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PubMed1997

Tissue-specific alterations in insulin-like growth factor-I concentrations in response to 3,3',5-triiodo-L-thyronine supplementation in the growth hormone receptor-deficient sex-linked dwarf chicken.

Insulin-like growth factor-I (IGF-I) mediates many of the effects of growth hormone (GH). The regulation of IGF-I, independent of GH, is methodologically difficult to assess in vivo, as hypophysectomy results in derangement of many pituitary hormone axes in addition to GH, and a gene knockout model is not available. The recessive sex-linked dwarfing (SLD) gene (dw) in chickens results in a lack of functional target tissue GH receptors due to a variety of molecular defects, which provides a unique model for evaluating GH-independent regulation of IGF-I. In the present study, the impact of 3,3', 5-triiodo-l-thyronine (T3) on circulating and tissue IGF-I was determined in normal versus SLD birds. Adult, nonovulatory female normal and SLD chickens were restrict-fed 40 g of feed/kg bw/day containing 0, 0.5, or 1.0 ppm T3, resulting in supplementation levels of 0 (control), 20 (low dose), or 40 (high dose) microg T3/kg bw/day for 10 days. Samples of GH target tissues including liver, abdominal fat pad, skeletal muscle (pectoralis major), and spleen were extracted and assayed for IGF-I. Plasma T3, T4, GH, and IGF-I were determined by homologous RIA. Tissue GH binding was determined for hepatic membranes by radioreceptor assay. Under control conditions, dwarf chickens were markedly hypersomatotropic (33.3 +/- 4.1 ng GH/ml plasma; mean +/- SEM) compared to normals (2.4 +/- 3.9 ng/ml), and T3 supplementation reduced this to normal levels. Despite the high circulating level of GH in dwarfs, plasma IGF-I was low compared to normal controls (dwarfs 1.5 +/- .9 ng/ml; normals 5. 3 +/- .9 ng/ml; P = 0.004), but this difference was eliminated with low-dose T3. In this study, tissue IGF-I was undetectable in liver and pectoralis muscle in adults (55 weeks of age) of both genotypes, under all treatments. In contrast, adipose tissue IGF-I was relatively high and did not differ (P = 0.84) between genotypes under control conditions (normals 776.5 +/- 236.7; dwarfs 844.6 +/- 236.7 pg/mg protein), but was increased in normals and decreased in dwarfs, resulting in higher levels (P = 0.02) in the normal (1249.9 +/- 200.0 pg/mg protein) than in the dwarf genotype (558.4 +/- 200.0 pg/mg protein) at the higher level of T3 supplementation. This relationship was somewhat reversed in spleen, where T3 tended to decrease tissue IGF-I concentration in normals and increase it in dwarfs. The low level of plasma IGF-I despite nonmeasureable hepatic IGF-I tissue concentrations suggests that IGF-I synthesis by extrahepatic tissues contributes to the circulating pool of IGF-I. The relatively high control levels of adipose tissue IGF-I in the dwarf genotype further suggest that considerable IGF-I synthesis exists that is GH-independent in this extrahepatic tissue. The presence of GH action, however, may mediate the effects of other hormones that can influence local IGF-I production in this tissue, as reflected by the differential response to T3 supplementation between genotypes. The tissue-specific nature of the effect of T3 on IGF-I production supports an additional point of regulation of hormone action at the target tissue level.

Adipose Tissue↗

PubMed2002

Recent developments in leishmaniasis.

PURPOSE OF REVIEW: The leishmaniases, caused by protozoan parasites of the genus Leishmania, are a significant health problem in many regions of the world. This review highlights the recent advances in the study of leishmaniasis related to parasite biology, disease pathogenesis, clinical evaluation and treatment, and prevention. RECENT FINDINGS: Genetic heterogeneity and clonal diversity is common among Leishmania strains. Gene knockout, overexpression, and re-introduction studies have identified a number of genes that play a role in parasite virulence. Surprisingly, the importance of the surface lipophosphoglycan in parasite virulence appears to differ among Leishmania spp. Studies in experimental animal models have further defined the roles of CD4 and CD8 T cells, IL-4, IL-10, and IL-12 in the control, maintenance, or progression of disease. The effect of Leishmania on dendritic cells and macrophage effector function has also been an important area of investigation. A number of new vaccine candidates have been identified through experimental animal studies. Clinical studies of leishmaniasis have focused on the host determinants of disease (most notably HIV co-infection), serological and DNA-based diagnostic assays, and treatment. Antimony-resistant cases of cutaneous and visceral leishmaniasis have become more common; liposomal amphotericin and oral miltefosine are promising alternative therapies. SUMMARY: Significant advances have been made in the areas of pathogenesis, host defence, and treatment of leishmaniasis. A number of new vaccine candidates and potential targets of drug therapy have been identified, but progress from preclinical studies to clinical trials has been slow. Translational research, built upon the solid foundation of existing and ongoing basic investigation, is a high priority.

