta3-ARs in brown adipocytes only (BAT-mice) failed to rescue, in any way, CL-mediated effects on insulin levels and food intake and only minimally restored effects on oxygen consumption, indicating that any effect on insulin secretion and food intake, and a full stimulation of oxygen consumption required the presence of beta3-ARs in white adipocytes. The mechanisms by which beta3-AR agonist stimulation of white adipocytes produces these responses are unknown but may involve novel mediators not previously known to effect these processes.

Adipocytes↗

PubMed1999

Essential role of inducible nitric oxide synthase in monophosphoryl lipid A-induced late cardioprotection: evidence from pharmacological inhibition and gene knockout mice.

BACKGROUND: Monophosphoryl lipid A (MLA), a nontoxic analogue of endotoxin, is a pharmacological agent that is known to have anti-ischemic effects. Mechanisms involved with the cardioprotection are still unclear. A role for inducible nitric oxide synthase (iNOS) was recently proposed. We tested this hypothesis using S-methylisothiourea (SMT), one of the specific pharmacological inhibitors of iNOS, as well as iNOS gene knockout mice. METHODS AND RESULTS: Adult male ICR or B6,129 mice were pretreated with either MLA 35 or 350 microg/kg IP (MLA35 or MLA350) or vehicle 24 hours before global ischemia/reperfusion, which was carried out in a Langendorff isolated perfused heart model (n=8 to 9 per group). Another group of MLA350 mice received SMT 3 mg/kg IP 30 minutes before heart perfusion. Ventricular contractile function and heart rate were not different between the groups during the preischemia and reperfusion periods (P>0.05). Preischemic basal coronary flow was significantly increased in all MLA350 but not MLA35 mice. Myocardial infarct size was reduced significantly, from 26.9+/-2.9% of risk area in vehicle-treated mice to 13.5+/-2.4% in the MLA350 group (mean+/-SEM, P<0.05). This reduction in infarct size was accompanied by augmented nitrite/nitrate accumulation, from 0.23+/-0. 05 nmol/mg protein in the vehicle group to 0.97+/-0.27 nmol/mg protein in MLA350 mice (P<0.01). Infarct size increased significantly, to 22.2+/-2.8% after treatment with SMT in the MLA350 group. Furthermore, MLA350 failed to reduce infarct size in iNOS knockout mice (25.5+/-3.6%). CONCLUSIONS: These results demonstrate a direct association of infarct size reduction with increased NO production with MLA350. An obligatory role for iNOS in mediating the cardioprotective effect induced by MLA was confirmed with the pharmacological inhibition and gene knockout mice.

Animals↗

PubMed2004

Enhanced gastric emptying of a liquid gastric load in mice lacking cholecystokinin-B receptor: a study of CCK-A,B, and AB receptor gene knockout mice.

BACKGROUND: Although cholecystokinin (CCK) has been shown to inhibit gastric emptying via CCK-A receptors (CCK-ARs), the role of CCK-B receptors (CCK-BRs) has not been verified. We examined whether gastric emptying of a nonnutrient liquid load was modified in CCK-AR, BR, and ARBR gene knockout mice. METHODS: A liquid gastric load prepared with phenol red was administered via an orogastric tube (0.15 ml/mouse). The animals were killed by decapitation, and gastric emptying was estimated at 10 and 30 min after ingestion. The effects of the sulfated form of CCK-8 (CCK-8S) and of graded doses of atropine were examined. In addition, a proton pump inhibitor was administered to wild-type mice to examine the contribution of gastric acid to emptying. RESULTS: Gastric emptying was significantly enhanced in mice lacking CCK-BR, as compared with wild-type and CCK-AR(-/-) mice. CCK-8S inhibited gastric emptying in mice with CCK-AR, but not in mice without CCK-AR. A proton pump inhibitor did not affect gastric emptying. Atropine dose dependently inhibited gastric emptying in all genotypes. The thickness of smooth muscle was comparable for all genotypes. CONCLUSIONS: The gastric emptying of a nonnutrient liquid load was enhanced in mice without CCK-BR, although the precise mechanism is not known. Although cholecystokinin (CCK) has been shown to inhibit gastric emptying via CCK-A receptors (CCK-ARs), the role of CCK-B receptors (CCK-BRs) has not been verified. We examined whether gastric emptying of a nonnutrient liquid load was modified in CCK-AR, BR, and ARBR gene knockout mice.

Animals↗

PubMed2005

Production of alpha 1,3-galactosyltransferase gene knockout pigs expressing both human decay-accelerating factor and N-acetylglucosaminyltransferase III.

