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PubMed2001

AAV-mediated delivery of ciliary neurotrophic factor prolongs photoreceptor survival in the rhodopsin knockout mouse.

Retinitis pigmentosa (RP), an inherited retinal degenerative disease causing blindness, is characterized by progressive apoptotic death of photoreceptors. Therapeutic modification of photoreceptor apoptosis may provide an effective therapy for this disorder. Ciliary neurotrophic factor (CNTF) has been shown to promote survival of a number of different neuronal cell types, including photoreceptors. The present study aimed to test whether adeno-associated virus (AAV)-mediated delivery of the gene encoding CNTF delays photoreceptor death in the rhodopsin knockout (opsin(-/-)) mouse, an animal model of RP. The vector was made to express a secretable form of CNTF in tandem with a marker GFP. Cultured 293 cells transduced with this virus expressed both CNTF and GFP. The conditioned media from such cells supported the survival of chick dorsal root ganglion neurons in the same manner as recombinant CNTF. Subretinal administration of this virus led to efficient transduction of photoreceptors as indicated by GFP fluorescence and CNTF immunostaining. Histologic examination showed significant photoreceptor preservation in the injected quadrant of the retina. This protection lasted through termination of the experiment (3 months). AAV-mediated delivery of CNTF may have implications for the treatment of human retinal degeneration.

Animals↗

PubMed1996

Dopamine neurons from transgenic mice with a knockout of the p53 gene resist MPTP neurotoxicity.

We have examined MPTP toxicity to dopamine neurons of mice homozygous for a transgenic knockout of the p53 growth control gene (p53-/-). MPTP at a total dose of 96 mg/kg administered in four doses over two days produced a non-homogeneous loss of striatal dopamine transport sites and quantitatively reduced 3H-mazindol binding to similar degrees in p53-/- and wild type controls 2 and 3 weeks after starting MPTP. Nigral DA neurons stained immunohistochemically for tyrosine hydroxylase were counted using both manual and automated methods and found to be reduced 29-34% in wild type controls but were not reduced in p53-/-. Mean DA neuronal surface areas were reduced 63-68% by MPTP in controls and 35-50% in p53-/-. We conclude that p53 protein appears necessary for complete expression of MPTP neurotoxicity to dopamine neurons. Our findings suggest that the p53 gene and other growth control genes may regulate dopamine neuronal death in PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2000

Inducible nitric oxide synthase knockout mouse macrophages disclose prooxidant effect of interferon-gamma on low-density lipoprotein oxidation.

To test our hypothesis that interferon-gamma (IFN-gamma) has a direct prooxidant effect on macrophage-mediated LDL oxidation behind its antioxidant effect via induction of inducible nitric oxide synthase (iNOS), we incubated LDL with wild-type (iNOS(+/+)) or iNOS knockout mouse (iNOS(-/-)) macrophages preincubated with IFN-gamma or IFN-gamma plus lipopolysaccharide (IFN-gamma/LPS) for 24 h. LDL oxidation was measured in terms of formation of thiobarbituric acid reactive substances (TBARS) and electrophoretic mobility. Thiol production, nitrite production, and superoxide production from macrophages were measured by using Ellman's assay, the Griess reagent, and the SOD-inhibitable cytochrome c reduction method, respectively. IFN-gamma alone or combined with LPS induced iNOS expression and increased nitrite production in iNOS(+/+) macrophages, but not in iNOS(-/-) macrophages. TBARS formation from LDL was suppressed in IFN-gamma- and IFN-gamma/LPS-treated iNOS(+/+) macrophages but was increased in IFN-gamma-treated iNOS(-/-) macrophages. In the presence of N(G)-monomethyl-l-arginine (l-NMMA), a NOS inhibitor, the suppressive effect of IFN-gamma and IFN-gamma/LPS was abolished and TBARS formation was even increased to a level above that of untreated iNOS(+/+) macrophage. NOC 18, an NO donor, dose dependently inhibited macrophage-mediated LDL oxidation. IFN-gamma increased superoxide and thiol productions in both types of macrophages. We conclude that IFN-gamma promotes macrophage-mediated LDL oxidation by stimulating superoxide and thiol production under conditions where iNOS-catalyzed NO release is restricted.

