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Results for “Mice, Knockout”

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PubMed1997

High incidence of T-cell tumors in E2A-null mice and E2A/Id1 double-knockout mice.

The basic-helix-loop-helix (bHLH) proteins encoded by the E2A gene are broadly expressed transcription regulators which function through binding to the E-box enhancer sequences. The DNA binding activities of E2A proteins are directly inhibited upon dimerization with the Id1 gene product. It has been shown that disruption of the E2A gene leads to a complete block in B-lymphocyte development and a high frequency of neonatal death. We report here that nearly half of the surviving E2A-null mice develop acute T-cell lymphoma between 3 to 10 months of age. We further show that disruption of the Id1 gene improves the chance of postnatal survival of E2A-null mice, indicating that Id1 is a canonical negative regulator of E2A and that the unbalanced ratio of E2A to Id1 may contribute to the postnatal death of the E2A-null mice. However, the E2A/Id1 double-knockout mice still develop T-cell tumors once they reach the age of 3 months. This result suggests that E2A may be essential for maintaining the homeostasis of T lymphocytes during their constant renewal in adult life.

Animals↗

PubMed1996

Polyoma tumor development in neonatally polyoma-virus-infected CD4-/- and CD8-/- single knockout and CD4-/-8-/- double knockout mice.

CD4-/- or CD8-/- single knockout as well as CD4-/-8-/- double knockout mice were infected with polyoma virus as newborns or 1 week after birth. The animals were followed for tumor development and virus persistence. Double knockout mice developed tumors at a higher incidence (29%) than either the CD8-/- or CD4-/- single knockout mice (11% and 2%, respectively). Persistence of polyoma virus was examined by PCR in one third of all animals included in the study. Seven of the 17 CD4-/-8-/- double knockout mice gave positive evidence of virus persistence up to 6 months p.i. where virus DNA was present in most organs. Corresponding tests in single knockout mice gave positive results of persistent viral DNA in 2 of the 19 CD8-/-and 2 of the 7 CD4-/-mice. In the single knockout mice polyoma DNA could only be detected in a more limited variety of organs compared to the double knockouts.

Animals↗

PubMed2001

Viability of Sarcocystis neurona sporocysts and dose titration in gamma-interferon knockout mice.

Gamma-interferon knockout mice have become the model animal used for studies on Sarcocystis neurona. In order to determine the viability of S. neurona sporocysts and to evaluate the course of the disease in these mice, sporocysts were collected from opossums (Didelphis virginiana), processed, and stored for varying periods of time. Gamma-interferon knockout mice were then inoculated orally with different isolates at different doses. These animals were observed daily for clinical signs until they died or it appeared necessary to humanely euthanize them. 15 of 17 (88%) mice died or showed clinical signs consistent with neurologic disease. The clinical neurologic symptoms observed in these mice appeared to be similar to those observed in horses. 15 of 17 (88%) mice were euthanized or dead by day 35 and organisms were observed in the brains of 13 of 17 (77%) mice. Dose appeared not to effect clinical signs, but did effect the amount of time in which the course of disease was completed with some isolates. The minimum effective dose in this study was 500 orally inoculated sporocysts. Efforts to titrate to smaller doses were not attempted. Direct correlation can be made between molecularly characterized S. neurona sporocysts and their ability to cause neurologic disease in gamma-interferon knockout mice.

Animals↗

PubMed2000

Differential expression of alternatively spliced isoforms of neuronal nitric oxide synthase (nNOS) and N-methyl-D-aspartate receptors (NMDAR) in knockout mice deficient in nNOS alpha (nNOS alpha(Delta/Delta) mice).

