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PubMed1999

The tenascin-C knockout revisited.

In the past seven years, two groups have independently produced tenascin-C-knockout mice. These mice are born alive and, originally, were described as showing no abnormalities. More recent studies, many involving pathological intervention, have shown that tenascin-C-knockout mice have several defects. The mice exhibit abnormal behaviour, as well as abnormalities in brain chemistry. They also show defects in structure and repair of neuromuscular junctions, in the ability to recover from snake-venom-induced glomerulonephritis and in chemically induced dermatitis. Healing of skin wounds is morphologically normal, but the mice exhibit defects in healing after suture injury of corneas. In both skin and corneal wounds, fibronectin expression is abnormally low in tenascin-C-knockout mice. Finally, in vitro studies indicate that haemopoietic activity is defective in bone marrow from these mice. When examined together, these studies provide evidence for precise functions for tenascin-C, as well as an explanation for why the sequence of tenascin-C is so highly phylogenetically conserved.

Amino Acid Sequence↗

PubMed2000

The p53 heterozygous knockout mouse as a model for chemical carcinogenesis in vascular tissue.

Heterozygous p53 knockout mice were investigated as a potential model for vascular tumor carcinogenesis. Groups of 20 male mice were exposed by gavage for 6 months to the vascular carcinogen urethane at 1, 10, or 100 mg/kg body weight/day. Wild-type and heterozygous p53 knockout control groups were exposed by gavage to the vehicle alone. Another group of 20 male mice received d-limonene by gavage (d-limonene is noncarcinogenic in mice). The high dose of urethane caused early mortality in the majority of mice associated with histopathologic evidence of toxicity and tumors, including a high incidence of benign and malignant vascular tumors, in all animals. At the intermediate dose, toxicity was less marked and 3 of 20 mice had tumors; mice that received the low dose did not have signs of toxicity or neoplasia. The two control groups had no tumors and the d-limonene group had one tumor of the prostate, which was considered spontaneous. We conclude that the p53 knockout mouse is a useful tool for investigating vascular tumorogenesis.

Animals↗

PubMed2003

A role for the aryl hydrocarbon receptor in cardiac physiology and function as demonstrated by AhR knockout mice.

The aryl hydrocarbon receptor (AhR), a ligand activated transcription factor, is the receptor for the polycyclic aromatic hydrocarbons found in tobacco smoke, polychlorinated biphenyls, and the environmental pollutant, dioxin. To better understand the role of the AhR in the heart, echocardiography, invasive measurements of aortic and left ventricular pressures, isolated working heart preparations, as well as morphological and molecular analysis were used to investigate the impact of AhR inactivation on the mouse heart using the AhR knockout as a model. Cardiac hypertrophy is an early phenotypic manifestation of the AhR knockout. Although the knockout animals were not hypertensive at the ages examined, cardiomyopathy accompanied by diminished cardiac output developed. Despite the structural left ventricular remodeling, the hearts of these animals exhibit minimal fibrosis and do not have the expected increases in surrogate molecular markers of cardiac hypertrophy. The anatomic remodeling without typical features of molecular remodeling is not consistent with hypertrophic growth secondary to pressure or volume overload, suggesting that increased cardiomyocyte size may be a direct consequence of the absence of the AhR in this cell type.

Actins↗

PubMed2006

Production of the butyrylcholinesterase knockout mouse.