Animals↗

PubMed2002

Nitric oxide affects the phosphorylation state of microtubule-associated protein 2 (MAP-2) and neurofilament: an immunocytochemical study in the brain of rats and neuronal nitric oxide synthase (nNOS)-knockouts.

Alterations in function and specific cellular location of cytoskeletal elements are characterized by changes in their phosphorylation state. On this background we studied immunocytochemically the distribution pattern of neurofilament (NF) in its phosphorylated (P-NF) and nonphosphorylated (NP-NF) form and of microtubule-associated protein-2 (MAP-2) in the rat and mouse brain. Neurons that are strongly positive for neuronal nitric oxide synthase (nNOS)-immunoreactivity (IR) showed, interestingly, neither P-NF- nor MAP-2-IR. In contrast, nNOS-negative neuronal cell elements exhibited an intense IR and specific location for both antigens throughout the brain. As a model we chose the dorsolateral tegmental nucleus (LDT) of knockout (nNOS(-/-)) mice in which the main splice isoform nNOSalpha is lacking, but a low nNOS-activity persists, apparently due to the splice isoforms nNOSbeta and gamma. The principal neurons of such nNOS(-/-)-mice, which are equivalent to the nNOS-containing neurons in the LDT of wild-type mice exhibited a decreased nitrotyrosine-IR and an increased phosphotyrosine-IR if compared to those of wild-type mice. The same neurons failed to show NF-IR and MAP-2-IR, though. When the residual nNOS activity in nNOS(-/-)-mice was inhibited by treatment with N-omega-nitro-L-arginine methyl ester (L-NAME) the principal neurons displayed a moderate MAP-2 and NF-staining. NO and NO-derived oxygen species are suggested to modulate the balance between the activities of kinases and phosphatases, thus changing phosphorylation levels for NF, MAP-2, and, possibly, other proteins in neurons and adjacent cell elements.

Animals↗

PubMed1996

Genes in the RB pathway and their knockout in mice.

The retinoblastoma susceptibility gene (RB), the first identified human tumor suppressor gene, has been shown to be directly involved in the genesis of a variety of human cancers. RB is actually one of a family of three closely related genes including p107 and p130. Many elegant biochemical studies have demonstrated that RB is a critical component of the cell cycle regulatory machinery and have characterized the downstream effectors which the RB gene product regulates. More recent advances have demonstrated that the function of RB and RB-related genes is positively and negatively regulated by an intricate network of cell cycle regulatory proteins, some of which have also been implicated as tumor suppressor genes. Despite the detailed understanding of these biochemical and genetic pathways, the full function of genes in the RB pathway in the context of a whole organism is only now being addressed. Using gene knockout technology, it is now known that RB, and RB-related proteins p107 and p130, have important functions during early mouse development. Furthermore, despite its ubiquitous expression, RB has tissue- and cell-type specific effects which account for its function as a tumor suppressor but may also be independent of its role as a cell cycle regulator. Analysis of mice lacking regulatory genes upstream of RB and effector genes downstream of RB have confirmed that other genes in this pathway have tissue-specific effects on development and tumor susceptibility in mice.

Animals↗

PubMed1999

Development of Peyer's patches, follicle-associated epithelium and M cell: lessons from immunodeficient and knockout mice.

Studies with immunodeficient and knockout mice have revealed that the development of mucosa-associated lymphoid tissues (MALT) and peripheral lymphoid nodes share common mechanisms, but also require distinct signals. Gene disruption of lymphotoxins or their cognate receptors affects both Peyer's patch and lymph node organogenesis. Disruption of the osteoprotegerin TNF-family member gene does not impair Peyer's patch development, but prevents formation of peripheral lymph nodes. Peyer's patch do not form in mice with a deleted gene encoding a B lymphocyte-specific chemokine receptor, while most peripheral lymph nodes, except inguinal, are normal in numbers and architecture. In B or T lymphocyte-deficient mice, Peyer's patches, with their overlying follicle-associated epithelium (FAE), are present although reduced in number and size. No Peyer's patches develop in RAG deficient mice. Formation of FAE with typical M cells has not been analyzed in these mice. M cell formation requires the close association of immune cells with differentiated enterocytes and their conversion appears to be transcriptionally regulated. The development of MALT, FAE and probably M cells is a multistep process that requires signalling pathways common to all secondary lymphoid tissues, but also MALT-specific factors.