Recent data suggest that the neuronal isoform of nitric oxide synthase (nNOS) and glutamate receptors of the N-methyl-D-aspartate (NMDA) type are physically coupled and, hence, functionally interrelated. Several alternatively spliced isoforms of the N-methyl-D-aspartate receptor 1 (NMDAR1) subunit and the neuronal nitric oxide synthase (nNOS) are known, and recent studies have shown that a spliced C-terminal may be responsible for the coupling of NMDAR's to nNOS via its PDZ domain and the postsynaptic density protein PSD95. However, little is known about whether and to what extent changes in nNOS expression influence NMDA receptor density or function. We have therefore compared the localization of nNOS alpha, beta and gamma with that of two relevant NMDAR1 splice variants in wild-type mice versus knockout mice deficient in nNOS alpha, generated by homologous recombination with a targeted deletion of exon 2, containing one PDZ domain (nNOS alpha(Delta/Delta) mice). Whereas nNOS alpha was completely absent in nNOS alpha(Delta/Delta) mice, nNOS beta and gamma were expressed in both wild-type and knockout animals. nNOS gamma mRNA, though, was hardly detectable, if at all, mainly within the olfactory bulb, the cerebellum and mesencephalic nuclei of knockout animals. The expression of the NMDAR1-1 splice variant (without any short carboxy-terminal amino acid motif, recognized by PDZ domains) was remarkably decreased in striatal, cortical, hippocampal and cerebellar tissue in nNOS alpha(Delta/Delta) animals, but no changes in NMDAR1-4 (with an alternatively spliced C-terminal and thus with a PDZ binding motif) mRNA and protein levels were observed. While NMDAR1-4 may be related to receptor targeting and clustering to PSD95 and to nNOS, our data suggest that differences in nNOS expression obviously do not directly influence gene expression of this particular NMDAR splice variant. Otherwise, the observed diminution of NMDAR1-1 splice variant mRNA and protein levels may, at least partially, explain the decreased vulnerability of nNOS alpha(Delta/Delta) mice to glutamate-mediated neurotoxicity.

Alternative Splicing↗

PubMed2006

Functional monoclonal antibodies to p75 neurotrophin receptor raised in knockout mice.

In this study, p75NTREXONIII knockout mice were used as immune-naive hosts to produce functional antibodies to human p75NTR. Three monoclonal antibodies were produced and named MLR1, MLR2 and MLR3, and isotyped as IgG1, IgG2a and IgG2a, respectively. MLR1 and MLR2 bound to human p75NTR with higher affinity than the well-characterized ME20.4 in ELISA and also recognized p75NTR present on neurons in both rat and mouse. MLR1 and MLR2 bound to nerves known to express p75NTR following injection into Balb/C mice but not p75NTREXONIII knockout mice, indicating the antibodies are directed against the ligand binding extracellular region absent in knockout mice. Both MLR1 and MLR2 partially blocked NGF induced cell death in a mouse cell-line that expresses p75NTR but not TrKA. Importantly, intracerebroventricular injections indicated MLR2 was internalized within the cell bodies of mouse basal forebrain neurons, further demonstrating that this antibody is biologically active.

Animals↗

PubMed2001

Cytochrome P450 and antioxidant activity in interleukin-6 knockout mice after induction of the acute-phase response.

Hepatic cytochrome P450 (CYP) expression and antioxidant activity have been shown to decrease following endotoxin (lipopolysaccharide [LPS]) or proinflammatory cytokine administration. Using mice deficient in interleukin-6 (IL-6), the role of IL-6 in the regulation of hepatic CYP activity, glutathione (GSH) metabolism, and catalase (CAT) activity was analyzed after LPS administration. Administration of LPS produced comparable decreases in hepatic CYP3A activity in WT B6x129 (WT) mice and IL-6 knockout mice. No decrease was observed for CYP2D9 activity after LPS administration in either WT or IL-6 knockout mice. LPS administration significantly increased hepatic and renal CYP2E1 and CYP4A activity in WT mice, with no effect in IL-6 knockout mice. CYP2A12 activity increased in IL-6 knockout, mice with no change in WT mice after LPS administration. LPS administration had no significant effect on hepatic GSH reductase, GST peroxidase, GSH-S-transferase (GST), or total GSH in either WT or IL-6 knockout. However, hepatic CAT activity was significantly reduced in WT mice after LPS administration, with no effect in IL-6 knockout mice. These results support IL-6 as a critical mediator of the effects of LPS on specific hepatic and renal CYP activities and hepatic CAT activity.

Acute-Phase Reaction↗

PubMed1999

[Dopamine receptor knockout mice].

