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PubMed2001

Zellweger syndrome knockout mouse models challenge putative peroxisomal beta-oxidation involvement in docosahexaenoic acid (22:6n-3) biosynthesis.

The putative involvement of peroxisomal beta-oxidation in the biosynthetic pathway of docosahexaenoic acid (22:6n-3, DHA) synthesis is critically reviewed in light of experiments with two recently developed knockout mouse models for Zellweger syndrome, a peroxisomal disorder affecting brain development. These mice were generated by targeted disruption of the PEX2 and PEX5 peroxisomal assembly genes encoding targeting signal receptor peroxins for the recognition and transport of a set of peroxisomal enzymes, including those of peroxisomal beta-oxidation, to the peroxisomal matrix. Analysis of esterified 22:6n-3 concentrations in PEX2-/- and PEX5-/- mice do not support the hypothesized requirement of peroxisomal beta-oxidation in 22:6n-3 synthesis, as only brain, but not liver or plasma, 22:6n-3 levels were decreased. Supplementation of PEX5+/- dams with 22:6n-3, although restoring the levels of brain 22:6n-3 in total lipids to that of controls, did not normalize the phenotype. These decreased brain 22:6n-3 concentrations appear to be secondary to impaired plasmalogen (sn-1-alkyl-, alkenyl-2-acyl glycerophospholipids) synthesis, probably at the level of the dihydroxyacetonephosphate acyltransferase (DHAP-AT), a peroxisomal enzyme catalyzing the first step in the synthesis of 22:6n-3-rich plasmalogens. To diminish the confounding effects of impaired plasmalogen synthesis in the brains of these Zellweger syndrome mouse models, kinetic experiments with labeled precursors, such as 18:3n-3 or 20:5n-3, in liver or isolated hepatocytes, which have negligible amounts of plasmalogens, are suggested to establish the rates of 22:6n-3 biosynthesis and precursor-product relationships. Similar experiments using brain of the acyl-CoA oxidase knockout mouse model are proposed to confirm the lack of peroxisomal beta-oxidation involvement in 22:6n-3 synthesis, since this mutation would not impair plasmalogen synthesis.

Animals↗

PubMed2001

Glycoconjugate metabolism in a cystic fibrosis knockout mouse model.

Cystic fibrosis knockout mice (cftr(-/-)) die prematurely of obstruction of the intestine which may result from accumulation of dehydrated glycoconjugate-containing mucus. We noted an increase in the specific activity of [(14)C]glucosamine-labeled high-molecular weight glycoconjugates, probably mucin, in the lumen of the intestine of cftr(-/-) (homozygous) mice compared to cftr(+/+) (wild-type) and cftr(+/-) (heterozygous) mice and a decrease in the turnover of glycoconjugates of several organs of the cftr(-/-) mice. No difference in the anionic composition of secreted intestinal glycoconjugates was detected and no difference in the amount of mucin 1 (Muc1) was found in the small intestine, colon, pancreas, and lungs of the different genotypes. In addition, the spleen of the cftr(-/-) mice was significantly smaller than that of control mice and the small intestine and colon were, respectively, longer and shorter compared to control mice. These results indicate modified glycoconjugate metabolism in cystic fibrosis knockout mice and morphologic changes to the spleen and intestine where the latter modifications are possibly related to the intestinal malabsorption associated with cystic fibrosis.

Animals↗

PubMed2001

Knockout mouse models and mammary tumorigenesis.

The generation of transgenic mice overexpressing activated forms of oncogenes has greatly advanced our understanding into their roles in mammary tumor initiation, promotion and progression. However, targeted disruption of tumor suppressor genes often results in lethality at stages prior to mammary tumor formation. This obstacle can now be overcome using several approaches including conditional knockouts that delete genes of interest in a spatial and temporal manner. This review summarizes recent studies on tumor suppressor genes, including APC, ATM, BRCA1, BRCA2, PTEN and p53, in knockout mouse models and our understanding of the possible mechanisms underlying mammary tumorigenesis.

Adenomatous Polyposis Coli Protein↗

PubMed1999

A chimeric aryl hydrocarbon receptor knockout mouse model indicates that aryl hydrocarbon receptor activation in hematopoietic cells contributes to the hepatic lesions induced by 2,3,7, 8-tetrachlorodibenzo-p-dioxin.