Heterozygous alpha 1,3-galactosyltransferase (GT) gene knockout pigs were produced with transgenic pig fetal cells expressing both human decay-accelerating factor (hDAF) and N-acetylglucosaminyltransferase III (GnT-III). In this study, we assessed the gene targeting efficiency in the transgenic pig fetal cells derived from different fetal tissues such as brain, skin, heart, and liver, or fetal carcass. Targeted cell colonies were selected by hygromycin B. The GT-knockout colonies (KO colonies) were obtained equally from the cells derived from all tissues except liver. Staining with five antibodies against intermediate filaments, all examined KO cell lines stained positive for vimentin with the exception of a colony that stained positive for both vimentin and glial fibrillary acidic protein simultaneously. This is the first study to produce KO cells from the astrocytes. Some of these KO cell lines were used for nuclear transfer (NT) to obtain KO pig fetuses. Fourteen fetuses were obtained from two recipients of the embryo transfer and eight of them had normal ploidy. The cells from the KO pig fetuses were also used for NT to produce cloned KO pigs. Two healthy clone pigs were born. These pigs were determined to have a heterozygous knockout GT gene and the two transgenes. The cells collected from the KO pigs were shown to have similar expression levels of hDAF and GnT-III compared to their original transgenic pigs and less than a half levels of the alphaGal epitopes existed in wild-type pig cells.

Animals↗

PubMed2001

Using gene knockouts to investigate plant metabolism.

Arabidopsis functional genomics resources now make the isolation of knockout mutants in any gene of choice both realistic and increasingly straightforward. Coupled with the completion of the genome sequence, this reverse genetics approach provides a platform facilitating dramatic progress in our understanding of fundamental aspects of plant metabolism. Recent experience shows that knockouts of genes encoding enzymes of primary metabolism can produce mutants with clear and sometimes unexpected phenotypes. They can provide new information about old pathways. Specific functions for individual members of multigene families can be revealed. Knockouts of enzymes of undefined function can lead to the discovery of those functions, and the analysis of enzymes which have previously never been studied at the biochemical level offers the potential to reveal new pathways of plant metabolism. Furthermore, the mutants isolated provide the starting point for genetic modification experiments to determine exactly how metabolism fuels growth and development, so providing a rational basis for the future modification of plant productivity.

Arabidopsis↗

PubMed2002

A robust inducible-repressible promoter greatly facilitates gene knockouts, conditional expression, and overexpression of homologous and heterologous genes in Tetrahymena thermophila.

The Cd(2+)-inducible metallothionein (MTT1) gene was cloned from Tetrahymena thermophila. Northern blot analysis showed that MTT1 mRNA is not detectable in the absence of Cd(2+), is induced within 10 min of its addition, is expressed in proportion to its concentration, and rapidly disappears upon its withdrawal. Similarly, when the neo1 gene coding region flanked by the MTT1 gene noncoding sequences was used to disrupt the MTT1 locus, no transformants were observed in the absence of Cd(2+), and the number of transformants was proportional to increased Cd(2+) concentration. The neo3 cassette, in which the MTT1 promoter replaced the histone gene HHF1 promoter of the previously used neo2 cassette, transformed cells at much higher frequencies than neo2 and produced germ-line knockouts where neo2 had failed. Rescuing the progeny of a mating of gamma-tubulin gene, GTU1, knockout heterokaryons with a GTU1 gene inserted into the MTT1 locus yielded >75 times more transformants than rescuing with the wild-type GTU1 gene itself. When cells rescued with the MTT1-GTU1 chimeric gene were transferred to medium lacking Cd(2+), they stopped growing and had phenotypic changes indistinguishable from cells containing only disrupted GTU1 genes. Thus, it is now possible to create conditional lethal mutants and study the terminal phenotypes of null mutations for essential genes by replacing the endogenous gene with one under the control of the MTT1 promoter. The MTT1 promoter also resulted in approximately 30 times more overexpression of the IAG48[G1] surface antigen gene of the ciliate fish parasite Ichthyophthirius multifiliis than the highly expressed BTU1 promoter, accounting for approximately 1% of the total cell protein. Thus, the MTT1 promoter should enable routine over-expression of endogenous and foreign genes in Tetrahymena.

Animals↗

PubMed2002

Disturbed tooth development in parathyroid hormone-related protein (PTHrP)-gene knockout mice.

