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Results for “Gene knockout”

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PubMed2004

Mitochondrial gene-knockout (rho0) cells: a versatile model for exploring the secrets of trans-plasma membrane electron transport.

Plasma membrane electron transport (tPMET) pathways have been identified in all living cells, and a wide variety of tools have been used to study these processes. In our laboratory we have used the cell-impermeable tetrazolium dye WST-1, together with the mitochondrial gene knockout (rho0) cell model, to investigate one of these pathways. We have shown that growth of HL60rho0 cells is dependent on oxygen, and that these cells consume oxygen at the cell surface. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and WST-1 reduction suggest that both systems share a common tPMET pathway. In support of this, oxygen was shown to compete with the intermediate electron acceptor that mediates WST-1 reduction, for reducing electrons. The observation that tPMET activity is higher in rho0 cells compared to their mitochondrially-competent counterparts was shown to be the result of competition between the mitochondrial and plasma membrane electron transport systems for intracellular reducing equivalents. Elevated rates of dye reduction appear to be mediated through increased expression of the key components of tPMET, which include the cell surface NADH oxidase, CNOX. These findings have played a critical role in shaping our current understanding of the mechanisms of this particular pathway of tPMET.

Cell Line, Tumor↗

PubMed2005

Blockage of histamine H1 receptor attenuates social isolation-induced disruption of prepulse inhibition: a study in H1 receptor gene knockout mice.

RATIONALE: Histaminergic neurotransmission has been implicated in the pathophysiology of stress-related psychiatric diseases. Although several atypical antipsychotics are potent H1 antagonists, the clinical significance of interaction between atypical antipsychotics and H1 receptors is still unknown. OBJECTIVE: In this study, we investigated the effects of H1 receptors blockage on social isolation-induced behavioral changes in H1 receptor gene knockout (H1KO) mice and their wild-type (WT) mice. METHODS: Both H1KO and their WT mice were subjected to 4-week social isolation rearing after weaning (21 postnatal days). After the 4-week isolation period, mice behavioral changes were evaluated using behavioral tests. RESULTS: Locomotor activity in home cages was significantly lower in isolation-reared WT mice than in socially reared WT mice. However, no change in locomotor activity was observed between socially and isolation-reared H1KO mice. Social isolation significantly impaired prepulse inhibition (PPI) of startle response in WT mice but not in H1KO mice. In addition, social isolation significantly impaired spatial learning and memory in WT mice but not in H1KO mice. Furthermore, H1KO mice treated with methamphetamine (METH) showed no enhancement in isolation-induced disruption of PPI. A neurochemical study revealed that isolation-reared WT mice had significantly lower dopamine (DA) levels and slightly increased DA turnover in the cortex than socially reared WT mice. Conversely, isolation-reared H1KO mice showed significantly higher DA contents as compared with socially reared H1KO mice. CONCLUSION: The results of our study indicate that blockage of H1 receptor-mediated neurotransmission attenuates social isolation-induced behavioral changes and that the therapeutic effects of atypical antipsychotics are mediated, at least in part, by interaction with H1 receptors in the brain.

Animals↗

PubMed2001

Determination of the activity of pyrantel tartrate against Sarcocystis neurona in gamma-interferon gene knockout mice.

Equine protozoal myeloencephalitis (EPM) is the most important protozoal disease of horses in the United States. Some horse owners and equine clinicians believe that horses which are on daily pyrantel tartrate at 2.64mg/kg for helminth prophylaxis are less likely to develop EPM. The present study examined the efficacy of pyrantel tartrate in preventing clinical disease in gamma-interferon gene knockout (BALB/c-Ifng(tm1ts)) mice. No activity was seen against sporocyst-induced Sarcocystis neurona infections in mice treated prophylacticly with 4-5mg pyrantel tartrate per mouse per day in the drinking water.

Animals↗

PubMed1999

Regulation of immune complexes during infection of mice with lactate dehydrogenase-elevating virus: studies with interferon-gamma gene knockout and tolerant mice.

