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PubMed2003

Family 1 G protein-coupled receptor function in the CNS. Insights from gene knockout mice.

Family 1 G protein-coupled receptors (GPCRs) are activated by a large number of ligands including photons, odorants, neurotransmitters and hormones and are involved in a wide variety of central and peripheral functions. Due to their wide distribution in the central nervous system (CNS), family 1 GPCRs play a major role in the regulation of neuronal activity and behaviour. In general, the lack of selective ligands for each member of the GPCR subfamilies has made it difficult to assign specific central functions to each receptor subtype. Advances in gene targeting techniques have allowed the inactivation of receptor genes in the mouse through homologous recombination leading to the generation of mouse 'knockout' models lacking one or more GPCRs. In this review, we have listed the family 1 GPCR knockout models produced in the past decade and we have summarized the findings obtained from studies on these mice with respect to CNS function.

Animals↗

PubMed1998

Requirement of the CD8+ or CD4+ T lymphocyte subsets for the rejection of lymphoma and fibrosarcoma grafts studied in gene knockout hosts.

Rejections of the retrovirus induced lymphomas (ALC and RBL-5) and the methylcholanthrene (MCA) induced fibrosarcoma (MC57X) grafts were tested in syngeneic CD8 and CD4 single and double knockout C57BL/6 mice. The results with the lymphomas showed that the CD8+ T cell deficiency prevented the development of rejection response induced by immunization. Deficiency of the CD4+ T subset abrogated also the rejection of ALC. Immunity against the fibrosarcoma cells developed in both type of single knockout mice, but not in the ones which lacked both CD4+ and CD8+ T cells. Thus CD8+ T cells were required for rejection of the lymphoma cells, while the CD4+ T cells only mediated a weak response. In absence of CD8+ T cells, CD4+ T cells were sufficient to reject the fibrosarcoma cells.

Animals↗

PubMed2003

Enhanced humoral response in kappa-opioid receptor knockout mice.

Opiates are major analgesics and addictive drugs described also as immunomodulators. Here, we investigated the contribution of kappa-opioid receptor (KOR) activity in immunity in vivo by studying immune responses in KOR knockout mice. These animals displayed a modest reduction in thymus cellularity and CD4(+) cell ratio, parallel to a slight increase in immature CD4(+)CD8(+) lymphocytes. In spleen, KOR null animals showed augmented cell number with no change in cell distribution. T and B lymphocyte proliferative capabilities in vitro, Natural Killer activity and steady-state Ig levels were unchanged in KOR-/- mice. We immunized the mice with the antigen keyhole limpet hemocyanin (KLH). Compared to wild-type (WT) mice, KOR-/- animals produced significant higher levels of antigen-specific total Ig, IgM, IgG1 and IgG2a antibodies. This enhancement of humoral activity was not observed in mu-opioid receptor and delta-opioid receptor knockout animals. These results show that endogenous activation of kappa-opioid receptors may exert a tonic inhibition of antibody (Ab) response.

Animals↗

PubMed2002

Knockout mice: simple solutions to the problems of genetic background and flanking genes.

Inducing null mutations by means of homologous recombination provides a powerful technique to investigate gene function and has found wide application in many different fields. However, it was realized some time ago that the specific way in which such knockout mutants are generated can be confounding, making it impossible to separate the effects of the induced null mutation from those of alleles originating from the embryonic stem cell donor. In addition, effects from null mutations can be altered on different genetic backgrounds. Here we present some simple breeding strategies to test for flanking gene effects that are compatible with the recommendations of the Banbury Conference on Genetic Background in Mice and with common practices of creating and maintaining mouse knockout lines.

3' Flanking Region↗

PubMed1997

Synaptic plasticity, place cells and spatial memory: study with second generation knockouts.

The use of genetically engineered mice has been a major development in neuroscience research. Genetic engineering is an undoubtedly powerful technique; however, the value of this approach has been debated, particularly in relation to its use to probe the underlying bases of complex behaviors, such as memory. A recent new development of the technique is the ability to target a specific gene knockout to a particular subregion or even to specific and limited cell types of the mouse brain. An example of this approach is the knockout of the NMDARI gene in only CAI-pyramidal cells of the hippocampus. The resulting animals can be tested by several methods, including in vivo multielectrode recording during behavioral tasks. The data provide strong evidence in favor of the notion that NMDA receptor-dependent synaptic plasticity at CAI synapses is required for both the acquisition of spatial memory and the formation of normal CAI place fields. This relationship suggests that robust place fields may be essential for spatial memory.