Animals↗

PubMed1998

Purification, gene cloning, targeted knockout, overexpression, and biochemical characterization of the major pyrazinamidase from Mycobacterium smegmatis.

The pyrazinamidase from Mycobacterium smegmatis was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of M. smegmatis. An open reading frame, designated pzaA, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the pzaA gene by homologous recombination yielded a mutant, pzaA::aph, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of M. smegmatis. Recombinant forms of the M. smegmatis PzaA and the Mycobacterium tuberculosis pyrazinamidase/nicotinamidase (PncA) were produced in Escherichia coli and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparable Km values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for M. tuberculosis was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of pzaA conferred PZA susceptibility on M. smegmatis by reducing the MIC of this drug to 150 micrograms/ml.

Amidohydrolases↗

PubMed1998

A disrupted cholecystokinin A receptor gene induces diabetes in obese rats synergistically with ODB1 gene.

Otsuka Long-Evans Tokushima fatty (OLETF) rats develop hyperglycemia, hyperinsulinemia, and mild obesity, which are characteristic of human non-insulin-dependent diabetes mellitus. We have shown that two recessive genes, ODB1 mapped on the X chromosome and ODB2 mapped on chromosome 14, are involved in the induction of the diabetes in OLETF rats. Recently we found that OLETF rats are the naturally occurring cholecystokinin type A receptor (CCKAR) gene knockout rats. In this study, we focused on the genotype of CCKAR gene and the ODB1 gene in regulation of glucose homeostasis in the F2 cross of the OLETF rats. Relatively high plasma glucose levels were observed in the F2 offspring with the homozygously disrupted CCKAR gene. A synergistic effect for increasing plasma glucose levels in F2 rats between disrupted CCKAR gene and the ODB1 gene was shown. The CCKAR gene was found to map very close to ODB2 by a linkage analysis using microsatellite markers. These results suggest that CCKAR gene maintains normoglycemia in rats.

Animals↗

PubMed2006

Pharmacogenetics: defining the genetic basis of drug action and inositol trisphosphate analysis.

Medicinal drugs do not always have clearly understood mechanisms of action, especially as regards psychiatric treatment. Identification of genes involved in drug resistance is an important step toward elucidating the genetic basis of disease and the molecular mechanism of drug action. However, this approach is impractical in higher animals, as ablation and screening of every gene in an animal is not currently possible. Dictyostelium has proven a good model system for molecular pharmacological research as a result of its genetic tractability, ease of gene knockout, and creation of isogenic lines. In this system, we have identified genes that confer resistance to bipolar disorder drugs. This work has implicated inositol (1,4,5) trisphosphate (InsP3) signaling as a common mechanism of action for these drugs in patients.

Animals↗

PubMed2026

Big data analytics for CLEC5A dynamics based on single cell genomics and proteomics reveal its diverse functions in human diseases.

BACKGROUND: CLEC5A (C-type lectin domain family 5 member A) is an innate immune receptor implicated in inflammatory signaling, contributing to hyperinflammatory responses in infections and sterile inflammation. However, CLEC5A dynamics in human diseases remain to be identified. Here, we systematically characterized CLEC5A dynamics in humans across cells, tissues, and disease states, and to explore the functional significance of CLEC5A in macrophage activation based on single-cell genomics. METHODS: With multi-omics (scRNA-seq, proteomics and big data analytics), we analyzed extensive human transcriptomic datasets (>42,000 samples) to profile CLEC5A expression by cell type, tissue, and disease. Single-nucleus RNA-seq (snRNA-seq) from pediatric congenital heart disease and a virtual CLEC5A gene knockout were also performed to characterize CLEC5A dynamics in humans. RESULTS: CLEC5A is highly enriched in innate immune cells, particularly in macrophages and neutrophils. Baseline CLEC5A in most tissues is low, but it is markedly upregulated in inflammatory and infectious diseases. CLEC5A expression has sex-specific differences in certain organs. Single-cell analysis showed that CLEC5A can be considered novel marker of proinflammatory macrophages with elevated cytokine production, antigen presentation, and impaired phagocytosis. Virtual CLEC5A knockout analysis identified coordinated perturbation of immune-regulatory pathways and overlapping genes linking CLEC5A to macrophage activation networks. CONCLUSION: CLEC5A is predominantly expressed in myeloid cells and acts as a key amplifier of inflammation in human diseases. Our findings highlight CLEC5A as a potential biomarker and therapeutic target in myeloid-driven hyperinflammatory conditions, warranting further experimental and translational validation.