Mice persistently infected with lactate dehydrogenase-elevating virus (LDV) develop circulating IgG-containing hydrophobic immune complexes, with a molecular mass of 150 to 300 kd, which bind to the surfaces of high-capacity enzyme-linked immunosorbent assay (ELISA) plates. LDV infection also stimulates polyclonal B-cell activation and autoimmunity. For this study, interferon-gamma gene knockout (GKO) mice were utilized to study circulating immune complexes and other parameters of LDV infection. The kinetics of LDV viremia, formation of plasma IgG anti-LDV antibodies, and LDV replication in the spleen and liver were essentially normal in GKO mice. Polyclonal activation of B cells, as reflected by increased total plasma IgG concentration during LDV infection, was found to be intact in GKO mice, although at a lower magnitude than in control mice. The plasma concentration of IgG-containing hydrophobic immune complexes was reduced about 75% in LDV-infected GKO mice relative to normal LDV-infected controls. Allogeneic tissue responses were also found to be reduced in LDV-infected GKO mice relative to those in normal LDV-infected controls. These results dissociate specific anti-LDV immunity from formation of hydrophobic immune complexes, show that the IgG anti-LDV response as well as LDV replication in the spleen and liver are insensitive to physiological levels of interferon (IFN)-gamma, and suggest that IgG-containing immune complexes stimulated by LDV infection are a marker for autoimmunity.

Animals↗

PubMed1999

Opioid analgesia: new information from gene knockout studies.

The cloning of opioid peptides and receptors has led to the development of knockout mice deficient in a gene encoding for each peptide and each receptor. The major advantage of the knockout approach is the complete selectivity that the removal of a protein encoded for by a gene provides. These knockout animals are adding to our knowledge of the physiology, pathophysiology and pharmacology of opioid systems. In addition, they are providing real impact in addressing the long-standing debates over receptor subtype heterogeneity and functional cooperativity between receptor subtypes.

Journal Article↗

PubMed1999

IL-10 gene knockout mice show enhanced Th1-like protective immunity and absent granuloma formation following Chlamydia trachomatis lung infection.

We previously reported that higher IL-10 production is correlated with lower IFN-gamma production, weaker delayed hypersensitivity (DTH), and slower organism clearance following chlamydial infection in mice. To assess more directly the role of IL-10, we examined protective immunity and pathological reaction in C57BL/6 IL-10 gene knockout (KO) and wild-type mice. The results showed that in the absence of endogenous IL-10, mice had significantly accelerated chlamydial clearance and developed significantly stronger DTH responses, which could be inhibited by local delivery of rIL-10. Consistent with the enhancement of DTH responses, IL-10 KO mice showed stronger and more persistent CD4 T cell-dependent IFN-gamma production and significant elevation of IL-12 and TNF-alpha production. Additionally, wild-type, but not IL-10 KO, mice showed granuloma formation that was correlated with higher levels of Th2 cytokine (IL-5) production at the later stages of infection. Moreover, chlamydial infection, unlike parasitic protozoan infection, did not induce significant acute toxicity in IL-10 KO mice, which may be due to the low (undetectable) levels of systemic release of proinflammatory cytokines. These results suggest that IL-10 inhibits the priming and expansion of Th1-like T cell responses and that IL-10 plays a role in the fibrotic reaction seen with chlamydial infection.

Animals↗

PubMed2003

Vegfb gene knockout mice display reduced pathology and synovial angiogenesis in both antigen-induced and collagen-induced models of arthritis.

OBJECTIVE: To determine the role of vascular endothelial growth factor B (VEGF-B) in 2 mouse models of arthritis, antigen-induced arthritis (AIA) and collagen-induced arthritis (CIA). METHODS: For AIA studies, monarticular AIA was induced by methylated bovine serum albumin (mBSA) priming of Vegfb gene knockout (Vegfb(-/-)) and wild-type (Vegfb(+/+)) mice, followed by intraarticular injection of mBSA or saline control 8 days later. CIA was induced in Vegfb(-/-) and Vegfb(+/+) mice by intradermal injection of chick type II collagen in adjuvant. Arthritis was monitored in both models using defined criteria (clinical and histologic). Angiogenesis was measured by synovial vessel density in diseased and control joints. RESULTS: In AIA studies, Vegfb(+/+) mice displayed significant knee joint swelling and synovial inflammation 7 days after intraarticular injection of antigen. Synovial inflammation was associated with angiogenesis, since vessel density in AIA synovium was significantly higher in arthritic than in control joints from the same animal. Knee joint swelling, synovial inflammation, and inflammation-associated vessel density in arthritic joints were reduced in Vegfb(-/-) mice compared with arthritic joints from Vegfb(+/+) mice. Similarly, in CIA, both disease incidence and mean clinical severity scores were significantly reduced in Vegfb(-/-) mice compared with Vegfb(+/+) mice. Mean histologic severity scores and mean synovial vessel density were reduced in diseased joints from Vegfb(-/-) mice when compared with diseased joints from Vegfb(+/+) mice. CONCLUSION: The reduction in inflammation-associated synovial angiogenesis in Vegfb(-/-) mice implicates VEGF-B in pathologic vascular remodeling in inflammatory arthritis. VEGF-B may be an attractive target in the design of anti-angiogenic therapies for rheumatoid arthritis.