Animals↗

PubMed2001

Measuring pain in the (knockout) mouse: big challenges in a small mammal.

The increasing popularity of the mouse as a subject in basic science studies of pain can largely be attributed to the development of transgenic "knockout" technology in this species only. To take advantage of this biological technique, many investigators are rushing to adapt to the mouse experimental protocols that were designed for the rat. However, the myriad physiological and behavioral differences between these two rodent species render such adaptations non-trivial and in many cases seriously problematic. In this article we review the basic nociceptive assays used in behavioral pain research (thermal, mechanical, electrical and chemical), and highlight how species differences affect their proper application. In addition, some of the issues specifically pertaining to the interpretation of such data in knockout studies are addressed.

Animals↗

PubMed2000

Involvement of mu-opioid receptors in potentiation of apomorphine-induced climbing behavior by morphine: studies using mu-opioid receptor gene knockout mice.

The present study examined the hypothesis that mu-opioid receptors contribute to a behavioral stimulation produced by stimulation of dopamine receptors by comparing responses in mu-opioid receptor knockout and wild type mice. Apomorphine-induced climbing behavior was augmented by 65%, in wild type mice, but not in mu-knockout, following subcutaneous administration of morphine (15 mg/kg). Moreover, pretreatment with either naloxone (an opioid receptor antagonist) or haloperidol (a mixed D(1)/D(2) receptor antagonist) eliminated the enhancement by morphine of climbing behavior in wild type mice. These results indicate that expression of mu-opioid receptors plays an important role in the enhancement of climbing behavior induced by the dopamine receptor agonist, apomorphine. Furthermore, this augmentation is mediated by interaction between dopamine and mu-opioid receptors.

Analgesics, Opioid↗

PubMed2001

Generation of dynorphin knockout mice.

The opioid system has important roles in controlling pain, reward and addiction, and is implicated in numerous other processes within and outside the nervous system, such as mood states, immune responses, and prenatal developmental processes. The effects of the opioid system are mediated by at least three ligands, enkephalin, endorphin, and dynorphin, which act through the opioid receptors mu, delta, and kappa. In order to dissect the roles of individual components of the opioid system, mutant mice lacking single ligands or receptors are instrumental. We report here on the generation and initial characterization of a mutant mouse strain lacking pre-prodynorphin. Dynorphin 'knockout' mice are viable, healthy, and fertile and show no overt behavioral differences to wildtype littermates. Dynorphin knockout mice constitute a valuable tool for many research areas, among them research into pain, substance abuse, and epilepsy.

Animals↗

PubMed1999

Region-specific up-regulation of opioid receptor binding in enkephalin knockout mice.

Mu and delta opioid receptors were labeled in enkephalin knockout mice by quantitative autoradiography. Discrete, large increases (100-300%) were found in limbic forebrain structures for mu binding and striatum and pallidum for delta binding. The up-regulation of opioid receptors may reflect a form of 'denervation supersensitivity. ' The receptor up-regulation in limbic areas is consistent with the increased emotional and aggressive behaviors observed in the enkephalin knockout mice.

Animals↗

PubMedDate not supplied

The role of follicle-stimulating hormone in spermatogenesis: lessons from knockout animal models.

The development of knockout mouse models for the FSH-beta subunit, the FSH receptor, and LH-receptor performed in different laboratories has confirmed and extended our knowledge concerning the critical role of these hormone-signaling systems in spermatogenesis. In this article, we summarize the phenotypic changes observed in male FSH receptor knockout (FORKO) mice. Young FORKO males have underdeveloped testis with 50% reduction in Sertoli cells, suggesting that FSH-R signaling is required very early for gonadal development, maturity, and function. These mice experience delayed puberty with postponement in the formation of round spermatids. Adult males show reduction in serum testosterone levels despite normal circulating LH concentration, indicating disturbances in Sertoli-Leydig cell communication. As a consequence of reduced sperm production and sperm quality, adult FORKO males have reduced fertility. Aberrant sperm from FORKO males have retention of cytoplasmic droplets and inadequate DNA compaction, hallmarks of infertility in many species including man. Interestingly, these changes are also experimentally inducible in FSH- and/or FSH-R-immunized male bonnet monkeys, creating a state of infertility. Reports of human mutations in FSH-beta and the FSH receptor also indicate that spermatogenesis is dependent on this system. Further investigations in FORKO males should be helpful in uncovering the downstream genes involved in sustaining Sertoli cell function and maintenance of the quantitative and qualitative aspects of spermatogenesis. This might pave the way for treatment of male infertility and contraception.