Animals↗

PubMed1999

Effects of CYP1A2 on disposition of 2,3,7, 8-tetrachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran, and 2,2',4,4',5,5'-hexachlorobiphenyl in CYP1A2 knockout and parental (C57BL/6N and 129/Sv) strains of mice.

TCDD is the prototype and most potent member of the highly lipophilic polyhalogenated aromatic hydrocarbons (PHAHs), which are persistent and ubiquitous environmental contaminants. In both acute and subchronic animal studies, there is a specific accumulation of TCDD in liver greater than in adipose tissue. The inducible hepatic binding protein responsible for this hepatic sequestration of TCDD and its congeners has been shown by our laboratory to be CYP1A2 (J. J. Diliberto, D. Burgin, and L. S. Birnbaum, 1997, Biochem. Biophys. Res. Commun. 236, 431-433). The present study was conducted using knockout (KO) mice lacking expression of CYP1A2 (CYP1A2-/-) in order to investigate the role of CYP1A2 gene on the disposition of TCDD, 4-PeCDF (a dioxin-like PHAH), and PCB 153 (a nondioxin-like PCB) in KO (CYP1A2-/-) mice and age-matched parental mice strains (C57BL/6N: CYP1A2+/+, Ah(b/b) and 129/Sv: CYP1A2+/+, Ah(d/d)). Mice were dosed (25 microgram [(3)H]TCDD/kg, 300 microgram [(14)C]4-PeCDF/kg, or 35.8 mg [(14)C]PCB 153/kg bw in a corn oil vehicle) orally and terminated after 4 days. Residues of administered compounds in collected tissues and daily excreta were quantitated using (3)H or (14)C activity. Results demonstrated differential effects in disposition for the various treatments within the three genetically different groups of mice. In KO mice, TCDD, 4-PeCDF, and PCB 153 had very little hepatic localization of chemical, and the major depot was adipose tissue. In contrast, parental strains demonstrated hepatic sequestration of TCDD and 4-PeCDF, whereas disposition of PCB 153 in parental strains was similar to that in KO mice. Another difference between KO mice and parental strains was the enhanced urinary excretion of 4-PeCDF. This study demonstrates the importance of CYP1A2 in pharmacokinetic behavior and mechanistic issues for TCDD and related compounds.

Animals↗

PubMed2002

Decrease in 4-aminobiphenyl-induced methemoglobinemia in Cyp1a2(-/-) knockout mice.

Methemoglobin formation, as well as hemoglobin or DNA adducts, are useful biomarkers of occupational exposure to certain arylamines. It has been suggested that, in liver from animals not treated with a cytochrome P450 (CYP) inducer, hepatic CYP1A2 is the major P450 involved in N-hydroxylation. This is the first step in the metabolic activation of many arylamines, such as the human urinary bladder carcinogen 4-aminobiphenyl (ABP). The product of this catalytic step, N-hydroxy-4-ABP, reacts in the blood with oxyhemoglobin to form methemoglobin and nitrosobiphenyl. We therefore examined the role of CYP1A2 in causing methemoglobinemia in ABP-treated Cyp1a2(-/-) knockout mice. Application of ABP (100 micromol/kg body wt) to the skin resulted in a marked depletion in the levels of the hepatic thiols (reduced glutathione and cysteine) after 2 h, which rebounded to basal levels 24 h later, and we found no differences between the Cyp1a2(-/-) and wild-type Cyp1a2(+/+) animals. Unexpectedly, the methemoglobin levels were significantly (p < 0.05) higher in Cyp1a2(-/-) than Cyp1a2(+/+) mice at 2, 7, and 24 h following topical ABP. Treatment with dioxin, 24 h prior to ABP, decreased methemoglobin levels by about half at each of the time points in both the Cyp1a2(-/-) and Cyp1a2(+/+) mice. These data suggest that CYP1A2 does not play a positive role in methemoglobin formation via the activation of ABP; rather, the absence of CYP1A2 enhances ABP-induced methemoglobinemia. Because liver CYP1A2 levels are known to vary more than 60-fold between humans, our findings may be relevant to patients who are exposed to arylamines in the workplace.