Angiogenesis Inducing Agents↗

PubMed2006

Gene knockout experiments to quantify a G2/M genetic network simulation for mammary cancer susceptibility.

A G2/M genetic network simulation is trained with tumor incidence data from knockout experiments. The genetic network is implemented using a neural network; knockout genotypes are simulated by removing nodes in the neural network. Two analyses are used to interpret the resulting network weights. We use a novel approach of fixing the network topology that allows knockout TSG (tumor suppressor gene) data from multiple studies to overlap and indirectly inform one another. The trained simulation is validated by reproducing qualitative mammary cancer susceptibilities of ATM, BRCA1, and p53 TSGs. The work described is valuable because it allows TSG mammary cancer susceptibility to be quantified using genetic network topology and in vivo knockout data.

Animals↗

PubMed2005

[Effects of carnitine acetyltransferase gene knockout on long chain dicarboxylic acid production and metabolism of Candida tropicalis].

Candida tropicalis can assimilate n-alkane as a sole carbon source and produce dicarboxylic acids (DCAs). The synthesis of DCAs is thought to be reduced by beta-oxidation. Carnitine acetyltransferase (CAT) is the major enzyme to transfer DCAs into beta-oxidative pathway, then DCAs would be catalyzed to generate ATP to supply cells with energy. A homologous recombination plasmid was constructed, in which CAT gene was disrupted by inserting hygromycin B resistance gene. This plasmid was used to transform Candida tropicalis wild type strain F10-1, and one single CAT gene knockout strain was obtained. Comparing with the wild type, the recombinant increased DCA13 yield and molar conversion of alkane by 13.0% and 11.8%, respectively, and decreased unnecessary consumption of DCAs in beta-oxidation.

Candida tropicalis↗

PubMed2005

Inhibition of scratching behaviour caused by contact dermatitis in histidine decarboxylase gene knockout mice.

A neuronal system dedicated to itch consists of primary afferent and spinothalamic projection neurons. Histamine is thought to be one of the main mediators for the transmission of itch sensation. However, there are little available information on the role of histamine in scratching behaviour and sensory transmission of atopic dermatitis and chronic eczema. In the present study, the role of histamine in scratching behaviour and neural conduction of sensation in the chronic eczema model was investigated by using l-histidine decarboxylase (HDC) gene knockout mice lacking histamine. The chronic contact dermatitis was induced with daily application of diphenylcyclopropenone (DCP) on a hind paw of HDC (+/+) and HDC (-/-) mice for 2 months. The observation of scratching behaviour and the hot-plate test were performed in both mice. Histological studies were performed in the skin and spinal cord tissues. Histological examination revealed that both HDC (+/+) and HDC (-/-) mice displayed the similar extent of inflammatory cell infiltration, hyperplastic epidermis and newly spreading of neuronal processes in the skin tissue. Scratching behaviour was exclusively induced in HDC (+/+) mice, whereas it was barely observed in HDC (-/-) mice. The expression of c-Fos was specifically upregulated in HDC (+/+) mice in lamina I of the spinal dorsal horn following repeated DCP application. Scratching behaviour in chronic contact dermatitis in mice was thought mainly mediated with histamine. The afferent pathway of sensation in chronic contact dermatitis model may connect with the central nervous system through lamina I of the spinal dorsal horn.

Animals↗

PubMed2004

Bone marrow transplantation from alpha1,3-galactosyltransferase gene-knockout pigs in baboons.