Parathyroid hormone-related protein (PTHrP) is involved in epithelial-mesenchymal cell interactions during development of various tissues and organs. Tooth germ development is a classical model for this interaction. In tooth germs, PTHrP is expressed in the enamel organ (epithelial component), whereas its major receptor, the type I PTH/PTHrP receptor is expressed in cells of the alveolar bone and dental follicle (mesenchymal components). To clarify the role of PTHrP during fetal tooth germ development, PTHrP gene-knockout mice were used for histochemical and ultrastructural analysis. In wild-type mice, osteoclastic cells were aligned predominantly in the inner aspects of the alveolar bone surrounding the developing tooth germs throughout the late embryonic (after embryonic, 17.5 days) and neonatal animals examined. In contrast, osteoblasts were predominant in corresponding areas of fetal homozygous PTHrP-gene knockout mice with only occasional osteoclasts. In such areas, cell-free surfaces showing cement line-like tartrate-resistant acid phosphatase (TRAP) reactions were frequently observed. In neonatal homozygous mice, bone spicules were often shown to penetrate and/or compress the enamel organ and caused partial destruction of the tooth germs. Osteoclasts were few in number in the inner aspects of the alveolar bone, and had poorly developed ruffled border. No morphological abnormality was noted in cells of the tooth germs proper. On bone surfaces away from developing tooth germs, functional osteoclasts with structural features similar to those in wild-type mice were observed in homozygous mice. These observations suggest that PTHrP is required to maintain an appropriate spatiotemporal arrangement of bone cells and osteoclast function, which are necessary for the normal development of tooth germ and alveolar bone encasing the tooth germ. The observation also demonstrates that PTHrP deficiency affects the structure and function of osteoclasts exclusively those located in the vicinity of the growing tooth germ.

Acid Phosphatase↗

PubMed2002

A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome.

We transposed Dissociation (Ds) elements from three start loci on chromosome 5 in Arabidopsis (Nossen ecotype) by using a local transposition system. We determined partial genomic sequences flanking the Ds elements and mapped the elements' insertion sites in 1,173 transposed lines by comparison with the published genomic sequence. Most of the lines contained a single copy of the Ds element. One-half of the lines contained Ds on chromosome 5; in particular, insertion "hot spots" near the three start loci were clearly observed. In the other lines, the Ds elements were transposed across chromosomes. We found other insertion hot spots at the tops of chromosomes 2 and 4, near nucleolus organizer regions 2 and 4, respectively. Another characteristic feature was that the Ds elements tended to transpose near the chromosome ends and rarely transposed near centromeres. The distribution patterns differed among the three start loci, even though they possessed the same Ds construct. More than one-half of the Ds elements were inserted irregularly into the genome; that is, they did not retain the perfect inverted repeat sequence of Ds nor leave perfect target site duplications. This precise analysis of distribution patterns will contribute to a comprehensive understanding of the transposing mechanism. From these Ds insertion sites, we have constructed a database for screening gene-knockout mutants in silico. In 583 of the 1,173 lines, the Ds elements were inserted into protein-coding genes, which suggests that these lines are gene-knockout mutants. The database and individual lines will be available freely for academic use from the RIKEN Bio-Resource Center (http://www.brc.riken.go.jp/Eng/index.html).

Arabidopsis↗

PubMed2002

Pathology of sarcocystis neurona in interferon-gamma gene knockout mice.

Pathologic changes were studied in 27 interferon-gamma gene knockout mice 34-54 days after being fed graded doses of Sarcocystis neurona sporocysts derived from a naturally infected opossum. The target tissue for S. neurona infection was the central nervous system. Characteristic histopathologic changes present in all mice consisted of an inflammatory infiltrate consisting of mostly neutrophils and macrophages, fewer eosinophils, and rare multinucleated giant cells. Intralesional protozoa and scattered subacute perivascular cuffs were present. Where the infiltrates were extensive, neuropil rarefaction was frequent. Pathologic changes were much more frequent and severe in the caudal portion of the brain, especially in the cerebellum, than in the middle and cranial portions. Changes were present in all spinal cords examined (10 of 10). Lesions were equally distributed in white and gray matter of the brain and spinal cord and their meningeal linings.

Animals↗

PubMed2005

Transcript profiling in plastid arginine tRNA-CCG gene knockout moss: construction of Physcomitrella patens plastid DNA microarray.

The moss Physcomitrella patens is a newly established model plant that is widely used for the characterization of gene function by targeted gene knockout or over-expression. The target gene disruption occurs in both the nuclear and chloroplast genomes. We applied DNA microarray technology to the P. patens plastid genome for large-scale analysis of transcripts. A microarray was constructed containing 108 DNA fragments to detect all annotated plastid genes. We analyzed the transcript profile in a knockout transformant for the arginine tRNA gene, trnR-CCG, and confirmed previous results that rbcL and psaI transcripts accumulate in similar levels to wild-type moss, and accD transcript level is higher than those of wild-type moss. Additionally, the plastid DNA microarray revealed that most plastid genes were expressed at similar levels in wild-type and transformant mosses. This indicates that trnR-CCG is not essential for the expression of plastid genes.

Base Sequence↗

PubMed2003

Proteotyping of mammary tissue from transgenic and gene knockout mice with immunohistochemical markers: a tool to define developmental lesions.