The butyrylcholinesterase (BChE [EC 3.1.1.8]) knockout mouse is a model for BChE deficiency in humans. The existence of genetic variants of human BChE was discovered after a new muscle relaxant, succinylcholine, was introduced into the practice of medicine in the late 1950s. People with the atypical variant were unable to breathe for 2 h after receiving a dose intended to paralyze for 3-5 min (Kalow and Gunn, 1957, 1959). The atypical variant was later found to have a single-amino-acid mutation at Asp-70 (McGuire et al., 1989), which decreased the affinity of BChE for all positively charged compounds. Though the atypical BChE mutant is the one most commonly encountered in cases of succinylcholine apnea, an additional 58 mutations in the BChE coding sequence have been reported. The frequency of BChE mutations in the American population is known (Lockridge, 1990). One person out of 25 carries one atypical allele (D70G), whereas 1 out of 2500 is homozygous for D70G. The most frequent mutation, A539T, is carried by 1 person out of every 4 and is found in homozygous form in 1 person out of 69 (Bartels et al., 1992). The homozygous A539T form is associated with a 33% decrease in plasma BChE activity. Some people have no detectable BChE activity in plasma, owing to a mutation that truncates the protein, or inactivates it. The frequency of silent BChE is 1 out of 160 for carriers, and 1 out of 110,000 for homozygotes. The BChE knockout mice are models for silent BChE in humans. The literature contains no documentation of the health of people with silent BChE, other than to say they are healthy. We know nothing about their life expectancy, fertility, risk of cognitive impairment, risk of heart disease, or susceptibility to toxins. The BChE knockout mouse will allow us to test the hypothesis that the function of BChE is to detoxify poisons and will allow us to test the role of BChE in other physiological functions.

Alternative Splicing↗

PubMed2003

Oestrogen receptor knockout mice: roles for oestrogen receptors alpha and beta in reproductive tissues.

Oestrogen is an essential component of female reproduction, with well-characterized functions in the uterus, ovaries, mammary gland and hypothalamic-pituitary axis. The mechanism of oestrogen action involves mediation of the rate of transcription by nuclear-localized oestrogen receptor molecules. Two oestrogen receptors are present in mouse tissues, oestrogen receptors alpha and beta. Each receptor exhibits differential tissue expression patterns. Mouse models with genetically engineered disruption or 'knockout' of the oestrogen receptors have been developed. Characterization of the resulting defects in reproductive tissues as well as alterations in physiological and genomic responses has given insight into the receptor-mediated effects of oestrogen in reproduction. Oestrogen receptor alpha knockout females are infertile because they are anovulatory, have disruption in LH regulation and have uteri that are insensitive to oestrogen. In contrast, oestrogen receptor beta knockout females are sub-fertile and primarily lack efficient ovulatory function. Mice with deletion of both oestrogen receptors alpha and beta are similar to those lacking oestrogen receptor alpha only, but exhibit a unique ovarian pathology. These observed phenotypes elucidate the relative roles of the oestrogen receptors in reproductive functions of female rodents.

Animals↗

PubMed1999

What knockout mice can tell us about parturition.

Many molecules, including steroid and peptide hormones, prostaglandins and cytokines, regulate the preparation, initiation and progression of parturition in mammals. Gene targeting studies show that, in the knockout mice of steroid 5alpha-reductase type 1 gene, prostaglandin F2alpha receptor gene and cytosolic phospholipase A2 gene, parturition was severely disturbed, although live offspring were delivered by Caesarean section. Relaxin gene-disrupted mice also showed protracted labour. However, most knockout mice in which the steroid hormone, prostaglandin, cytokine or peptide hormone (for example, oxytocin, corticotrophin releasing hormone and endothelin) endocrine-paracrine systems are disrupted are inadequate for analysis of the mechanism of parturition because they die before reaching reproductive age or are infertile, or because they reproduce normally. A conditional knockout strategy, for example, using the Cre-LoxP system, should be considered for investigating the biochemical background of parturition to overcome these problems.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed2005

Change in prostaglandin E synthases (PGESs) in microsomal PGES-1 knockout mice in a preterm delivery model.