BACKGROUND: Successful hematopoietic cell allotransplantation results in donor-specific tolerance, but this approach has been unsuccessful in the wild-type pig-to-baboon xenotransplantation model, as pig cells were lost from the circulation within 5 days. However, after cessation of immunosuppressive therapy on day 28, all baboons demonstrated non-specific unresponsiveness on mixed leukocyte reaction (MLR) for at least 30 days. We have now investigated the transplantation of bone marrow (BM) cells from miniature swine homozygous for alpha1,3-galactosyltransferase gene-knockout (GalT-KO). METHODS: Baboons (n = 3) were pre-treated with whole body and thymic irradiation, anti-thymocyte globulin, and splenectomy, and received immunosuppressive and supportive therapy for 28 days. BM was harvested from GalT-KO swine (n = 3). The baboons were monitored for the presence of pig cells by flow cytometry and colony-forming units (CFUs), and for cellular reactivity by MLR. RESULTS: A mean of 11 x 10(8) BM cells/kg was infused into each baboon. The mean absolute numbers and percentages of pig cells detected in the blood at 2 h and on days 1, 2 and 4, respectively, were 641/microl (9.5%), 132/microl (3.4%), 242/microl (3.9%), and 156/microl (2.9%). One baboon died (from accidental hemorrhage) on day 6, at which time chimerism was present in the blood (2.0%) and BM (6.4%); pig cell engraftment in the BM was confirmed by polymerase chain reaction (PCR) of CFUs. In the two other baboons, blood chimerism was lost after day 5 but returned at low levels (<1%) between days 9 to 16 and 7 to 17, respectively, indicating transient BM engraftment. Both surviving baboons showed non-specific unresponsiveness on MLR until they were euthanized on days 85 and 110, respectively. CONCLUSIONS: By using BM cells from GalT-KO pigs, chimerism was detected at levels comparable with previous studies when 30-fold more growth factor-mobilized peripheral blood progenitor cells had been transplanted. In addition, cellular hyporesponsiveness was prolonged. However, long-term engraftment and chimerism were not achieved.

Animals↗

PubMed2004

The cytotoxic potential of regulatory T cells: what has been learned from gene knockout model systems?

The mechanisms of T-cell regulation are difficult to elucidate because of their complexity and the numerous subcategories of cell populations. There are two fundamental approaches to address this conundrum. First, it is possible to use a purified cell population and submit these cells to various assays. The second approach is to manipulate a target molecule and determine what effect this has on T-cell homeostasis in vitro and in vivo. This molecular strategy may help characterize multiple regulatory populations that use the same pathway for controlling T-cell function. Through a concerted two-pronged effort, the authors' laboratory and others have attempted to decipher different molecular pathways for regulatory cell function. Several gene knockout mouse models display a phenotype of profound lack of homeostasis in which T cells accumulate, presumably because of a defect in regulation. Dependent on the molecule disrupted, the immune cell subset being examined may no longer be appropriately regulated. Accordingly, the phenotype of exogenously added "putative" regulatory cells can then be examined by assessing their ability to control this aberrant accumulation. By using co-transplantation techniques, much information can be postulated regarding potential regulatory cell phenotype and function. Model systems with target gene manipulations involving Fas ligand, Fas, perforin, interleukin-2, and cytotoxic T-lymphocyte-associated antigen-4 exist and in all cases appear to disrupt critical cytotoxic regulatory cell function.

Animals↗

PubMed2000

Experimental histoplasmosis in mice treated with anti-murine interferon-gamma antibody and in interferon-gamma gene knockout mice.

Histoplasma capsulatum is an important fungal pathogen in immunocompromised hosts, including AIDS patients. Experimental evidence suggests interferon-gamma (IFN) plays a role in host defense against H. capsulatum. In these studies we sought to demonstrate the importance of IFN in innate resistance to systemic histoplasmosis. The possible exacerbation of infection in BALB/c mice was assessed by administering 200 microg of hamster anti-IFN antibody prior to infection with H. capsulatum (2 x 10(6) yeasts, i.v.) and by comparing the severity of infection between BALB/c IFN gene knockout mice (GKO) and congenic control animals. In two separate studies, we found that anti-IFN treatment caused a dramatic loss of resistance to lethal infection and resulted in earlier mortality of IFN-depleted animals compared with normal IgG or no treatment (P<0.001). GKO mice were significantly (P<0.001) more susceptible to lethal infection than were control animals, and histological studies corroborated this. These studies clearly demonstrate that IFN is a vital part of the host's innate resistance to systemic infection with H. capsulatum and provide an additional rationale for studying IFN as an immunomodulatory therapeutic for the treatment of this disease.