Humans↗

PubMed2004

A role for nuclear factor I in the intrinsic control of cerebellar granule neuron gene expression.

Nervous system formation requires the elaboration of a complex series of differentiation events in both a spatially and maturation-regulated manner. A fundamental question is how neuronal subtype specification and developmental gene expression are controlled within maturing neurons. The alpha6 subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor (GABRA6) is preferentially expressed in cerebellar granule neurons and is part of an intrinsic program directing their differentiation. We have employed a lentiviral approach to examine the transcriptional mechanisms controlling neuronal subtype-selective expression of this gene. These studies demonstrated that nuclear factor I (NFI) proteins are required for both transgenic GABRA6 promoter activity as well as endogenous expression of this gene in cerebellar granule neurons. Chromatin immunoprecipitation also showed that NFI proteins are bound to the GABRA6 promoter in these cells in vivo. Furthermore, analyses of gene knockout mice revealed that Nfia is specifically required for normal expression of the GABRA6 gene in cerebellar granule neurons. NFI expression and DNA binding activity are highly enriched in granule neurons, implicating this transcription factor family in the neuronal subtype-selective expression of the GABRA6 gene. These studies define a new role for NFI proteins as neuronal subtype-enriched transcriptional regulators that participate in an intrinsic transcriptional program directing the differentiation of cerebellar granule neurons.

Animals↗

PubMed1995

Cytochrome P450 1A1 promoter as a genetic switch for the regulatable and physiological expression of a plasma protein in transgenic mice.

Transgenic and gene knockout techniques allow for in vivo study of the consequences of adding or subtracting specific genes. However, in some instances, such as the study of lethal mutations or of the physiological consequences of changing gene expression, turning on and off an introduced gene at will would be advantageous. We have used cytochrome p450 1A1 promoter to drive expression of the human apolipoprotein E (apoE) gene in transgenic mice. In six independent lines, robust expression of the transgene depended upon injection of the inducer beta-naphthoflavone, whereas the seventh line had high basal expression that was augmented further by the inducer. The low level of basal expression in an inducer-dependent line was confirmed upon breeding the transgene onto the hypercholesterolemic apoE-deficient background. In the basal state transgene expression was physiologically insignificant, as these mice were as hypercholesterolemic as their nontransgenic apoE-deficient littermates. When injected with the inducer, plasma cholesterol levels of the transgenic mice decreased dramatically as apoE expression was induced to yield greater than physiological levels in plasma. The inducer could pass transplacentally from an injected mother to her fetuses with concomitant induction of fetal transgene mRNA. Inducer could also pass via breast milk from an injected mother to her suckling neonatal pups, giving rise to the induction of human apoE in neonate plasma. These finding suggest a strategy to temporarily ameliorate genetic deficiencies that would otherwise lead to fetal or neonatal lethality.

Animals↗

PubMed1995

Genetic approaches to the study of cytokine regulation of mucosal immunity.

Efforts to design effective mucosal vaccines have been hampered by an incomplete understanding of factors controlling the development of mucosal immunity. It is now clear, however, that T cell-derived cytokines play a major role. Recent developments in 'gene knockout' technology have allowed the generation of strains of mice in which particular genes have been inactivated. The availability of mice rendered deficient for production of Th2 cytokines has facilitated studies of the induction and development of mucosal immune responses in the absence of these factors. We have used several genetic approaches, including cytokine-deficient mice and recombinant vectors constructed to express genes for a range of different cytokines, to demonstrate the importance of these factors in the mucosa. Such genetic approaches appear to represent powerful tools for in vivo studies of the influence of cytokines in mucosal immunoregulation.

Animals↗

PubMed1999

Insight into prostaglandin, leukotriene, and other eicosanoid functions using mice with targeted gene disruptions.

In recent years, there has been an exponential increase in the number of targeted gene disruptions performed in mice. At least 18 different gene knockouts have now been reported that have direct relevance to eicosanoid biology. These include genes that influence substrate availability (phospholipases), metabolism to eicosanoids (e.g., prostaglandin H synthases, lipoxygenases), and eicosanoid action (e.g., receptors for various prostaglandins). This minireview will outline the phenotype of these knockout mice and what has been learned about eicosanoid functions through use of this novel methodology.