Most preterm deliveries are associated with infection and inflammation. Prostaglandin E2 (PGE2) is one of the most important mediators in the processes of inflammation, and is converted from PGH2 by various kinds of PGE synthases (PGESs). Among PGESs, microsomal PGES-1 (mPGES-1) is known to be the most important subtype in the processes of inflammation. To evaluate the role of PGESs in preterm delivery, we used mPGES-1 knockout mice in a lipopolysaccharide (LPS)-induced preterm labor model. Unexpectedly, the duration of labor after LPS treatment was not statistically different between C57BL6 wild-type mice and mPGES-1 knockout mice. In wild-type mice, mPGES-1 mRNA and protein expression increased in the myometrium and fetal membrane after LPS treatment. In contrast, the expression of mPGES-2 or cytosolic PGES was not changed by LPS treatment. On mPGES-1 knockout mice, mPGES-2 increased by LPS treatment in myometrium. The present data indicate that mPGES-1 may be involved in LPS-induced preterm labor, but inhibition of mPGES-1 alone may not prevent preterm delivery, because mPGES-2 might compensate for the role of mPGES-1.

Animals↗

PubMed2005

Hypervitaminosis D mediates compensatory Ca2+ hyperabsorption in TRPV5 knockout mice.

Vitamin D plays an important role in Ca(2+) homeostasis by controlling Ca(2+) (re)absorption in intestine, kidney, and bone. The epithelial Ca(2+) channel TRPV5 mediates the Ca(2+) entry step in active Ca(2+) reabsorption. TRPV5 knockout (TRPV5(-/-)) mice show impaired Ca(2+) reabsorption, hypercalciuria, hypervitaminosis D, and intestinal hyperabsorption of Ca(2+). Moreover, these mice demonstrate upregulation of intestinal TRPV6 and calbindin-D(9K) expression compared with wild-type mice. For addressing the role of the observed hypervitaminosis D in the maintenance of Ca(2+) homeostasis and the regulation of expression levels of the Ca(2+) transport proteins in kidney and intestine, TRPV5/25-hydroxyvitamin-D(3)-1alpha-hydroxylase double knockout (TRPV5(-/-)/1alpha-OHase(-/-)) mice, which show undetectable serum 1,25(OH)(2)D(3) levels, were generated. TRPV5(-/-)/1alpha-OHase(-/-) mice displayed a significant hypocalcemia compared with wild-type mice (1.10 +/- 0.02 and 2.54 +/- 0.01 mM, respectively; P < 0.05). mRNA levels of renal calbindin-D(28K) (7 +/- 2%), calbindin-D(9K) (32 +/- 4%), Na(+)/Ca(2+) exchanger (12 +/- 2%), and intestinal TRPV6 (40 +/- 8%) and calbindin-D(9K) (26 +/- 4%) expression levels were decreased compared with wild-type mice. Hyperparathyroidism and rickets were present in TRPV5(-/-)/1alpha-OHase(-/-) mice, more pronounced than observed in single TRPV5 or 1alpha-OHase knockout mice. It is interesting that a renal Ca(2+) leak, as demonstrated in TRPV5(-/-) mice, persisted in TRPV5(-/-)/1alpha-OHase(-/-) mice, but a compensatory upregulation of intestinal Ca(2+) transporters was abolished. In conclusion, the elevation of serum 1,25(OH)(2)D(3) levels in TRPV5(-/-) mice is responsible for the upregulation of intestinal Ca(2+) transporters and Ca(2+) hyperabsorption. Hypervitaminosis D, therefore, is of crucial importance to maintain normocalcemia in impaired Ca(2+) reabsorption in TRPV5(-/-) mice.

Animals↗

PubMed1998

Lessons from gene knockouts.

The authors describe the technique for the application of homologous recombination in embryonic stem cells, which is now widely used to engineer mice which carry specific knockouts of genes. A summary is given of some of the knowledge of the pathogenesis of and resistance to infections with parasites, bacteria, or viruses which has accumulated during recent years, based on the investigation of knockout mice. Special emphasis is placed on knockout animals which lack components of the cytokine network, lack genes which are critical for the correct presentation of antigens or are deficient in different immune cell subsets. In addition, a brief explanation is offered of the possibilities for inducing targeted deletions or mutations in genes of livestock species (e.g., by nuclear transfer or by mutagenesis using the alkylating agent N-ethyl-N-nitrosourea) which could lead to the breeding of animals which are resistant to infectious diseases in the future.