Animals↗

PubMed2000

IL-12 plasmid-enhanced DNA vaccination against carcinoembryonic antigen (CEA) studied in immune-gene knockout mice.

Intramuscular (i.m.) injection of a plasmid encoding human carcinoembryonic antigen (CEA) elicited immunity against transplanted syngeneic (C57BL/6) CEA-positive Lewis lung carcinoma (CEA/LLC) cells, but tumors still appeared in all mice. In wild-type mice, coinjection of an IL-12 plasmid markedly enhanced anti-CEA humoral, T-helper-1 and cytotoxic T lymphocyte (CTL) responses, and resistance to a CEA/LLC tumor challenge such that 80% of mice remained tumor free. Injection of the IL- 12 plasmid alone was not protective. To analyze immune requirements, we immunized gene knockout (KO) mice of C57BL/6 background, deficient in either CD3, CD4, CD8, interferon gamma (IFNgamma), perforin or Fas ligand (FasL). Only CD3+ mice expressing both CD4 and CD8, which appear equally important, as well as IFNgamma and perforin, could fully resist a tumor challenge. IL-12 stimulated CTL activity, which was strictly CD3/CD8/perforin-dependent. FasL-KO mice had normal CTL activity and tumor resistance, indicating that only the perforin lytic pathway was involved. CD4-KO and IFNgamma-KO mice still generated CTLs. CEA-stimulated IFNgamma production occurred in both CD4- or CD8-KO mice and in both cases was augmented by IL-12. In IFNgamma-KO mice, IL-12 still enhanced anti-CEA antibody production but only moderately restored impaired DTH and tumor resistance. We conclude that the immune requirements for tumor rejection are stringent, involving multiple mechanisms which are all enhanced by IL-12.

Animals↗

PubMed1999

Susceptibility of immunodeficient gene-knockout mice to urinary tract infection.

PURPOSE: Clinical and experimental evidence indicates that mucosal immunity to urinary tract infection involves B and T-cell functions. The present study was conducted to assess the susceptibility of immunocompetent and immunodeficient mice with genetically engineered deletions in T and B-cell functions to experimentally induced urinary tract infections (UTI) with Escherichia coli. MATERIALS AND METHODS: Interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-10, inducible nitric oxide synthase, T cell receptor (TCR) delta-chain and JHD B cell-deficient gene knockout mice and their immunocompetent controls were challenged with uropathogenic Escherichia coli. The bladders and kidneys were cultured for viable E. coli at time intervals after intraurethral challenge to assess susceptibility to an experimentally induced UTI. RESULTS: Knockout mice with gammadelta-T cell or IFN-gamma deficiencies were more susceptible to UTI than immunocompetent mice or mice with immunodeficiencies in IL-10, IL-4, inducible nitric oxide synthase or antibody production (JHD). CONCLUSIONS: These data support an important role for gammadelta-T cells and IFN-gamma in resistance to UTI in mice.

Animals↗

PubMed2005

Limited exercise capacity in heterozygous manganese superoxide dismutase gene-knockout mice: roles of superoxide anion and nitric oxide.