Animals↗

PubMed1999

Inducible nitric oxide synthase (iNOS) gene deficiency increases the mortality of sepsis in mice.

BACKGROUND: Nitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS or NOS2) has been implicated in the hypotension, organ failure, and death that complicate sepsis. To avoid the confounding effects and limitations of iNOS inhibitors, we used iNOS gene "knockout" mice to examine the effect of inducible NO production in a model of polymicrobial abdominal sepsis treated with antibiotics. We hypothesized that iNOS gene deficiency would significantly alter outcome. METHODS: C57BL6 wild-type (control) and congenic iNOS knockout mice were studied concurrently. Under halothane anesthesia, the ceca were ligated with 4-0 silk suture and punctured twice with a 26-gauge needle (cecal ligation and puncture, CLP). Survival was followed for 7 days, after which necropsies were performed in surviving animals. In an accompanying study examining the acute effects of sepsis, organ injury at 18 hours after CLP as determined by histology and the degree of cell death by apoptosis were examined with the use of hematoxylin and eosin (H&E) and TUNEL staining and two-channel fluorescence-activated cell sorter (FACS) analysis. RESULTS: Sham laparotomy produced no lethality in either knockout (n = 3) or wild-type (n = 3) animals. Compared with survival in controls (n = 20), survival after CLP in iNOS knockout mice (n = 21) was significantly decreased (P < .01 at 2 days, P = .080 at 7 days, Mantel-Haenszel log-rank test). CLP-induced apoptotic cell death was significantly less in the thymus of iNOS knockout mice compared with wild-type mice. CONCLUSIONS: We conclude that iNOS gene function provides a survival benefit in septic mice and is associated with increased sepsis-induced thymocyte apoptosis. To our knowledge, this is the first survival study examining the effect of iNOS gene deficiency in a clinically relevant model of sepsis.

Animals↗

PubMedDate not supplied

Use of gene-manipulated models to study the physiology of lipid transport.

1. In vivo and in vitro gene-manipulated models were used to study the metabolism of chylomicron remnants. Transgenic mice expressing human apolipoprotein (Apo) A1 or E4, gene knockout mice deficient in ApoE or low density lipoprotein (LDL) receptors and antisense gene inhibition in HepG2 cells were used to evaluate the effect of gene manipulations on the metabolism of chylomicron remnants. 2. Mice transgenic for human ApoE4 showed accelerated clearance of chylomicron-like emulsions when animals were fed a low-fat diet. When challenged by a high-fat diet, remnant clearance in ApoE4 transgenic mice was delayed, as in normal or non-transgenic controls. However, unlike normal nontransgenic controls, in ApoE4 transgenic mice high density lipoprotein (HDL)-cholesterol levels remained high after high-fat feeding, which probably protected the animals from the development of atherosclerosis. In contrast, clearance of chylomicron-like lipid emulsions was not affected by the over-expression of human ApoAI in transgenic mice. 3. Gene knock-out mice deficient in ApoE or deficient in the LDL receptor were used to show that ApoE and LDL receptors are both essential for the normal, fast catabolism of chylomicron remnants by the liver. In the absence of the LDL receptor, an alternative ApoE-dependent pathway operates to clear chylomicrons from the plasma, with significantly delayed catabolism. 4. Antisense gene inhibition techniques were used to suppress the expression of syndecan, a core protein of heparan sulfate proteoglycan, in HepG2 cells. Remnant uptake in cells transfected with the antisense oligodeoxynucleotide complementary to a 20 nucleotide sequence upstream of the initiation site of syndecan cDNA markedly reduced the uptake of chylomicron remnant.

Animals↗

PubMed2002

Progress towards cell-mediated gene transfer in zebrafish.

Although the zebrafish possesses several favourable characteristics that make it an ideal model for genetic studies of vertebrate development, one disadvantage of this model system is the absence of methods for the production of gene knockouts. The authors' laboratory, and others, are working to develop zebrafish pluripotent embryonic stem (ES) and primordial germ cell (PGC) cultures that can be used for cell-mediated gene transfer and the production of knockout mutant lines of fish. Progress has been made in developing short-term cell cultures that possess the ability to contribute to multiple tissues, including the germ line of a host embryo, and transgenic lines of zebrafish have been established using the embryo cell cultures. Work is in progress to extend the length of time that the embryo cells can be maintained in culture without losing their ability to generate germ-line chimeras.

Animals↗

PubMed2001

The pharmacology of mu analgesics: from patients to genes.