Pathologic changes associated with 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) exposure have been reported in the livers of a wide range of species. While these changes have been extensively described, the mechanisms of toxic interaction(s) that produce these lesions remain unclear. Using an aryl hydrocarbon receptor (Ahr) knockout male mouse chimeric model, we investigated whether the presence of this receptor in hematopoietic and/or parenchymal cells affects TCDD-induced hepatotoxicity. Bone marrow chimeras were produced by hematopoietic reconstitution of irradiated mice. Specifically, chimeras were generated with aryl hydrocarbon receptor (AHR) positive hematopoietic and parenchymal cells (Ahr+/+ animal bone marrow cells into irradiated Ahr+/+ animals), AHR positive hematopoietic and negative parenchymal cells (Ahr+/+ into Ahr-/-), AHR negative hematopoietic and positive parenchymal cells (Ahr-/- into Ahr+/+), and AHR negative hematopoietic and parenchymal cells (Ahr-/- into Ahr-/-). Male wild-type (Ahr+/+) and knockout (Ahr-/-) animals were used as nonchimeric controls. Following TCDD treatment (30 microg/kg body wt), liver sections from mice in each control and chimeric group were histologically evaluated for necrotic and inflammatory changes. TCDD treatment produced moderate inflammation in Ahr+/+ controls and Ahr+/+ into Ahr+/+ chimeras. This response was mild in TCDD-treated Ahr-/-, Ahr-/- into Ahr-/-, Ahr+/+ into Ahr-/-, and Ahr-/- into Ahr+/+ animals and was not different from the corresponding vehicle-treated groups. Moderate necrosis was observed in all TCDD-treated controls or chimeras with AHR-positive parenchyma. No or mild necrosis was observed in TCDD- and vehicle-treated animals containing AHR-negative parenchyma. These data indicate that the presence of AHR in hepatic parenchyma alone is sufficient for TCDD induction of hepatic necrosis, and its presence in hematopoietic cells is necessary for the inflammatory response to TCDD-induced hepatic lesions.

Animals↗

PubMed2001

Accelerated DNA adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust.

Diesel exhaust (DE) has been recognized as a noxious mutagen and/or carcinogen, because its components can form DNA adducts. Mechanisms governing the susceptibility to DE and the efficiency of such DNA adduct formation require clarification. The transcription factor Nrf2 is essential for inducible and/or constitutive expression of a group of detoxification and antioxidant enzymes, and we hypothesized that the nrf2 gene knockout mouse might serve as an excellent model system for analyzing DE toxicity. To address this hypothesis, lungs from nrf2(-/-) and nrf2(+/-) mice were examined for the production of xenobiotic-DNA adducts after exposure to DE (3 mg/m(3) suspended particulate matter) for 4 weeks. Whereas the relative adduct levels (RAL) were significantly increased in the lungs of both nrf2(+/-) and nrf2(-/-) mice upon exposure to DE, the increase of RAL in the lungs from nrf2(-/-) mice exposed to DE were approximately 2.3-fold higher than that of nrf2(+/-) mice exposed to DE. In contrast, cytochrome P4501A1 mRNA levels in the nrf2(-/-) mouse lungs were similar to those in the nrf2(+/-) mouse lungs even after exposure to DE, suggesting that suppressed activity of phase II drug-metabolizing enzymes is important in giving rise to the increased level of DNA adducts in the Nrf2-null mutant mouse subjected to DE. Importantly, severe hyperplasia and accumulation of the oxidative DNA adduct 8-hydroxydeoxyguanosine were observed in the bronchial epidermis of nrf2(-/-) mice following DE exposure. These results demonstrate the increased susceptibility of the nrf2 germ line mutant mouse to DE exposure and indicate the nrf2 gene knockout mouse may represent a valuable model for the assessment of respiratory DE toxicity.

Animals↗

PubMed1999

Rationale for a conditional knockout mouse model to study carnitine palmitoyltransferase I deficiencies.