Animals↗

PubMed2006

A simulation study to rescue the Na+/Ca2+ exchanger knockout mice.

The Na(+)/Ca(2+) exchanger (NCX) is the major Ca(2+) efflux system in cardiac myocytes, and thereby its global knockout is embryonically lethal. However, Henderson et al. (2004) found that mice with the cardiospecific knockout of NCX1 lived to adulthood. No adaptation was detected in expression levels of other proteins except for a 50% reduction in the L-type Ca(2+) current (I(CaL)) as revealed in electrophysiological studies. To predict mechanisms of survival, we simulated cardiac myocyte activity in the absence of NCX using a mathematical model of guinea pig ventricular myocytes. The NCX knockout resulted in contracture of the model cell because of a rise in the cytoplasmic Ca(2+) ([Ca(2+)](i)). However, up-regulation of the sarcolemmal Ca(2+) pump (PMCA) and/or down-regulation of I(CaL) enables steady rhythmic contractions even if NCX is totally excluded. The simulation predicted that the steady activities are maintained by a functional up-regulation of PMCA by about 2.3 times in addition to the down-regulation of I(CaL) to a half, as observed in the experiment. However, the model analysis predicted that the myocyte depending on PMCA for Ca(2+) extrusion is unstable against any changes in ionic fluxes and energetically unfavorable in comparison with the control. The reason for the instability is that the activity of PMCA driven by the ATP hydrolysis is hardly affected by changes in [Ca(2+)](i), but NCX has a reversal potential in the middle level of the action potential and is immediately affected by the Ca(2+) flux via NCX itself. The source code of the model is available at http://www.sim-bio.org/.

Action Potentials↗

PubMed2004

Hepatic PTP-1B expression regulates the assembly and secretion of apolipoprotein B-containing lipoproteins: evidence from protein tyrosine phosphatase-1B overexpression, knockout, and RNAi studies.

Protein tyrosine phosphatase-1B (PTP-1B) plays an important role in regulation of insulin signal transduction, and modulation of PTP-1B expression seems to have a profound effect on insulin sensitivity and diet-induced weight gain. The molecular link between PTP-1B expression and metabolic dyslipidemia, a major complication of insulin resistance, was investigated in the present study using PTP-1B knockout mice as well as overexpression and suppression of PTP-1B. Chronic fructose feeding resulted in a significant increase in plasma VLDL in wild-type mice but not in PTP-1B knockout mice. Lipoprotein profile analysis of plasma from PTP-1B knockout mice revealed a significant reduction in apolipoprotein B (apoB100) lipoproteins, associated with reduced hepatic apoB100 secretion from isolated primary hepatocytes. In addition, treatment of cultured hepatoma cells with PTP-1B siRNA reduced PTP-1B mass by an average of 41% and was associated with a 53% decrease in secretion of metabolically labeled apoB100. Conversely, adenoviral-mediated overexpression of PTP-1B in HepG2 cells downregulated the phosphorylation of insulin receptor and insulin receptor substrate-1 and caused increases in cellular and secreted apoB100 as a result of increased intracellular apoB100 stability. Collectively, these findings suggest that PTP-1B expression level is a key determinant of hepatic lipoprotein secretion, and its overexpression in the liver can be sufficient to induce VLDL overproduction and the transition to a metabolic dyslipidemic state.

Animals↗

PubMed2005

Novel testis/sperm-specific contraceptive targets identified using gene knockout studies.

The population explosion and unintended pregnancies resulting in elective abortions continue to be the major public health issues in spite of availability of current methods of contraception. There is an urgent need for a better method of contraception that is accepted, effective, and available. Various targets are being investigated that can be used for contraception. The gene knockout technology is a powerful approach to identify such novel targets. Using search in the database, at least 93 genes were identified in the literature whose deletion demonstrated an effect on fertility in male mice. However, majority of these knockouts also demonstrated an effect on non-reproductive organs concomitant with an effect on fertility. The knockouts of only a few genes/proteins induced a specific effect on fertility without a serious side effect. The potential role of these novel genes/proteins in contraception/contraceptive vaccine development is discussed.