Amino Acid Substitution↗

PubMed2003

Lack of susceptibility of heterozygous p53-knockout CBA and CIEA mice to phenolphthalein in a 6-month carcinogenicity study.

Phenolphthalein has carcinogenic activity, causing malignant lymphomas in B6C3F1 mice at a dietary dose of 3000 ppm in a 2-year carcinogenicity study and in female heterozygous p53-knockout TSG mice (C57BL/6 background) at the same dose in a 6-month study. To examine whether carcinogenic potential of phenolphthalein can be detected in other p53-knockout mouse [p53 (+/-)] strains such as p53 (+/-) CBA mice or p53 (+/-) CIEA mice (C57BL/6 background) and their littermates, they were given a diet containing 0, 6000 or 12000 ppm for 6 months. Unequivocal induction of neoplastic lesions was not apparent, suggesting that p53 (+/-) CBA mice and p53 (+/-) CIEA mice are resistant to the induction of malignant lymphomas by the treatment of phenolphthalein.

Administration, Oral↗

PubMed1997

Physiological role of brain endothelin in the central autonomic control: from neuron to knockout mouse.

Although endothelin (ET) was discovered as a potent vascular endothelium-derived constricting peptide, its presumed physiological and pathophysiological roles are now considered much more diverse than originally though. Endothelin in the brain is thought to be deeply involved in the central autonomic control and consequent cardiorespiratory homeostasis, possibly as a neuromodulator or a hormone that functions locally in an autocrine/paracrine manner or widely through delivery by the cerebrospinal fluid (CSF). This notion is based on the following lines of evidence. (1) Mature ET, its precursors, converting enzymes, and receptors all are detected at strategic sites in the central nervous system (CNS), especially those controlling the autonomic functions. (2) The ET is present in the CSF at concentrations higher than in the plasma. (3) There is a topographical correspondence of ET and its receptors in the CNS. (4) The ET is released by primary cultures of hypothalamic neurons. (5) When ET binds to its receptors, intracellular calcium channels. (6) An intracerebroventricular or topical application of ET to CNS sites elicits a pattern of cardiorespiratory changes accompanied by responses of vasomotor and respiratory neurons. (7) Recently generated knockout mice with disrupted genes encoding ET-1 exhibited, along with malformations in a subset of the tissues of neural crest cell lineage, cardiorespiratory abnormalities including elevation of arterial pressure, sympathetic overactivity, and impairment of the respiratory reflex. Definitive evidence is expected from thorough analyses of knockout mice by applying conventional experimental methods.

Amino Acid Sequence↗

PubMed2001

Studying TGF-beta superfamily signaling by knockouts and knockins.

The transforming growth factor beta (TGF-beta) superfamily has profound effects on many aspects of animal development. In the last decade, our laboratory and others have performed in vivo functional studies on multiple components of the TGF-beta superfamily signal transduction pathway, including upstream ligands, transmembrane receptors, receptor-associated proteins and downstream Smad proteins. We have taken gene knockout approaches to generate null alleles of the genes of interest, as well as a gene knockin approach to replace the mature region of one TGF-beta superfamily ligand with another. We found that activin betaB, expressed in the spatiotemporal pattern of activin betaA, can function as a hypomorphic allele of activin betaA and rescue the craniofacial defects and neonatal lethal phenotype of activin betaA-deficient mice. With the knockout approach, we have shown that the expression pattern of a component in the TGF-beta superfamily signal transduction cascade does not necessarily predict its in vivo function. Two liver-specific activins, activin betaC and activin betaE are dispensable for liver development, regeneration and function, whereas ubiquitously expressed Smad5 has specific roles in the development of multiple embryonic and extraembryonic tissues.

Activins↗

PubMed1997

Lipid metabolism in tumour-bearing mice: studies with knockout mice for tumour necrosis factor receptor 1 protein.

The implantation of the Lewis lung carcinoma (a fast-growing mouse tumour that induces cachexia) to both wild-type and gene-deficient mice for the tumour necrosis factor (TNF) receptor type I protein (Tnfr1(0)/Tnfr1(0)), resulted in a considerable loss of carcass (26%) and white (77%) and brown adipose (37%) tissue weights in the wild-type mice, while it induced much less marked effects in the gene-deficient mice. Tumour burden also inflicted an important decrease in total lipoprotein lipase (LPL) activity in epididymal white adipose tissue (50%) in the wild-type mice while no changes were observed in the knockout mice. In addition, all tumour-bearing animals were clearly hypertriglyceridaemic (80% increase in circulating triacylglycerols in wild-type and 36% in knockout mice). It is concluded that although TNF seems to be to some extent responsible for adipose waste, LPL changes and hyperlipaemia (via receptor I), the role of other cytokines (alone or in combination with TNF) in promoting changes in lipid metabolism during cancer cachexia cannot be discarded.