Several severe congenital cardiomyopathies are known to be associated with deficiencies in long-chain fatty acid transport and oxidation. Our studies are focused on a key enzyme in the regulation of intracellular long-chain fatty acid transport: carnitine palmitoyltransferase 1. Of this enzyme, two isoforms are expressed in the neonatal heart: L-CPT1 (the "liver-type" isoform) and M-CPT1 (the "muscle-type" isoform). It is known from studies in rats that chemical inhibition of both CPT1 isoforms results in hypertrophy of the cardiomyocytes, leading to an increase in heart-weight of up to 25%. With the aid of expressed sequence tag database analyses, cDNA- and genomic sequence information, we analysed the human gene for M-CPT1 in detail, and obtained partial clones of the murine genes for both CPT1 isoforms. We now started the development of a conditional knockout model to analyse and dissect deficiencies in these genes. While of the other mitochondrial components of the carnitine system deficiencies are known, some with severe cardiac consequences, M-CPT1 deficiencies have never been described. This suggests that M-CPT1 deficiency either (1) has not been recognised within the pool of congenital disorders, (2) is detrimental in an early stage of reproduction or embryogenesis, or (3) does not lead to physiological problems, probably due to the existence of a rescue system. If (1) is the case, the phenotypic effects of M-CPT1 deficiency have to be studied in order to generate criteria for clinical decision making and diagnosis. Option (2) demonstrates the necessity to use novel vector systems to create conditional gene disruptions. Hypothesis (3) implies a possible role for L-CPT1, and a knockout model allows a study of the interaction between the genes for L-CPT1 and M-CPT1. Applicable strategies to develop such a model system will be discussed.

Animals↗

PubMed2006

Canavan disease: studies on the knockout mouse.

Canavan disease (CD) is an autosomal recessive disorder, characterized by spongy degeneration of the brain. Patients with CD have aspartoacylase (ASPA) deficiency, which results accumulation of N-acetylaspartic acid (NAA) in the brain and elevated excretion of urinary NAA. Clinically, patients with CD have macrocephaly, mental retardation and hypotonia. A knockout mouse for CD which was engineered, also has ASPA deficiency and elevated NAA. Molecular studies of the mouse brain showed abnormal expression of multiple genes in addition to ASPA deficiency. Adenoassociated virus mediated gene transfer and stem cell therapy in the knockout mouse are the latest attempts to alter pathophysiology in the CD mouse.

Amidohydrolases↗

PubMed2006

Lessons learned from Myc/Max/Mad knockout mice.

The past two decades of gene targeting experiments have allowed us to make significant strides towards understanding how the Myc/Max/Mad network influences multiple aspects of cellular behavior during development. Here we summarize the findings obtained from the myc/max/mad knockout mice generated to date, namely those in which the N-myc, c-myc, L-myc, mad1, mxi1, mad3, mnt, or max genes have been targeted. A compilation of lessons we have learned from these myc/max/mad knockout mouse models, and suggestions as to where future efforts could be focused, are also presented.

Animals↗

PubMed1996

Mean corpuscular hemoglobin is not increased in Fmr1 knockout mice.

A slight increase in mean corpuscular hemoglobin (MCH) has been reported in erythrocytes from human fragile X patients. As it is difficult to perform case-controlled studies in patients with fragile X syndrome, we studied MCH in erythrocytes from transgenic mice with an Fmr1 knockout. None of the knockout mice showed increased MCH levels when compared with normal littermates. We conclude that it is unlikely that the FMR1 gene product has an effect on MCH.

Animals↗

PubMed1995

Genetic knockouts in mice: an update.

Gene disruption technology in mammals, by homologous recombination in embryonic stem cells, is a powerful method to manipulate the mouse germ line. In the past decade it has produced a wealth of knowledge concerning neuronal development, neurodegenerative disorders and the roles of oncogenes, Hox genes and growth factors during development. A surprising variety of genes, however, have given unexpected and disappointing results. A gene/function redundancy theory proposed by many investigators to explain the unexpected results has been supported in certain cases by the generation of double knockout mice. Modification of the basic technology now allows the investigators to carry out a variety of manipulations including conditional or tissue-specific knockouts. This may provide a better opportunity in the future for the gene therapy approach to correct the genetic disorder.

Animals↗

PubMedDate not supplied

An HLA-restricted, p53 specific immune response from HLA transgenic p53 knockout mice.