Dopaminergic systems are transmitted by dopamine receptors which couple to GTP binding proteins. Five subtypes of dopamine receptors were so far cloned. To study the functions of each dopamine receptors, mice lacking each of D1R, D2R, D3R and D4R dopamine receptors have been generated. Histological analyses of D1R knockout mice indicated that the expression of dynorphin is reduced in the striatum. In contrast with wild-type mice, D1R knockout mice exhibit a dose dependent decrease in locomotion. D2R knockout mice display a hypoactivity. The expression of enkephalin mRNA in the striatum is increased in the D2R knockout mice. D2R knockout mice showed hyperplastic changes of intermediate lobe of the pituitary and the increased expression of POMC in the pituitary in D2R knockout mice. D3R knockout mice are more active than wild-type mice in a novel environment and they exhibit enhanced behavioral sensitivity to cocaine and amphetamine. D4R knockout mice show a hypoactivity, but they display locomotor supersensitivity to ethanol, cocaine, and methamphetamine. Dopamine synthesis and its conversion to DOPAC are elevated in the dorsal striatum from D4R knockout mice.

Animals↗

PubMed1997

Hair follicle dermal papilla cell lines from p53-knockout mice.

We have attempted to establish a cell line from hair follicle dermal papillae microdissected from vibrissa of mice lacking p53 tumor suppressor gene. The hair follicle dermal papillae were obtained from three types of mice: null knockout, hetero-knockout and wild type litters. Continuous cell lines with short doubling time were obtained only from null knockout litter-mates. Dermal papilla cells from either the hetero-knockout or wild type mice did not multiply well, and died within three passages; provided, however, that in only three cases out of 40 primary dermal papilla implants from two hetero-knockout mice, the cell culture reached 29 passages with a much slower growth rate than the cell lines from the null-knockout mice. These data are in accordance with the notion that the loss of p53 function is closely related to perpetuation of cell cycles and or immortalization.

Animals↗

PubMed2002

Peroxisome proliferator-activated receptor alpha,gamma coagonist LY465608 inhibits macrophage activation and atherosclerosis in apolipoprotein E knockout mice.

The apolipoprotein E (apoE) knockout mouse has provided an approach to the investigation of the effect of both cellular and humoral processes on atherosclerotic lesion progression. In the present study, pharmacologic modulation of both interferon gamma (IFNgamma)-inducible macrophage effector functions, and atherosclerotic lesions in the apoE knockout mouse were investigated using the peroxisome proliferator-activated receptor (PPAR) alpha,gamma coagonist LY465608. LY465608 inhibited, in a concentration-dependent manner, IFNgamma induction of both nitric oxide synthesis and the beta 2 integrin CD11a in elicited peritoneal macrophages from apoE knockout mice. Similar effects were observed ex vivo following 10 d of treating mice with 10 mg/kg of LY465608. Treatment of apoE knockout mice for 18 wk with LY465608 resulted in a statistically significant 2.5-fold reduction in atherosclerotic lesion area in en face aorta preparations. These effects were apparent in the absence of any reduction in total serum cholesterol or in lipoprotein distribution. Finally, treatment of apoF knockout mice with established atherosclerotic disease resulted in a modest but not statistically significant decrease in aortic lesional surface area. These results demonstrate the utility of PPAR coagonists in reducing the progression of the atherosclerotic lesion.

Animals↗

PubMed1997

Glutathione peroxidase knockout mice are susceptible to myocardial ischemia reperfusion injury.