Morphine and most clinical opioids act through mu opioid receptors. Yet, their pharmacological profiles differ. The presence of incomplete cross-tolerance among these drugs clinically was one of the first indications that these mu opioids differed in their receptor mechanisms of action. This was followed by similar studies in preclinical models, which also found genetic differences in sensitivity toward morphine and other mu opioids. This concept of mu receptor multiplicity is now supported by antisense and gene knockout models. Although all the mu opioids are sensitive to antisense probes against the mu opioid receptor gene MOR-1, the sensitivity profiles of the drugs to the antisense probes differ based on the exon being targeted. Knockout mice also reveal striking differences. In one knockout mouse, morphine analgesia is completely lost while the potent mu drugs morphine-6beta-glucuronide and heroin both retain analgesic activity. Finally, cloning studies have identified at least seven different splice variants of the MOR-1 gene, with more likely. These studies illustrate the complexity of mu opioid pharmacology.

Analgesics, Opioid↗

PubMed2006

Susceptibility genes for schizophrenia: characterisation of mutant mouse models at the level of phenotypic behaviour.

A wealth of evidence indicates that schizophrenia is heritable. However, the genetic mechanisms involved are poorly understood. Furthermore, it may be that genes conferring susceptibility interact with one another and with non-genetic factors to modulate risk status and/or the expression of symptoms. Genome-wide scanning and the mapping of several regions linked with risk for schizophrenia have led to the identification of several putative susceptibility genes including neuregulin-1 (NRG1), dysbindin (DTNBP1), regulator of G-protein signalling 4 (RGS4), catechol-o-methyltransferase (COMT), proline dehydrogenase (PRODH) and disrupted-in-schizophrenia 1 (DISC1). Genetic animal models involving targeted mutation via gene knockout or transgenesis have the potential to inform on the role of a given susceptibility gene on the development and behaviour of the whole organism and on whether disruption of gene function is associated with schizophrenia-related structural and functional deficits. This review focuses on data regarding the behavioural phenotype of mice mutant for schizophrenia susceptibility genes identified by positional candidate analysis and the study of chromosomal abnormalities. We also consider methodological issues that are likely to influence phenotypic effects, as well as the limitations associated with existing molecular techniques.

Animals↗

PubMed2006

Differential proteome profiles in E2F2-deficient T lymphocytes.

E2F transcription factors are important regulators of proliferation, differentiation and apoptosis. We have previously shown that E2F2-/- mice develop late-onset autoimmune features, similar to systemic lupus erythematosus. E2F2-deficient T lymphocytes exhibit enhanced T cell receptor (TCR)-stimulated proliferation, which is presumably responsible for causing autoimmunity in E2F2-deficient mice. The comparison of E2F2-/- and wild-type T lymphocyte expression profiles by 2-DE followed by MS identification has revealed a set of deregulated proteins involved in TCR-mediated signaling, cell survival and stress responses. The deregulation of these proteins may account for the hyperproliferative phenotype that characterizes E2F2-/- T cells. Our work shows that proteomic analysis of gene-knockout strains can be a useful methodology to study the functional role of specific genes.

Amino Acid Sequence↗

PubMed2003

Odontoblast-specific expression of cre recombinase successfully deletes gene segments flanked by loxP sites in mouse teeth.

Embryonic or neonatal lethality of mice with targeted disruption of critical genes preclude them from further characterization of specific roles of these genes during postnatal development and aging. In order to study the molecular roles of such genes in teeth, we generated transgenic mouse lines expressing bacteriophage Cre recombinase under the control of the mouse dentin sialophosphoprotein (dspp) gene promoter. The expression of Cre recombinase protein was mainly detected in the nucleus of the odontoblasts. The efficiency of Cre activity was analyzed by crossing the Dspp-Cre mice with ROSA26 reporter (R26R) mice. The offspring with both genotypes have shown specific deletion of intervening sequences flanked by loxP sites upstream of the reporter gene, thereby facilitating the expression of the beta-galactosidase (beta-gal) gene in the teeth. The activity of beta-gal was initially observed in the odontoblasts of 1-day-old mice and increased with tooth development. Almost all of the odontoblasts have shown lacZ activity by 3 weeks of age. We could not detect Cre recombinase activity in any other cells, including ameloblasts. These studies indicate that the Dspp-Cre transgenic mice will be valuable to generate odontoblast-specific gene knockout mice so as to gain insight into the molecular roles of critical genes in the odontoblasts during dentinogenesis.

Animals↗

PubMed2025

Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).

Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.

Animals↗