BACKGROUND: p53 is over-expressed in most human malignancies and is therefore an attractive target for immunotherapy. Unfortunately, a human cytotoxic T cell response to p53 is difficult to generate. p53 knockout transgenic mice may provide a model to circumvent immunologic tolerance to p53 and develop high-affinity p53-specific cytotoxic T lymphocytes (CTL). METHODS: p53 knockout, HLA A2.1 transgenic mice were generated and immunized with the immunodominant wild-type p53 nonamer peptide epitope p53149-157. Two weeks later splenocytes were harvested and stimulated in vitro with acid-treated, p53 peptide-pulsed syngeneic blast cells. Cultures were restimulated weekly with acid-treated, p53 peptide-pulsed Jurkat cells transfected with the HLA A2.1 gene. Peptide-specific cytotoxic activity was measured by chromium release assay, and the resulting CD8+ effectors were cloned via limiting dilution. RESULTS: P53 peptide-specific CTL were generated against p53149-157. Clones generated from the p53149-157 cell line demonstrated high affinity and specificity for p53149-157 when presented by HLA A2.1+ antigen-presenting cells. The p53149-157 CTL killed only cells overexpressing p53 cells that were HLA A2.1+ and did not kill cells with normal levels of p53 expression or those that were HLA A2.1-. CONCLUSION: HLA transgenic mice not previously exposed to the p53 protein provide a useful model for generating high-affinity p53-specific CTL.

Animals↗

PubMed2000

Lessons from knockout and transgenic mice for infertility in men.

This review concentrates on the clear cases where knocking out a gene in mice has caused male infertility and thus comes near to proving that the gene plays a role in the development of sperm. Knockout mice have been created with primary defects at every stage of spermatogenesis thus creating a framework for decoding the genetic hierarchy that causes male germ cell differentiation. As well as defining essential genes in vivo experiments have defined promoter and untranslated sequences responsible for the expression of proteins at all the spermatogenic stages. In conclusion knockout mice remain the ultimate test of spermatogenic hypotheses as well as providing detailed information about this complex process.

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↗

PubMed2003

Role of phosphoinositide 3-kinase signaling in mast cells: new insights from knockout mouse studies.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases essential for diverse physiological reactions. In recent years a series of gene-targeted mice lacking different types of PI3Ks and related molecules have been generated which enable us to understand the role of PI3K pathways, particularly class I members, in vivo. Analyses of such gene-targeted mice have led to major discoveries in the physiological roles of PI3K signaling in mast cell biology. In particular the role of PI3Ks has been extensively studied in signaling through the high-affinity IgE receptor (FcepsilonRI), since mast cells are the main effector cells in type I allergic reaction associated with IgE-dependent mechanisms. Furthermore, the knockout mice have provided significant information concerning the role of PI3K signals in mast cell differentiation. This review presents several new insights into mast cell biology, which have been elucidated by the analyses of these knockout mice.

Animals↗

PubMed2006

Absence of quasi-morphine withdrawal syndrome in adenosine A2A receptor knockout mice.

RATIONALE: Caffeine and other methylxanthines induce behavioral activation and anxiety responses in mice via antagonist action at A2A adenosine receptors. When combined with the opioid antagonist naloxone, methylxanthines produce a characteristic quasi-morphine withdrawal syndrome (QMWS) in opiate-naive animals. OBJECTIVES: The aim of this study was to establish the role of A2A receptors in the quasi-morphine withdrawal syndrome induced by co-administration of caffeine and naloxone and in the behavioral effects of caffeine. METHODS: We have used A2A receptor knockout (A(2A)R(-/-)) mice in comparison with their wild-type and heterozygous littermates to measure locomotor activity in the open field and withdrawal symptoms induced by caffeine and naloxone. Naïve wild-type and knockout mice were also examined for enkephalin and dynorphin mRNA expression by in situ hybridization and for mu-opiate receptor by ligand binding autoradiography to check for possible opiate receptor changes induced by A2A receptor inactivation. RESULTS: Caffeine increases locomotion and anxiety in wild-type animals, but it has no psychomotor effects in A(2A)R(-/-) mice. Co-administration of caffeine (20 mg/kg) and naloxone (2 mg/kg) resulted in a severe quasi-morphine withdrawal syndrome in wild-type mice that was almost completely abolished in A(2A)R(-/-) mice. Heterozygous animals exhibited a 40% reduction in withdrawal symptoms, suggesting that there is no genetic/developmental compensation for the inactivation of one of the A(2A)R alleles. A(2A)R(-/-) and wild-type mice have similar levels of striatal mu-opioid receptors, thus the effect is not due to altered opioid receptor expression. CONCLUSIONS: Our results demonstrate that A2A receptors are required for the induction of quasi-morphine withdrawal syndrome by co-administration of caffeine and naloxone and implicate striatal A2A receptors and mu-opiate receptors in tonic inhibition of motor activity in the striatum.