BACKGROUND: To test our hypothesis that intracellular antioxidant enzymes constitute a cellular defense against acute stress, we studied myocardial ischemia reperfusion injury in the setting of reduced level of glutathione peroxidase using GSHPx-1 gene knockout mice. METHODS: Knockout mice were developed by disrupting the coding sequence of GSHPx-1 gene after inserting a neomycin resistance gene derived from pMCIpol A into the EcoRI site located in exon 2. Isolated perfused hearts were prepared from two groups of mice-knockout and nontransgenic controls. A 4-0 silk was attached to the apex of the heart which in turn was attached to a force transducer. Hearts were perfused by the Langendorff mode, and after 20 minutes of stabilization subjected to 30 minutes of ischemia followed by 2 hours of reperfusion. The force developed by the heart (DF) and the first derivative of DF (dF/dt) were recorded. Creatine kinase (CK) release was measured in the perfusate and the infarct size was measured at the end of each experiment. RESULTS: For both GSHPx-1 knockout and nontransgenic control groups, DF and dF/dt were significantly lower during early postischemic reperfusion compared with baseline, but these values were significantly higher for the control group than the knockout mice throughout most of the reperfusion period. CK release from the heart increased during reperfusion for both groups, but this increase was significantly lower for the control group. The infarct size was also smaller for the control mice as compared with knockouts. CONCLUSIONS: The results indicate that the knockout mice are more susceptible to ischemia reperfusion injury, suggesting the importance of GSHPx-1 gene in myocardial protection from ischemic reperfusion injury.

Animals↗

PubMed2002

MCMV infection increases early T-lymphocyte influx in atherosclerotic lesions in apoE knockout mice.

BACKGROUND: Multiple epidemiological studies have suggested that cytomegalovirus (CMV) infection is associated with atherosclerotic disease. However, conclusive proof that the virus is directly related to the progression of the disease is still lacking. OBJECTIVES: The goal of this study was to investigate whether MCMV is able to exacerbate the atherosclerotic process in atherosclerosis-susceptible mice. STUDY DESIGN: apoE knockout mice kept on a chow diet were sacrificed at both 2 and 20 weeks post infection (p.i.). C57Bl/6J mice fed an atherogenic diet were sacrificed at 2 weeks p.i. Lesion area, lesion composition (endothelial cells and smooth muscle cells) and inflammatory influx (T-lymphocytes and macrophages) in lesions were determined. The former one was determined by means of a microscope coupled to a computer-assisted morphometry system. The latter ones were scored after immunohistochemical staining. RESULTS: In the chronic phase of the infection mean lesion size was significantly increased after MCMV infection in the apoE knockout mice. This increase could to a large extent be attributed to a significant increase in type V lesion area after MCMV infection. Also, a significant increase in T-lymphocyte influx was observed in the acute phase of the infection in lesions from apoE knockout mice after MCMV infection while this effect was absent in C57Bl/6J mice. After MCMV infection no increase was observed in macrophage, smooth muscle cell and endothelial cell number in lesions from both mice strains. CONCLUSIONS: MCMV infection may exacerbate the atherosclerotic process in apoE knockout mice by means of an acute lymphocytic inflammatory response. In contrast to the MCMV induced effect in apoE knockout mice, MCMV infection did not increase the influx of T-lymphocytes in atherosclerotic lesions of C57Bl/6J mice.

Acute Disease↗

PubMed2005

Dense core vesicle proteins IA-2 and IA-2beta: metabolic alterations in double knockout mice.

IA-2 and IA-2beta are members of the transmembrane protein tyrosine phosphatase family located in dense core vesicles of neuroendocrine cells, including the beta-cells of pancreatic islets. In the present study, by mating C57BL/6Nci IA-2(+/-) with IA-2beta(+/-) mice, we generated double knockout mice (IA-2(-/-)/IA-2beta(-/-)) to study the effect of the combined deletion of these two proteins on insulin secretion and blood glucose levels. The double knockout mice appeared healthy at birth and showed normal growth and development. Histological examination and immunostaining for insulin, glucagon, somatostatin, and pancreatic polypeptide revealed no difference between the double knockout and wild-type mice. Nonfasting blood glucose and insulin levels also were within the normal range. However, compared with the wild-type mice, the double knockout mice showed glucose intolerance and an absent first-phase insulin release curve. No evidence of insulin resistance was observed nor were there alterations in fasting blood glucose, insulin, or leptin levels in the double knockout mice maintained on a high-fat diet compared with the wild-type mice maintained on the same diet. In addition, to determine whether the combined deletion of IA-2 and IA-2beta played any role in the development of diabetes in NOD mice, we generated double knockout mice on the NOD/LtJ background. The incidence of diabetes in these mice was not significantly different than that in the wild-type mice. Taken together, our experiments show that the dense core vesicle proteins IA-2 and IA-2beta, alone or in combination, are involved in insulin secretion, but neither alone nor in combination are they required for the development of diabetes in NOD mice.

