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At least 757 records · Page 42Linked to original sources
PubMed1995

Xenobiotic receptor knockout mice.

Administration of certain foreign chemicals to animals elicits responses that are due to receptor-mediated activation of gene expression. Among the most well studied receptors are the Ah receptor (AHR) that binds 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds and the peroxisome proliferator-activated receptors, PPARs, that mediate gene activation by the diverse group of peroxisome proliferators. These receptors may also have critical roles in development or physiological homeostasis in addition to their abilities to allow animals to interact with exogenous chemicals or xenobiotics. To explore the function of AHR and PPAR alpha and to determine whether they participate in the adverse effects of dioxins and peroxisome proliferators, gene knockout mice were developed.

Animals↗
PubMed2006

Reconstructing gene regulatory networks: from random to scale-free connectivity.

The manipulation of organisms using combinations of gene knockout, RNAi and drug interaction experiments can be used to reveal regulatory interactions between genes. Several algorithms have been proposed that try to reconstruct the underlying regulatory networks from gene expression data sets arising from such experiments. Often these approaches assume that each gene has approximately the same number of interactions within the network, and the methods rely on prior knowledge, or the investigator's best guess, of the average network connectivity. Recent evidence points to scale-free properties in biological networks, however, where network connectivity follows a power-law distribution. For scale-free networks, the average number of regulatory interactions per gene does not satisfactorily characterise the network. With this in mind, a new reverse engineering approach is introduced that does not require prior knowledge of network connectivity and its performance is compared with other published algorithms using simulated gene expression data with biologically relevant network structures. Because this new approach does not make any assumptions about the distribution of network connections, it is suitable for application to scale-free networks.

Cell Physiological Phenomena↗
PubMed2004

Crystallization of YloQ, a GTPase of unknown function essential for Bacillus subtilis viability.

YloQ is a putative ATP/GTP-binding protein of unknown function identified from the complete sequence of the Bacillus subtilis genome. A gene-knockout programme established that yloQ is one of a set of some 270 indispensable genes for the viability of this organism. Crystals of YloQ have been grown from HEPES-buffered solutions at pH 7.5 containing polyethylene glycol and diffraction data have been collected extending to 2.5 A spacing.

Adenosine Triphosphate↗
PubMed2005

The contribution of epidermal stem cells to skin cancer.

Tumors arising from the skin are of multiple phenotypes, with differing degrees of malignant potential. In mouse models of skin carcinogenesis, tumors of squamous phenotype are the most common; however, human disease indicates that multiple phenotypes may arise from a common pool of stem cells that are then influenced by epigenetic factors. The use of transgenic and knockout gene technologies with mice is unraveling some of the specific genes regulating fate determination in stem cells other than squamous lineage, including basal cell carcinoma and sebaceous adenomas. The following review examines the evidence for the stem cell origin of epidermal tumors and the contribution of some specific gene families toward stem cell fate decisions during epidermal tumor progression.

Adenocarcinoma, Sebaceous↗
PubMed2001

Identification of genes that mediate sensitivity to cisplatin.

Cisplatin (cDDP) is effective against some human tumors, but many are intrinsically resistant and, even among initially sensitive tumors, acquired resistance develops commonly during treatment. It has not been possible to prove which biochemical mechanisms control sensitivity to cDDP. Gene knockout studies in yeast, Dictyostelium discoideum, and mammalian cells have begun to unambiguously identify genes whose products function to modulate the cytotoxicity of cDDP. This review summarizes information currently available about the function of these genes. This comprehensive compilation points to the involvement of regulatory pathways known to mediate apoptosis, cell cycle checkpoint activation, and transcriptional rescue as regulators of cDDP sensitivity. Elucidation of the molecular mechanisms that mediate cDDP resistance holds promise for the design of pharmacological strategies for preventing, overcoming, or reversing this form of drug resistance.

Antineoplastic Agents↗
PubMed2005

Factors involved in the migration of neuroendocrine hypothalamic neurons.