Administration, Topical↗

PubMed2005

Mutagenesis and knockout models: NK1 and substance P.

Tachykinins play an important role as peptide modulators in the CNS. Based on the concentration and distribution of the peptides and their receptors, substance P (SP) and its cognate receptor neurokinin 1 (NK1R) seem to play a particularly important role in higher brain functions. They are expressed at high levels in the limbic system, which is the neural basis of emotional responses. Three different lines of evidence from physiological studies support such a role of SP in the regulation of emotionality: (1) stress is often associated with elevated level of SP in animals and humans; (2) systematic and local injections of SP influence anxiety levels in a dose-dependent and site-specific manner; (3) NK1 receptor antagonists show anxiolytic effects in different animal models of anxiety. Although these studies point to the NK1 receptor as a promising target for the pharmacotherapy of anxiety disorders, high affinity antagonists for the human receptors could not be studied in rats or mice due to species differences in the antagonist binding sites. However, studies on anxiety and depression-related behaviors have now been performed in mouse mutants deficient in NK1 receptor or SP and NKA. These genetic studies have shown that anxiety and depression-related phenotypes are profoundly affected by the tachykinin system. For example, NK1R-deficient mice seem to be less prone depression-related behaviors in models of depression, and one study also provided evidence for reduced anxiety levels. Mice deficient in SP and NKA behaved similarly as the NK1R knockouts. In animal models of anxiety they performed like wildtype mice treated with anxiolytic drugs. In behavioral paradigms related to depression they behaved like wildtype animals treated with antidepressants. In summary, the genetic studies clearly show that the SP/NK1 system plays an important role in the modulation of emotional behaviors.

Animals↗

PubMed1999

Interferon-gamma-dependent/independent expression of indoleamine 2,3-dioxygenase. Studies with interferon-gamma-knockout mice.

The role of IFN-gamma in the expression of indoleamine 2,3-dioxygenase (IDO), a tryptophan oxidizing enzyme, in mouse tissues under physiological and pathological conditions was investigated using IFN-gamma-knockout mice. The results revealed that i) the expression of IDO in the large intestine or in the cecum is mediated by IFN-gamma, ii) for the systemic IDO induction under endotoxin shock, IFN-gamma is a dominant inducer but not essential, and an IFN-gamma-independent mechanism is also operative, iii) the systemic induction of IDO caused by IL-12 or Pokeweed mitogen is mediated by IFN-gamma, and iv) the constitutive IDO expression in the epididymis is IFN-gamma-independent.

Animals↗

PubMed1997

Edema formation exacerbates neurological and histological outcomes after focal cerebral ischemia in CuZn-superoxide dismutase gene knockout mutant mice.

In a variety of studies. CuZn-superoxide dismutase (CuZn-SOD) has been shown to protect against ischemic brain injury. A possible role for CuZn-SOD-related modulation of neuronal viability has been suggested by the finding that CuZn-SOD inhibits brain edema formation following various kinds of neurological insults. We have evaluated the role of CuZn-SOD on brain edema formation following focal cerebral ischemia in mice bearing a disruption of the CuZn-SOD gene (Sod1). Homozygous mutants (Sod1-/-) had no detectable CuZn-SOD activity and heterozygous mutants (Sod1+/-) showed a 50% decrease compared to wild-type mice. Sod1-/- mice showed a high level of blood-brain barrier (BBB) disruption shortly after 1 hr of middle cerebral artery occlusion and 100% mortality at 24 hr following ischemia. Sod1+/- mice showed a moderate level of BBB disruption and 30% mortality. The Sod1+/- animals had increased infarct volume and brain swelling, accompanying exacerbated neurological deficits at 24 hr following ischemia. These results indicate the important role of superoxide anions in the development of brain edema after focal cerebral ischemia and suggest the possibility that brain edema formation may contribute to the exacerbation of ischemic brain injury and neurological deficits in knockout mutant mice.