Animals↗

PubMed2005

Gene knockouts that cause female infertility: search for novel contraceptive targets.

The gene knockout technology has revolutionized the fertility/infertility field. It has revealed several essential previously undiscovered molecules, new insights and novel mechanisms involved in steps of the fertility cascade in females. Using database and literature search, knockouts of at least 83 genes were discovered that demonstrated an effect on fertility of female mice. These effects ranged from abnormality in reproductive structure, ovarian function, oocyte, fertilization, embryonic and fetal development, implantation and pregnancy to delivery. However, only a few of these knockout of genes such as encoding oocyte glycoprotein coat comprised of zona pellucida (ZP) 1, ZP2 and ZP3 and oocute plasma membrane specific proteins showed a specific and exclusive target infertility effect without concomitant effects on the non-reproductive organ system. These molecules will provide novel targets of contraception including contraceptive vaccine development.

Animals↗

PubMed1999

Induction of intratumoral tumor necrosis factor (TNF) synthesis and hemorrhagic necrosis by 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in TNF knockout mice.

5,6-Dimethylxanthenon-4-acetic acid (DMXAA) is a new antitumor drug currently undergoing clinical trial. Administration of DMXAA to mice with tumors leads to cessation of tumor blood flow and the onset of tumor hemorrhagic necrosis, accompanied by the production of the cytokine tumor necrosis factor (TNF). Previous studies have shown that DMXAA induces both tumor and host cells to synthesize TNF and that induced intratumoral TNF production correlates with the antitumor activity of DMXAA. To explore the hypothesis that TNF production by tumor cells contributed to the induction of hemorrhagic necrosis by DMXAA, TNF-/- (C57Bl/6 background) mice were used as recipients for the s.c. implantation of (TNF positive) colon 38 adenocarcinoma. Tumors removed 24 h after treatment with DMXAA (66 or 100 micromol/kg) were found to be hemorrhagic and necrotic. Cells expressing TNF mRNA in tumors removed 2 h after treatment with DMXAA (160 micromol/kg) were found by in situ hybridization to be comparable in frequency and distribution with those in tumors from C57Bl/6 TNF-positive mice. However, the amount of TNF protein extracted from tumors from TNF knockout mice was lower than that from TNF-positive mice. Spleen and liver tissue from TNF knockout mice, in contrast to that from TNF-positive mice, produced no TNF mRNA. TNF protein was undetectable in liver and spleen tissue from TNF knockout mice, but was evident in tissue from TNF-positive mice. These results confirm that DMXAA has the novel ability of inducing tumors to synthesize TNF in situ.

Adenocarcinoma↗

PubMed1999

Overexpressed lipoprotein lipase protects against atherosclerosis in apolipoprotein E knockout mice.

Lipoprotein lipase (LPL) is known to play a crucial role in lipoprotein metabolism by hydrolyzing triglycerides; however its role in atherogenesis has yet to be determined. We have previously shown that low density lipoprotein receptor knockout mice overexpressing LPL are resistant to diet-induced atherosclerosis due to the suppression of remnant lipoproteins. Plasma lipoproteins and atherosclerosis of apolipoprotein (apo) E knockout mice which overexpress the human LPL transgene (LPL/APOEKO) were compared with those of control apoE knockout mice (APOEKO). On a normal chow diet, LPL/APOEKO mice showed marked suppression of the plasma triglyceride levels compared with APOEKO mice (54 vs. 182 mg/dl), but no significant changes in plasma cholesterol and apoB levels. Non-high density lipoproteins (HDL) from LPL/APOEKO mice had lower triglyceride content, a smaller size, and a more positive charge compared with those from APOEKO mice. Cholesterol, apoA-I, and apoA-IV were increased in HDL. Although both groups developed hypercholesterolemia to a comparable degree in response to an atherogenic diet, the LPL/APOEKO mice developed 2-fold smaller fatty streak lesions in the aortic sinus compared to the APOEKO mice. In conclusion, overproduction of LPL is protective against atherosclerosis even in the absence of apoE.