Neuroendocrine control of physiological functions needs a complex developmental organisation of the hypothalamic parvicellular neurons, which synthesise and release hypophysiotropic hormones. Among the hypothalamic neuroendocrine cells, Gonadotropin-releasing hormone (GnRH) neurons represent a unique class; they are generated in the olfactory placode and, during embryonic life, migrate to the septo/hypothalamic region along terminal and vomeronasal nerves. At this level GnRH neurons undergo terminal differentiation and start to release GnRH to modulate the secretion of pituitary gonadotropins. All these steps are under the strict control of several developmental cues and their defect might represent a cause of clinical disorders. A number of factors have been proposed to be involved in the migration of GnRH neurons, but their role is still unclear. By using gene knockout techniques it has been found that mice carrying a targeted deletion of Ebf2 gene, a component of Olf/Ebf bHLH transcription factors, show a defective migration of GnRH neurons, providing the first evidence of a mouse model of such defect. Since the investigation of GnRH neurons is hindered by their peculiar anatomical distribution, other studies has been forwarded by the availability of immortalized GnRH-expressing neurons (GN11 cells) that retain a strong chemomigratory response "in vitro". Among the factors analysed, we found that hepatocyte growth factor/scatter factor (HGF/SF) and vascular endothelial growth factor (VEGF) induce specific chemotaxis of GN 11 neurons, suggesting that migratory signals can arise from nasal mesenchyme and from the concomitant vasculogenesis. Finally, anosmin-1 (the product of the gene responsible of the X-linked form of Kallmann's disease) was found to induce a significant chemotactic response of GN11 cells, confirming a permissive/instructive role of KAL1 gene product in the migratory behaviour of GnRH neurons. In conclusion, the migration of the GnRH neurons appears to be a complex process, which involves the interplay of multiple molecular cues. These studies may provide new insights on the etiopathogenesis of the large proportion of reproductive dysfunctions that affect humans and could provide novel insights on common biochemical events controlling neuronal development and migration.

Animals↗
PubMed2001

The pharmacological phenotype of combined multidrug-resistance mdr1a/1b- and mrp1-deficient mice.

Two major classes of plasma membrane proteins that actively extrude a wide range of structurally diverse hydrophobic amphipathic antineoplastic agents from cells, with different mechanisms of action, lead to multidrug resistance. To study the importance of these ATP-binding cassette transporters to the toxicity of cancer chemotherapy agents, we have used mice genetically deficient in both the mdr1a and mdr1b genes [mdr1a/1b(-/-) mice], the mrp1 gene [mrp1(-/-) mice], and the combined genes mdr1a/1b and mrp1 [mdr1a/1b(-/-), mrp1(-/-) mice] and embryonic fibroblasts derived from wild-type mice and from the three gene knockout animals. The consequences of export pump deficiencies were evaluated primarily using vincristine and etoposide. Mice deficient in the three genes, mdr1a/1b and mrp1, exhibited a 128-fold increase in toxicity to vincristine and a 3-5-fold increase in toxicity to etoposide; increased toxicity to embryonic fibroblast cells from triple knockout mice also occurred with vincristine and etoposide. Vincristine, which normally does not express toxicity to the bone marrow and to the gastrointestinal mucosa when used at therapeutic doses, caused extensive damage to these tissues in mdr1a/1b(-/-), mrp1(-/-) mice. The findings indicate that the P-glycoprotein and mrpl are compensatory transporters for vincristine and etoposide in the bone marrow and the gastrointestinal mucosa and emphasize the potential for increased toxicities by the combined inhibition of these efflux pumps.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Transcription factors and apoptosis in kidney development.

The study of kidney development at the cellular and molecular levels remains an active area of nephrologic research. This review highlights recent advances in two specific areas: transcriptional control of nephrogenesis and the role of cell death in normal kidney development. The mesenchymal-epithelial conversion that occurs during kidney development requires alterations in gene expression. Some transcription factors involved in early steps in nephrogenesis have recently been identified from the effects of gene "knockout" or dysregulated expression in transgenic mice. These include the product of the Wilms' tumor suppressor gene (WT-1), a mammalian paired homologue (Pax-2), and the N-myc oncoprotein. Other proteins, including Pax-8, hepatocyte nuclear factor-1, hepatocyte nuclear factor-4, Kid-1, and formins, exhibit spatiotemporally restricted patterns of expression, homology to products of developmental control genes in Drosophila, or mutant phenotypes consistent with possible roles in nephrogenesis. During the past year, another important observation was that apoptosis (programmed cell death) occurs during normal kidney development. Studies of knockout mice suggest that Bcl-2, which protects against death in many contexts, is also involved in kidney development.