BACKGROUND: We have reported that there is a limitation of exercise capacity in mice with defects in the expression of endothelial nitric oxide (NO) synthase, which is associated with a greater increase in whole-body oxygen consumption (VO2). We hypothesized that in states in which superoxide anion (O2-) is increased, especially in the mitochondria, whole-body VO2 will be increased because of the inactivation of NO, and consequently, exercise capacity will be reduced. METHODS AND RESULTS: Heterozygous manganese superoxide anion dismutase (SOD2) gene-knockout mice (SOD2+/-), in which SOD2 activity is reduced by 30% to 80%, and wild-type control mice (SOD2+/+) were treadmill-tested to measure indices defining exercise capacity. Tempol was given to each mouse for 7 days by an intraperitoneal injection to scavenge O2- before a second treadmill testing. VO2 and carbon dioxide production (VCO2) at rest were increased in SOD2+/-. The work (vertical distance run x body weight) to exhaustion was decreased in SOD2+/-. When the maximum VO2 and VCO2 were corrected to per work unit, they were increased in SOD2+/-. Tempol normalized basal VO2 and VCO2 and improved the work to exhaustion and corrected VO2 and VCO2 in SOD2+/-. VO2 of skeletal muscle was measured in vitro. Bradykinin-induced reduction in VO2 in vitro was attenuated in SOD2+/-, and was acutely restored by Tempol. There was a decrease in SOD2 protein level and a concomitant increase in lucigenin-detectable O2- production in skeletal muscle from SOD2+/-. CONCLUSIONS: These results suggest that exercise capacity is reduced in conditions in which superoxide anion is increased, and this is associated with a greater increase in whole-body oxygen consumption in SOD2+/- compared with SOD2+/+.

Animals↗

PubMed2001

Lack of effects of transforming growth factor-alpha gene knockout on peripheral nerve regeneration may result from compensatory mechanisms.

Transforming growth factor-alpha (TGF-alpha), previously identified as a major member of the epidermal growth factor (EGF) family of growth factors, plays a role in proliferation, differentiation, and survival of neuronal and glial precursors and is implicated in development of the nervous system. However, its roles in nerve injury-induced responses remain obscure. The current study examined roles of endogenous TGF-alpha in peripheral nerve regeneration using sciatic nerve injury models with TGF-alpha knockout mice. Three weeks after a sciatic nerve crush, no significant differences were found between TGF-alpha wild-type and mutant mice in the number of retrogradely labeled L5 dorsal root ganglion (DRG) sensory neurons and L5 spinal cord motor neurons and in the morphology of myelinated regenerating nerve fibers, indicating that TGF-alpha is not essential for sensory and motor nerve regeneration. To assess a possible functional redundancy among TGF-alpha-related ligands in response to a nerve injury, mRNA expression of the EGF family was analyzed by RT-PCR in L4/L5 DRG pools and distal degenerating sciatic nerve segments after sciatic nerve ligation. Prior to and 1 day after ligation, there was a higher level of EGF-R mRNA in DRGs and in nerve in TGF-alpha null mice compared to wild types, and there was an induction of ligand amphiregulin mRNA in DRGs in mutant mice in place of the TGF-alpha upregulation present in wild types. These results indicate that TGF-alpha gene knockout does not affect peripheral nerve regeneration, probably due to a functional redundancy within the EGF family through a compensatory expression mechanism at both the receptor and ligand levels in TGF-alpha knockout mice.

Amphiregulin↗

PubMedDate not supplied

IL-4-dependent effector phase in autoimmune exocrinopathy as defined by the NOD.IL-4-gene knockout mouse model of Sjögren's syndrome.

NOD mice manifest many features of autoimmune exocrinopathy (Sjögren's syndrome), a disease generally characterized by a chronic, progressive immunological attack against the exocrine tissues of the salivary and lacrimal glands. Previous studies using the NOD congenic partner strain, NOD.Igmu(null), defined an important role for B lymphocytes in the development of xerostomia, implicating autoantibodies reactive with the acetylcholine muscarinic receptor (M3R) as the possible effector mechanism. In the present study, we have examined the impact of the cytokine, interleukin (IL)-4, on autoimmune exocrinopathy by using the IL-4 gene knockout (KO) NOD mouse strain, NOD.IL-4-/-. Despite manifesting the physiological aberrations and marked leukocytic infiltration of the salivary glands characteristic of autoimmune xerostomia in NOD mice, the NOD.IL-4-/- mice do not develop xerostomia. However, NOD.IL-4-/- mice that received adoptively transferred T lymphocytes derived from NOD.Igmu-/- mice progress to xerostomia, thereby reversing the defect. While progression or lack of progression to xerostomia correlated with the ability of the NOD.IL-4-/- mice to express detectable anti-M3R autoantibodies, the precise mechanism of how IL-4 influences the development of autoimmune xerostomia remains speculative.

Adoptive Transfer↗