Adenosine A2 Receptor Antagonists↗

PubMed2006

Impaired performance of alpha7 nicotinic receptor knockout mice in the five-choice serial reaction time task.

RATIONALE: Nicotinic receptors have been implicated in attentional performance. Nicotine can improve attention in animals and humans, but knowledge about relevant receptor subtypes is very limited. OBJECTIVES: The aim was to examine the role of alpha7 receptors in attentional performance of mice and in effects of nicotine. MATERIALS AND METHODS: Mice with targeted deletion of the gene coding for the alpha7 subunit of nicotinic receptors and wild-type controls were trained on a five-choice serial reaction time task with food reinforcers presented under varying parametric conditions. Nicotine was administered in a range of doses (0.001-1.0 mg/kg sc), including those reported to enhance attentional performance. RESULTS: Initially the alpha7(-/-) (knockout) mice responded less accurately and made more anticipatory responses. After task parameters were altered so that the time allowed for responding was reduced and anticipatory (impulsive) responses were punished by a time-out, the pattern of performance deficits changed; there were increased omission errors in alpha7(-/-) mice but normal levels of accuracy and anticipatory responding. Nicotine did not improve any measure of performance, either with the original training parameters or after retraining; the largest dose used (1.0 mg/kg) produced a general impairment of responding in alpha7(-/-) and wild-type mice. CONCLUSIONS: alpha7 nicotinic receptor knockout mice are impaired in performance of the 5-CSRTT, suggesting a possible role for alpha7 receptors in attentional processing. However, identification of a protocol for assessing attention-enhancing effects of nicotine in mice may require further modifications of test procedures or the use of different strains of animal.

Animals↗

PubMed2001

Dissecting the role of the serotonin system in neuropsychiatric disorders using knockout mice.

RATIONALE: The serotonin system has an important role in the modulation of several processes relevant to psychiatry such as anxiety, affect, aggression, and drug abuse. This review summarizes the recent progress in elucidating the function of the serotonergic system using knockout mice. This review while not exhaustive, highlights recent findings of relevance to psychopharmacology. OBJECTIVES: To familiarize the reader with the technique and the findings from serotonergic knockout mice. METHODS: Information included in this review was drawn from our own experience in this field and relevant publications from other investigators. RESULTS: We have focused on three main themes that have emerged from studies with mice bearing single-gene mutations of serotonergic genes: anxiety, aggression, and drug abuse. Mice lacking the 5-HT1A have been found to be more anxious in several behavioral paradigms. Elevated levels of aggression have been reported in mice lacking the monoamine oxidase A and the 5-HT1B receptor genes. The mice lacking the 5-HT1B receptor have also been reported to exhibit an increased vulnerability to cocaine. The molecular basis of this enhanced vulnerability has been linked to compensatory changes in the nucleus accumbens. These results and their correlation with pharmacological studies will be discussed. CONCLUSION: Mice lacking key components of the serotonin system have provided us with important animal models of genetic vulnerability to conditions such as anxiety disorders, aggression, and drug abuse. Ongoing research with these mice may help elucidate the mechanistic functioning of this complex system.

Animals↗

PubMed2000

List of transgenic and knockout mice: behavioral profiles.

A list of transgenic and knockout mice, having behavioral profiles, has been compiled. Behavioral and genetic information has been assembled in a simplified fashion so that the reader can easily determine the possible usefulness of each transgenic or knockout strain. Brief descriptions of the behavioral tests are given. A discussion is also presented of the effects of genetic backgrounds on these behavioral profiles.

Animals↗