Animals↗
PubMed2026

A User-Friendly Protocol for Microinjection into Teleost Embryos to Study Gene Function.

Zebrafish (Danio rerio) and medaka (Oryzias latipes) are popular teleost models used in developmental biology and functional genomics. To achieve high-quality and reproducible microinjections, it is essential to have robust protocols for breeding, egg collection, and the precise delivery of genetic material. In this protocol, we present a comprehensive and optimized methodology for setting up breeding tanks under controlled photoperiod conditions to maximize egg yield while minimizing contamination. We provide detailed procedures for sex identification, pair selection, the use of grated breeding inserts, and methods to increase egg collection efficiency. We outline procedures for making injection gel beds, pulling needles, and calibration using one-microliter microcapillaries to achieve consistent nanoliter-scale injections. Our protocol outlines settings for the pico-liter injector that are optimized to deliver a precise amount per pulse with minimal variability. Finally, we demonstrate the application of these methods for gene knockdown using morpholino antisense oligonucleotides, gene knockout using CRISPR-Cas9, and gain-of-function mRNA overexpression experiments. Phenotypic assessments conducted at various developmental stages to evaluate gene-specific effects reveal consistent phenotypic outcomes between the morpholino and CRISPR-Cas9 approaches. This easy and comprehensive protocol enables efficient, precise, and scalable genetic manipulation of zebrafish and medaka embryos, thereby supporting advanced functional studies in developmental biology and disease modeling. To our knowledge, this is the first unified protocol for both zebrafish and medaka microinjection systems achieving 97.7% phenotype penetrance in CRISPR-Cas9 knockouts with precision together with a triple validation approach that confirms gene function across multiple techniques.

Animals↗
PubMed1996

The unique role of interferon-gamma in the regulation of MHC expression on arterial endothelium.

We examined the expression of MHC class I and II in the arterial endothelium of interferon-gamma (IFN-gamma, GKO) and IFN-gamma-R (IFN-gamma-R, GRKO) gene knockout mice in comparison with mice with intact IFN-gamma and IFN-gamma-R genes, BALB/c and 129Sv/J wild-type, respectively. The GKO and GRKO were produced by gene targeting. MHC class I and II expression was assessed by mAb binding to frozen tissue (kidney, spleen, heart, liver) sections by immunoperoxidase staining in the basal state and after various stimuli: allogeneic cells, oxazolone skin sensitization, LPS, and rIFN-gamma. As controls, we also examined the expression of two other IFN-gamma inducible genes present in the endothelium, Ly-6 and ICAM-1. We found that basal class I expression was present in the small arteries and arterioles of BALB/c and 129Sv/J wild-type mice but absent from arterial endothelium of GKO and GRKO mice. Class I was induced in the endothelium of BALB/c and 129Sv/J wild-type mice by three in vivo stimuli: allogeneic, LPS, and oxazolone, whereas class II was only induced after allogeneic stimulus. Administration of rIFN-gamma induced class I in the endothelium of GKO and BALB/c wild-type mice. The basal expression of Ly-6 and ICAM-1 was similar in the arteries of GKO and BALB/c wild-type mice, indicating that, the basal expression of these proteins in endothelium is IFN-gamma independent, unlike class I. In summary, basal class I expression in arterial endothelium is not constitutive as previously believed, but is dependent on basal IFN-gamma production. IFN-gamma has an essential role in the induction of class I and II expression in arterial endothelium. The fact that MHC class I is induced in endothelium may be useful therapeutically for reduction of immune recognition in transplantation.