Adipose Tissue↗

PubMed1998

Role of TNF receptor 1 in protein turnover during cancer cachexia using gene knockout mice.

The implantation of the Lewis lung carcinoma (a fast-growing mouse tumour that induces cachexia) to both wild-type and gene-deficient mice for the TNF-alpha receptor type I protein (Tnfr1 degree/Tnfr1 degree), resulted in a considerable loss of carcass weight in both groups. However, while in the wild-type mice there was a loss of both fat and muscle, in the gene-knockout mice muscle wastage was not affected to the same extent. In both groups, tumour burden resulted in significant increases in circulating TNF-alpha, a cytokine which, as we have previously demonstrated, can induce protein breakdown in skeletal muscle. Muscle wastage in wild-type mice was accompanied by an increase in the fractional rate of protein degradation, while no changes were observed in protein synthesis. The result is a decreased rate of protein accumulation that accounts for the muscle weight loss observed as a result of tumour burden. In contrast, gene knockout mice did not have significantly lower rates of protein accumulation as a result of tumour implantation. The increase in protein degradation in the tumour-bearing wild mice was accompanied by an enhanced expression of both ubiquitin and proteasome subunit genes, all of them related to the activation of the ATP-dependent proteolytic system in skeletal muscle. Tumour-bearing gene-deficient mice did not show any increase in gene expression. It is concluded that TNF-alpha (alone or in combination with other cytokines) is responsible for the activation of protein breakdown in skeletal muscle of tumour-bearing mice.

Animals↗

PubMed2000

Successful generation of peripheral neuropathy with onion-bulb formation in the macrophage scavenger receptor classA knockout mouse treated with isoniazid.

We previously reported successful generation of many onion-bulbs (OBs) and formation of oxidized phosphatidylcholine after compression injury to the peripheral nerve of mice deficient of macrophage scavenger receptor classA (MSR-A). In the present study, we employed chemical injury with isoniazid to the peripheral nerve of the MSR-A knockout mice to investigate the role of the MSR-A in toxic neuropathy. Peripheral neuropathy has not previously been generated with isoniazid in mice. In the present study, we also noted little histological change after isoniazid administration not only to control littermates but also to the A/J strain mice known to be slow acetylators. Surprisingly, however, we were successful in generating peripheral neuropathy with isoniazid in the MSR-A knockout mice. Histologically, the predominant feature was the presence of many thinly myelinated fibers with some OBs, which have not been observed in rats with isoniazid neuropathy. Deficiency of the MSR-A appears to have played an important role in generation of peripheral neuropathy with isoniazid in mice.

Animals↗

PubMed2002

Cyclooxygenase-1 gene knockout does not alter middle cerebral artery occlusion in a mouse stroke model.

Cyclooxygenase (COX) is crucial in inflammation; COX-1 is constitutional, and COX-2 is inducible. In this study, neurological function and infarct volume were evaluated at 24 h after permanent endovascular middle cerebral artery occlusion (MCAO) in both COX-1-gene knockout (homozygous or heterozygous) and wide-type mice. Similar infarct volumes and neurological deficits were seen among mice of different genotypes. There was no difference among the groups in arterial blood pressure and regional cerebral blood flow during the first 30 min of ischemia. Our results failed to confirm the harmful effect of losing COX-1 activity due to gene knockout in a permanent endovascular MCAO mouse stroke model.

Animals↗

PubMed1999

Differential loss of spinal sensory but not motor neurons in the p75NTR knockout mouse.

Sensory neurons in the dorsal root ganglia (DRG) and motor neurons in the spinal cord express the 75 kDa low-affinity neurotrophin receptor (p75NTR) during prenatal development. The p75NTR gene knockout mouse provides a unique opportunity to assess the role of p75NTR during this period. Quantitative analysis of the p75NTR knockout mouse revealed a significant developmental loss of sensory neurons. In the cervico-thoracic ganglia approximately 75% of the neurons are lost, while in the lumbar ganglia the loss is approximately 50%. In contrast, motor neurons were not lost in either the cervical or lumbar spinal cord. These data suggest that p75NTR is essential for the prenatal survival of a significant number of sensory, but not motor neurons.

Animals↗