Animals↗

PubMed2002

Characterization of lpa(2) (Edg4) and lpa(1)/lpa(2) (Edg2/Edg4) lysophosphatidic acid receptor knockout mice: signaling deficits without obvious phenotypic abnormality attributable to lpa(2).

Lysophosphatidic acid (LPA), a bioactive lipid produced by several cell types including postmitotic neurons and activated platelets, is thought to be involved in various biological processes, including brain development. Three cognate G protein-coupled receptors encoded by lpa(1)/lp(A1)/Edg-2/Gpcr26, lpa(2)/lp(A2)/Edg-4, and lpa(3)/lp(A3)/Edg-7 mediate the cellular effects of LPA. We have previously shown that deletion of lpa(1) in mice results in craniofacial dysmorphism, semilethality due to defective suckling behavior, and generation of a small fraction of pups with frontal hematoma. To further investigate the role of these receptors and LPA signaling in the organism, we deleted lpa(2) in mice. Homozygous knockout (lpa(2)((-/-))) mice were born at the expected frequency and displayed no obvious phenotypic abnormalities. Intercrosses allowed generation of lpa(1)((-/-)) lpa(2)((-/-)) double knockout mice, which displayed no additional phenotypic abnormalities relative to lpa(1)((-/-)) mice except for an increased incidence of perinatal frontal hematoma. Histological analyses of lpa(1)((-/-)) lpa(2)((-/-)) embryonic cerebral cortices did not reveal obvious differences in the proliferating cell population. However, many LPA-induced responses, including phospholipase C activation, Ca(2+) mobilization, adenylyl cyclase activation, proliferation, JNK activation, Akt activation, and stress fiber formation, were absent or severely reduced in embryonic fibroblasts derived from lpa(1)((-/-)) lpa(2)((-/-)) mice. Except for adenylyl cyclase activation [which was nearly abolished in lpa(1)((-/-)) fibroblasts], these responses were only partially affected in lpa(1)((-/-)) and lpa(2)((-/-)) fibroblasts. Thus, although LPA(2) is not essential for normal mouse development, it does act redundantly with LPA(1) to mediate most LPA responses in fibroblasts.

Adenylyl Cyclases↗

PubMed1996

Testing NF-kappa B1-p50 antibody specificity using knockout mice.

Cell extracts from knockout mice can provide definitive proof of antibody specificity. Two NF-kappa B1-p50 antibodies, sc-114 (a commercial antibody) and NR1157, were observed to recognize proteins having distinct electrophoretic mobilities of 52-55 kD and 50 kD, respectively, by Western blot analysis. In order to discriminate the specificity of these antibodies for NF-kappa B1-p50, whole cell extracts derived from NF-kappa B1-p105 knockout mice were employed. While the NR1157 antibody completely failed to recognize its 50 kD product in p105-/- knockout extracts, the sc-114 antibody still strongly recognized its 52-55 kD product. These data demonstrate that NR1157, but not sc-114, is highly specific for NF-kappa B1-p50 by Western blot analysis. In addition, these results highlight the utility of knockout cell extracts for discerning antibody specificity.

Amino Acid Sequence↗

PubMed2002

Sex-dependent modulation of ethanol consumption in vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) knockout mice.

Several lines of evidence suggest that monoaminergic systems, especially dopaminergic and serotoninergic systems, modulate ethanol consumption. Humans display significant differences in expression of the vesicular and plasma membrane monoamine transporters important for monoaminergic functions, including the vesicular monoamine transporter (VMAT2, SLC18A2) and dopamine transporter (DAT, SLC6A3). In addition, many ethanol effects differ by sex in both humans and animal models. Therefore, ethanol consumption and preference were compared in male and female wild-type mice, and knockout (KO) mice with deletions of genes for DAT and VMAT2. Voluntary ethanol (2-32% v/v) and water consumption were compared in two-bottle preference tests in wild-type (+/+) vs heterozygous VMAT2 KO mice (+/-) and in wild-type (+/+) vs heterozygous (+/-) or homozygous (-/-) DAT KO mice. Deletions of either the DAT or VMAT2 genes increased ethanol consumption in male KO mice, although these effects were highly dependent on ethanol concentration, while female DAT KO mice had higher ethanol preferences. Thus, lifetime reductions in the expression of either DAT or VMAT2 increase ethanol consumption, dependent on sex.