Animals↗
PubMed1999

CD8+ CTLs are essential for protective immunity against Encephalitozoon cuniculi infection.

Encephalitozoon cuniculi is a protozoan parasite that has been implicated recently as a cause of opportunistic infection in immunocompromised individuals. Protective immunity in the normal host is T cell-dependent. In the present study, the role of individual T cell subtypes in immunity against this parasite has been studied using gene knockout mice. Whereas CD4-/- animals resolved the infection, mice lacking CD8+ T cells or perforin gene succumbed to parasite challenge. The data obtained in these studies suggest that E. cuniculi infection induces a strong and early CD8+ T response that is important for host protection. The CD8+ T cell-mediated protection depends upon the CTL activity of this cell subset, as the host is rendered susceptible to infection in the absence of this function. This is the first report in which a strong dependence upon the cytolytic activity of host CD8+ T cells has been shown to be important in a parasite infection.

Animals↗
PubMed2001

Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.

Mouse genetic manipulation has provided an excellent system to characterize gene function in numerous contexts. A number of mutants have been produced by using transgenic, gene knockout, and mutagenesis techniques. Nevertheless, one limitation is that it is difficult to express a gene in vivo in a restricted manner (i.e., spatially and temporally), because the number of available enhancers and promoters which can confine gene expression is limited. We have developed a novel method to introduce DNA into in/exo utero embryonic mouse brains at various stages by using electroporation. More than 90% of operated embryos survived, and more than 65% of these expressed the introduced genes in restricted regions of the brain. Expression was maintained even after birth, 6 weeks after electroporation. The use of fluorescent protein genes clearly visualized neuronal morphologies in the brain. Moreover, it was possible to transfect three different DNA vectors into the same cells. Thus, this method will be a powerful tool to characterize gene function in various settings due to its high efficiency and localized gene expression.

Animals↗
PubMed1995

Recent advances in the Laboratory of Molecular and Cellular Cardiology.

This article highlights some of the research in cardiac molecular biology in progress in the Department of Cardiology at Children's Hospital. It provides a sampling of investigative approaches to key questions in cardiovascular development and function and, as such, is intended as an overview rather than a comprehensive treatment of these problems. The featured projects, encompassing four different "model" systems, include (1) genetic analysis of the mef2 gene required for fruit fly cardial cell differentiation, (2) cardiac-specific homeobox factors in zebrafish cardiovascular development, (3) mouse transgenic and gene knockout models of cardiac potassium ion channel function, and (4) mapping and identification of human gene mutations causing long QT syndrome.

Animals↗
PubMed2004

Gene expression in Plasmodium: from gametocytes to sporozoites.

Completion of the complex developmental program of Plasmodium in the mosquito is essential for parasite transmission, yet this part of its life cycle is still poorly understood. In recent years, considerable progress has been made in the identification and characterization of genes expressed during parasite development in the mosquito. This line of investigation was greatly facilitated by the availability of the genome sequence of several Plasmodium, and by the application of approaches such as proteomics, microarrays, gene disruption by homologous recombination (gene knockout) and by use of subtraction libraries. Here, we review what is presently known about genes expressed in gametocytes and during the Plasmodium life cycle in the mosquito.

Animals↗
PubMed2005

The crystal structure of TrpD, a metabolic enzyme essential for lung colonization by Mycobacterium tuberculosis, in complex with its substrate phosphoribosylpyrophosphate.

Mycobacterium tuberculosis, the cause of tuberculosis, presents a major threat to human health worldwide. Biosynthetic enzymes that are essential for the survival of the bacterium, especially in activated macrophages, are important potential drug targets. Although the tryptophan biosynthesis pathway is thought to be non-essential for many pathogens, this appears not to be the case for M.tuberculosis, where a trpD gene knockout fails to cause disease in mice. We therefore chose the product of the trpD gene, anthranilate phosphoribosyltransferase, which catalyses the second step in tryptophan biosynthesis, for structural analysis. The structure of TrpD from M.tuberculosis was solved by X-ray crystallography, at 1.9 A resolution for the native enzyme (R = 0.191, Rfree = 0.230) and at 2.3 A resolution for the complex with its substrate phosphoribosylpyrophosphate (PRPP) and Mg2+ (R = 0.194, Rfree = 0.255). The enzyme is folded into two domains, separated by a hinge region. PRPP binds in the C-terminal domain, together with a pair of Mg ions. In the substrate complex, two flexible loops change conformation compared with the apo protein, to close over the PRPP and to complete an extensive network of hydrogen-bonded interactions. A nearby pocket, adjacent to the hinge region, is postulated by in silico docking as the binding site for anthranilate. A bound molecule of benzamidine, which was essential for crystallization and is also found in the hinge region, appears to reduce flexibility between the two domains.