Animals↗

PubMed1995

Transgenic and gene knockout mice in cancer research.

Transgenic animal technology, and the use of germline manipulation for the creation of targeted gene mutations, has resulted in a plethora of murine models for cancer research. Our understanding of some of the important issues regarding the mechanisms controlling cell division, differentiation and death has dramatically advanced in recent years through exploitation of these techniques to generate transgenic mice. In particular, the generation of mice with targeted mutations in genes encoding proteins of oncological interest has proved to be a useful way of elucidating the function of these gene products in vivo. Transgenic mouse models have provided some insight into the complex oncogenic events contributing to cellular dysregulation and the loss of growth control that can lead to tumorigenesis. These animal studies have highlighted the fact that there are many different stages at which the loss of cell cycle control can occur, as a result of mutations affecting proteins anywhere from the cell surface to the nucleus. Although mutations affecting growth factors, growth factor receptors, signal transduction molecules, cytoplasmic proteins or nuclear proteins might appear to be very distinct, the end result of these changes may be accelerated and unchecked cell growth ultimately leading to cancer. It is beyond the scope of this review to mention every animal model that has been developed for cancer research, especially since many of the early studies have been covered extensively in previous reviews. This article will instead focus on a small selection of transgenic and knockout animal models which exemplify how proteins from distinct localisations along multiple pathways can contribute to loss of cell cycle control and the pathogenesis of cancer.

Animals↗

PubMed1994

More to learn from gene knockouts.

Gene targeting by homologous recombination in mouse embryonic stem cells is a powerful technique to determine the physiological function of any gene product in embryonic and postnatal development and in molecular pathogenesis. Although the technique is very demanding and still in its developing stage several knockout mice carrying disrupted genes, which were once thought important for the development or molecular pathogenesis of certain tissues, have given unexpected results. A gene/function redundancy or superfluous and on-functional theory has been advanced by many investigators to explain the unexpected results. These surprising results may teach us a new lesson and lead to a revision of the strongly held view that highly conserved and abundantly expressed genes have a prominent role and function in cell physiology and development. Additional, they may also support the notion that molecular cross-talk among the genes may play an important role in determining the minimal phenotype.

Animals↗

PubMed2001

Biological and clinical significance of the JAK-STAT pathway; lessons from knockout mice.

Originally described as the signal-transducing pathway of interferons, the JAK-STAT pathway soon turned out to participate in the signalling of numerous other immune and even non-immune mediators. Several murine knockout models have been described that underline the biological significance of this signalling system. Some human diseases (mainly neoplastic) are also known where malfunctioning of the JAK-STAT pathway is considered to participate in the pathogenesis. In this brief review article we will try to make a synopsis of its biological and clinical significance.

Animals↗

PubMed2003

Neuronal nicotinic receptors: insights gained from gene knockout and knockin mutant mice.

Neuronal nicotinic acetylcholine receptors are ligand-gated ion channels that subserve a range of functions in the brain and peripheral nervous system. They are pentamers variously composed of alpha (alpha2-alpha10) and beta subunits (beta2-beta4). Pharmacological and ligand-binding studies have shown that the different subunits vary in their distribution and channel properties, but precise delineation of the in vivo function of individual subunits has been hampered by lack of subunit-specific antagonists. The development of transgenic mice with targeted deletions of specific subunits (knockout mice) or mutations in critical receptor domains (knockin mice) has extended understanding of nicotinic receptors, revealing that some subunits are necessary for viability, whereas others mediate modulatory effects on learning and memory, locomotion, anxiety, nociception, dopaminergic neurotransmission, seizure threshold, development of the visual system and autonomic function. In some cases, studies of transgenic mice have confirmed expectations derived from pharmacological and expression studies, but in other cases, compensation by related subunits has revealed a degree of functional redundancy not predicted by previous approaches.

Animals↗

PubMed2005

Mast cell deficient and neurokinin-1 receptor knockout mice are protected from stress-induced hair growth inhibition.