Through the use of transgenic and gene knockout mice, several studies have identified specific genes required for the functional development of mammary epithelium. Although histological and milk protein gene analyses can provide useful information regarding functional differentiation, they are limited in their ability to precisely define the molecular lesions. For example, mice that carry a mutation in one of the subunits of the IkappaB kinase, IKKalpha, cannot lactate despite the presence of histologically normal alveolar compartment and the expression of milk protein genes. To further define and understand such lesions on a molecular level, we sought evidence for proteins that are differentially expressed during mammary gland development with a view to generating a tissue proteotype. Using database screens and immunohistochemical analyses, we have identified three proteins that exhibit distinct profiles. Here, using mouse models as test biological systems, we demonstrate the development and application of mammary tissue proteotyping and its use in the elucidation of specific developmental lesions. We propose that the technique of proteotyping will have wide applications in the analyses of defects in other mouse models.

Animals↗

PubMed2006

Spontaneous recovery of injured Achilles tendon in inducible nitric oxide synthase gene knockout mice.

OBJECTIVE AND DESIGN: To determine if inducible nitric oxide synthase (iNOS) gene could affect Achilles tendon healing using iNOS gene knockout mice. METHODS: 21 iNOS knockout (iNOS(-/-)) mice and 8 of the wild type (iNOS(+/+)) mice were utilized in this study. Group 1: iNOS(+/+) mice (n = 8), group 2: iNOS(-/-) mice (n = 11) and group 3: iNOS(-/-) with a NOS inhibitor, (aminoguanidine, 500 mg/kg/day, via an intraperitoneal mini-osmotic pump for 7 days, n = 10). The right Achilles tendon was transected in all mice and harvested on day 7 for cross-sectional area and biomechanical properties. Serum nitrate concentration of the mice was measured by gas chromatography mass spectrometry (GC/MS). RESULTS: A significant reduction in cross-sectional area of the healing Achilles tendon was observed in group 3 mice compared to group 2 mice (p < 0.01). The serum nitrate concentration in both group 2 and group 3 mice was lower than that in group 1 mice (p < 0.01) iNOS gene deletion and inhibition of NOS did not affect the biomechanical properties of the healing tendons. CONCLUSIONS: iNOS gene is not solely responsible for the beneficial effects of nitric oxide (NO) on tendon healing.

Achilles Tendon↗

PubMedDate not supplied

Potential of aspirin to inhibit thrombotic microangiopathy in alpha1,3-galactosyltransferase gene-knockout pig hearts after transplantation in baboons.

Hearts from alpha1,3-Galactosyltransferase gene-knockout (GaIT-KO) pigs were transplanted heterotopically into 8 baboons that received an anti-CD154 monoclonal antibody (mAb)-based immunosuppressive regimen and heparin. Three baboons died or were euthanized with beating grafts on 16, 23, and 56 days, respectively, and the remaining 5 grafts functioned for 59-179 days. Hyperacute rejection did not occur, and classical features of acute humoral xenograft or acute cellular rejection were rare. However, thrombotic microangiopathy (TM) developed in all cases; its onset was delayed in 2 baboons that received aspirin. Function of a pig organ in a baboon for a period approaching 6 months has not been reported previously and lends encouragement that the barriers to xenotransplantation will be overcome, but TM requires investigation.

Animals↗

PubMed2006

Generation of medaka gene knockout models by target-selected mutagenesis.

We have established a reverse genetics approach for the routine generation of medaka (Oryzias latipes) gene knockouts. A cryopreserved library of N-ethyl-N-nitrosourea (ENU) mutagenized fish was screened by high-throughput resequencing for induced point mutations. Nonsense and splice site mutations were retrieved for the Blm, Sirt1, Parkin and p53 genes and functional characterization of p53 mutants indicated a complete knockout of p53 function. The current cryopreserved resource is expected to contain knockouts for most medaka genes.

Adenosine Triphosphatases↗

PubMed2005

Transposon-mediated generation of targeting vectors for the production of gene knockouts.

Vectors used for gene targeting experiments usually consist of a selectable marker flanked by two regions of homology to the targeted gene. In a homologous recombination event, the selectable marker replaces an essential element of the target gene rendering it inactive. Other applications of gene targeting technology include gene replacement (knockins) and conditional vectors which allow for the generation of inducible or tissue-specific gene-targeting events. The assembly of gene-targeting vectors is generally a laborious process requiring considerable technical skill. The procedures presented here report the application of transposons as tools for the construction of targeting vectors. Two mini-Mu transposons were sequentially inserted by in vitro transposition at each side of the region targeted for deletion. One such transposon carries an antibiotic resistance marker suitable for selection in mammalian cells. A deletion is then generated between the two transposons either by LoxP-induced recombination or by restriction digestion followed by ligation. This deletion removes part of both transposons plus the targeted region in between, leaving a transposon carrying the selectable marker flanked by two arms which are homologous to the targeted gene. Targeting vectors constructed using these transposons were electroporated into embryonic stem cells and shown to be effective in gene-targeting events.

Animals↗