Amino Acid Sequence↗
PubMed2003

Characterization of the mupirocin biosynthesis gene cluster from Pseudomonas fluorescens NCIMB 10586.

The polyketide antibiotic mupirocin (pseudomonic acid) produced by Pseudomonas fluorescens NCIMB 10586 competitively inhibits bacterial isoleucyl-tRNA synthase and is useful in controlling Staphylococcus aureus, particularly methicillin-resistant Staphylococcus aureus. The 74 kb mupirocin biosynthesis cluster has been sequenced, and putative enzymatic functions of many of the open reading frames (ORFs) have been identified. The mupirocin cluster is a combination of six larger ORFs (mmpA-F), containing several domains resembling the multifunctional proteins of polyketide synthase and fatty acid synthase type I systems, and individual genes (mupA-X and macpA-E), some of which show similarity to type II systems (mupB, mupD, mupG, and mupS). Gene knockout experiments demonstrated the importance of regions in mupirocin production, and complementation of the disrupted gene confirmed that the phenotypes were not due to polar effects. A model for mupirocin biosynthesis is presented based on the sequence and biochemical evidence.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1998

Advances and challenges in the prevention and treatment of Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia and accounts for one-half to three-fourths of all cases of dementia. In the United States, AD is the leading cause of a loss of independent living and subsequent institutionalization. Approximately 4 million Americans are currently diagnosed with Alzheimer's disease-which results in greater than $100 billion dollars in health care costs. This review provides a description of the cognitive and neuropathological features of AD and the challenge that aging populations around the globe pose to health care systems and to societies. A review of new and promising therapeutic strategies for the prevention of AD is discussed which includes estrogen replacement therapy and anti-inflammatory therapeutics. Pharmaceutical approaches that delay the progression of the disease, such as antioxidants, are discussed as well as therapeutic strategies for improvement of cognitive function in AD patients, including the new generation of compounds aimed at enhancing cholinergic function. This section is followed by a review of the current status on nerve growth factor trials. The final section addresses the issue of the genetic linkages of AD, the impact of transgenic and gene knockout mouse models of AD on research in the field and the potential use of gene therapy to treat AD.

Alzheimer Disease↗
PubMed2003

Gene loss, protein sequence divergence, gene dispensability, expression level, and interactivity are correlated in eukaryotic evolution.

Lineage-specific gene loss, to a large extent, accounts for the differences in gene repertoires between genomes, particularly among eukaryotes. We derived a parsimonious scenario of gene losses for eukaryotic orthologous groups (KOGs) from seven complete eukaryotic genomes. The scenario involves substantial gene loss in fungi, nematodes, and insects. Based on this evolutionary scenario and estimates of the divergence times between major eukaryotic phyla, we introduce a numerical measure, the propensity for gene loss (PGL). We explore the connection among the propensity of a gene to be lost in evolution (PGL value), protein sequence divergence, the effect of gene knockout on fitness, the number of protein-protein interactions, and expression level for the genes in KOGs. Significant correlations between PGL and each of these variables were detected. Genes that have a lower propensity to be lost in eukaryotic evolution accumulate fewer substitutions in their protein sequences and tend to be essential for the organism viability, tend to be highly expressed, and have many interaction partners. The dependence between PGL and gene dispensability and interactivity is much stronger than that for sequence evolution rate. Thus, propensity of a gene to be lost during evolution seems to be a direct reflection of its biological importance.

Amino Acid Substitution↗