Alcohol Drinking↗

PubMed2003

G-protein coupling of delta-opioid receptors in brains of mu-opioid receptor knockout mice.

Mu-opioid receptor knockout mice have been reported to show loss of some delta-opioid receptor function. We hypothesised that this is due to some delta-opioid receptors being uncoupled from G-proteins in the absence of the mu-opioid receptor. To address this possibility, we have used quantitative autoradiography to determine the binding of three delta-opioid receptor agonist ligands ([3H]deltorphin I, [3H] [R-Atc(3), Ile(5,6)]deltorphin II, [3H] 4-[(alpaR)-alpha-((2S,5R)-4-propyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-121)) and the delta-opioid receptor antagonist, [3H]naltrindole in the presence and absence of a GTP analogue, guanylylimidodiphosphate (GMPPNP) in the brains of mice lacking the mu-opioid receptor gene. Guanylylimidodiphosphate caused a decrease in the binding of the agonist ligands (to differing extents) and an increase in binding for the antagonist in wild-type controls. Overall, there were no major differences in the effects of guanylylimidodiphosphate for the agonist ligands in mu-knockout mice although a few structures showed a smaller sensitivity to the effects of this GTP analogue most notably for [3H]naltrindole. These findings suggest that in the majority of brain regions, G-protein coupling is unaltered in mu-opioid receptor knockout mice.

Animals↗

PubMed2004

Light deprivation slows but does not prevent the loss of photoreceptors in taurine transporter knockout mice.

UNLABELLED: Taurine transporter knockout mice show severe retinal degeneration at an early age. The study was designed to determine whether degeneration also takes place in the absence of light. Mice were maintained up to 6 weeks of age in cyclic lighting or in total darkness. Degeneration took place in both groups, but was more rapid in animals exposed to standard cyclic illumination. At the ultrastructural level the retinas showed features characteristic of apoptosis but not of necrosis. CONCLUSIONS: Cell differentiation is not seriously affected by the lack of a functional taurine transporter but mature photoreceptor cells do not survive without an intact transporter, even in the dark.

Animals↗

PubMed2003

Effect of a single dose of ponazuril on neural infection and clinical disease in Sarcocystis neurona-challenged interferon-gamma knockout mice.

Interferon gamma-knockout mice were challenged with 5000 Sarcocystis neurona sporocysts acquired from a naturally infected opossum. Ponazuril was administered once, by gavage, at day 1, 3, 7, 10, or 14 post-infection (pi). Ponazuril was given at either 20 or 200mg/kg. Mice that survived to day 30 pi were euthanized. Severity of CNS infection was quantified as schizont density in the cerebellum. Unchallenged mice in treatment and non-treatment groups remained free of disease and gained weight throughout the experiment. All challenged mice, regardless of treatment, developed histologic evidence of CNS infection even though clinical signs were prevented in some groups. The greatest treatment benefits were seen in mice given 200mg/kg ponazuril between days 4 and 14 pi. Weight gain over the course of the experiment occurred only in mice that were given 200mg/kg ponazuril on day 7 or 10 pi. With the exception of groups given 200mg/kg ponazuril on day 7 or 14 pi, mice in groups that got sporocysts developed abnormal neurologic signs. No deaths before day 30 pi occurred in mice given ponazuril at 20mg/kg on day 7 pi or 200mg/kg on day 1, 7, 10, or 14 pi. This effect was not significant. Mice given 200mg/kg on day 7 pi had significantly fewer cerebellar schizonts than did those of the control group that was not given ponazuril. These results indicate that single-dose administration of ponazuril for prevention of CNS infection is partially protective when given between days 4 and 14 pi.

Animals↗