Despite the lack of insight on distinct mediators in the skin orchestrating the pathophysiological response to stress, hair loss has often been reported to be caused by stress. Recently we revealed the existence of a "brain-hair follicle axis" by characterizing the neurokinin (NK) substance P (SP) as a central element in the stress-induced threat to the hair follicle, resulting in premature onset of catagen accompanied by mast cell activation in the skin. However, our understanding of possible SP-mast cell interactions in the skin in response to stress was limited since the receptor by which SP activates skin mast cells and the extent of mast cell mediated aggravation of SP remained to be elucidated. We now employed NK-1 receptor knockout mice (NK-1R(-/-)) and mast cell deficient W/W(v) mice and observed that stress-triggered premature induction of catagen and hair follicle apoptosis does not occur in NK1(-/-) and W/W(v) mice. Furthermore, the activation status of mast cells was less in stressed NK1(-/-) mice than in wild-type control. Additionally, stress-induced upregulation of SP positive nerve fibers was absent in both NK-1R and W/W(v) mice. These results indicate that the cross-talk between SP and mast cell activation via NK-1R appears to be the most important pathway in the regulation of hair follicle cycling upon stress response.

Animals↗

PubMed2003

Development of autoimmune diabetes in glutamic acid decarboxylase 65 (GAD65) knockout NOD mice.

AIMS/HYPOTHESIS: Type 1 diabetes mellitus, a T-cell-mediated autoimmune disease, results from the selective destruction of insulin-producing pancreatic beta cells. Autoantibodies against beta-cell components are used clinically as sensitive markers of this disease; however, their physiological role has not been clear. To investigate the role of glutamic acid decarboxylase 65 (GAD65) in the development of the Type 1 diabetes of non-obese diabetic (NOD) mice, we analysed and characterised NOD mice with targeted disruption of the GAD65 gene. METHODS: GAD65-deficient mice were previously established. After backcrossing the knockout mutation onto the NOD genetic background for up to eight generations, female littermates of the three resulting genotypes were produced by intercrossing: GAD65 +/+ (n=23), GAD65 +/- (n=62), and GAD65 -/- (n=31). RESULTS: The cumulative incidence of autoimmune diabetes showed no significant difference among the three groups in longitudinal studies using the Kaplan-Meier method. Islet morphology showed that the progression of islet infiltration did not differ significantly between the three groups. CONCLUSION/INTERPRETATION: The cumulative incidence of autoimmune diabetes was not influenced by the GAD65 deficiency. These data suggest that GAD65 is not a major regulatory target of beta-cell autoimmunity in NOD mice.

Age Factors↗

PubMed2006

Glucagon receptor knockout mice are resistant to diet-induced obesity and streptozotocin-mediated beta cell loss and hyperglycaemia.

AIMS/HYPOTHESIS: Under normal physiological conditions, glucagon signalling is important in glucose homeostasis. Hyperglucagonaemia or altered insulin:glucagon ratio plays a role in maintaining hyperglycaemia in subjects with type 2 diabetes. It has been reported that glucagon receptor knockout (Gcgr (-/-)) mice develop normally and have lower plasma glucose on a normal diet. The goal of the current research was to further investigate the role of glucagon signalling in metabolic control and glucose homeostasis. METHODS: Gcgr (-/-) mice were challenged with a high-fat diet (HFD) and with streptozotocin, which induces beta cell damage. They were then analysed for whole-body and serum metabolic phenotypes as well as pancreatic islet morphology. RESULTS: In comparison with wild-type mice, Gcgr (-/-) mice exhibited decreased body weight and food intake, reduced plasma glucose levels, and improved oral and intraperitoneal glucose tolerance. Elevated glucagon-like peptide-1 levels and reduced gastric emptying were also observed in Gcgr (-/-) mice, which also had reduced HFD-induced hyperinsulinaemia and hyperleptinaemia, and were resistant to the development of hepatic steatosis. In addition, Gcgr (-/-) mice were resistant to STZ-induced hyperglycaemia and pancreatic beta cell destruction. CONCLUSIONS/INTERPRETATION: This study demonstrates that blocking glucagon signalling by targeted Gcgr gene deletion leads to an improvement in metabolic control in this mouse model.

Animals↗