Animals↗

PubMed1999

Reduction of 5-hydroxytryptamine (5-HT)(1A)-mediated temperature and neuroendocrine responses and 5-HT(1A) binding sites in 5-HT transporter knockout mice.

The aim of the present study was to determine whether alterations in 5-hydroxytryptamine (5-HT)(1A) receptors would be found in knockout mice lacking the serotonin transporter (5-HTT). Hypothermic and neuroendocrine responses to the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT) were used to examine the function of 5-HT(1A) receptors. Initial studies evaluated the dose-response and time course of 8-OH-DPAT-induced hypothermia and hormone secretion in normal CD-1 mice (the background strain of the 5-HTT knockout mice). 8-OH-DPAT dose-dependently produced hypothermic responses that peaked at 20 min postinjection. 8-OH-DPAT-induced hypothermia was blocked by the 5-HT(1A) antagonist WAY-100635. 8-OH-DPAT dose-dependently increased the concentrations of plasma oxytocin, corticotropin, and corticosterone. In the 5-HTT knockout (-/-) mice, the hypothermic response to 8-OH-DPAT (0.1 mg/kg s.c.) was completely abolished. Furthermore, 5-HTT-/- mice had significantly attenuated plasma oxytocin and corticosterone responses to 8-OH-DPAT. No significant changes in the hypothermic or hormonal responses to 8-OH-DPAT were observed in heterozygous (5-HTT+/-) mice. [(3)H]8-OH-DPAT- and [(125)I]MPPI [4-(2'-methoxyphenyl)-1-[2'-[N-(2"-pyridinyl)-iodobenzamido]ethyl] pip erazine]-binding sites in the hypothalamus and [(125)I]MPPI-binding sites in the dorsal raphe were significantly decreased in 5-HTT-/- mice. The results indicate that lack of the 5-HTT is associated with a functional desensitization of 5-HT(1A) receptor responses to 8-OH-DPAT, which may be a consequence, at least in part, of the decrease in density of 5-HT(1A) receptors in the hypothalamus and dorsal raphe of 5-HTT-/- mice.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed2000

Characterization of antibodies induced by human factor VIII in a murine knockout model of hemophilia A.

To investigate the usefulness of factor VIII (FVIII) knockout mice as an animal model of hemophilia A, we characterized the antibody response in FVIII knockout mice to recombinant human FVIII, administered intravenously or subcutaneously with or without adjuvant, and compared results to those in normal mice. Anti-factor VIII antibodies were detected after both intravenous and subcutaneous administration, with the highest titers after subcutaneous administration plus adjuvant. Depending on the administration strategy. knockout mice formed antibodies more rapidly and developed higher titers of inhibitory antibodies (Bethesda) than normal mice, suggesting differences in epitope specificity. Blotting thrombin cleavage products separated by gel electrophoresis showed that both strains developed antibodies against the nonfunctional B domain as well as against functional domains of factor VIII. The antibodies were mainly of the IgG1 subclass and resembled type I antibodies in hemophilia A.

Animals↗

PubMed2001

A simplified method to prepare PCR template DNA for screening of transgenic and knockout mice.

Polymerase chain reaction (PCR) amplification of DNA is the most widely used technique for screening of large numbers of genetically engineered transgenic or knockout mice (Mus musculus). In this report, we present a new DNA preparation procedure for running diagnostic PCR. In this procedure, mouse ear tissue was used directly for PCR after the tissue underwent brief digestion in a solution containing only proteinase K. Using this method, we have successfully screened several lines of single, double, and triple transgenic and knockout mice. The results are reliable and reproducible. The advantage of this new method is that DNA purification by organic extraction or isolation kit was omitted. DNA purification is the limiting factor in terms of time and money when screening transgenic and knockout mice by PCR. In addition, using ear instead of tail tissue can reduce distress of animals because the samples can be obtained when the mice are labeled by ear punch.

Animal Identification Systems↗