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PubMed2001

Genetics and systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex, multifactorial autoimmune disease. Genetic factors are believed to contribute to its pathogenesis. There have been numerous recent advances in the study of murine and human lupus genetics. In well-defined, experimental, transgenic or gene-knockout mouse models, the development of lupus-like disease has implicated specific genes and pathways in the disease pathogenesis. Linkage analyses have mapped multiple susceptibility loci and disease suppressive loci using inbred strains of mice that spontaneously develop lupus-like disease. Elegant genetic dissection has demonstrated that a component phenotype of SLE is displayed by each congenic strain carrying a single susceptibility locus on a resistant genetic background, whereas polycongenic strains exhibit fatal lupus nephritis. These studies suggest that genes in separate pathways can interact to augment or suppress the initiation and progression of systemic autoimmunity. In association studies of human lupus, the contributions of the MHC loci, Fcg receptors, various cytokines, components of the complement cascade, and proteins involved in apoptosis have been explored. Most recently, linkage analyses have been performed and provide many chromosomal regions for further exploration for susceptibility genes. Studies to identify the genes in the susceptibility regions are underway. An understanding of the genes involved in the development of lupus should provide targets for more focused therapy in lupus.

Animals↗
PubMed2002

An update on genetic studies of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex, multifactorial autoimmune disease. Genetic factors are thought to contribute to its pathogenesis. There have been numerous recent advances in the study of murine and human lupus genetics. In well-defined experimental transgenic or gene-knockout mouse models, the development of lupus-like disease has implicated specific genes and pathways in the disease pathogenesis. Linkage analyses have mapped multiple susceptibility loci and disease suppressive loci using inbred strains of mice that spontaneously develop lupus-like disease. Elegant genetic dissection and function studies have led to the recent identification of two murine candidate susceptibility genes, Ifi202 (encoding an interferon-inducible protein) and Cr2 (encoding complement receptors 1 and 2). In human lupus, case- control studies have established associations of SLE with certain major histocompatibility class II alleles, complement deficiencies, and polymorphisms of Fc gamma receptor genes, a complement-related gene, and cytokine genes. During the past several years, linkage analyses using SLE multiplex families have provided many chromosomal regions for further exploration of susceptibility genes. Six regions exhibiting significant linkage to SLE are promising. Studies are underway to fine map these linked regions and to identify the genes in the susceptibility regions. An understanding of the genes involved in the development of lupus should provide targets for more focused therapy in lupus.

Alleles↗
PubMed1995

Immunogenetics in the analysis of resistance to intracellular pathogens.

Recent studies have identified genes involved in resistance to intracellular pathogens. Such genes include the murine MHC class I gene, Ld (toxoplasmosis), HLA-BW53, HLA DRB1* 1302-DQ B10s01 and TNF2 (malaria), murine Nramp (toxoplasmosis, leishmaniasis and tuberculosis), gene(s) modulating the T-helper type 1 and type 2 dichotomy (leishmaniasis, leprosy and HIV infection) and the natural killer cell complex (cytomegalovirus infection). There also have been other advances in immunogenetics that have led to a better understanding of resistance to intracellular pathogens. These include effector mechanisms of immune response genes and factors modulating genetic susceptibility. Identification of genes that determine resistance/susceptibility (and their effector mechanisms) has impacted on vaccine development. Immunogenetics has been important in characterizing roles of TCR genes, superantigens, and host genes that play a role in molecular mimicry in disease pathogenesis. In addition, recent work with gene knockout, recombinant inbred or congenic, mutant, consomic, and transgenic mice, positional cloning, mouse/human gene homologies to identify candidate human resistance genes, and the rapid expansion of the gene transcription maps of the human genome, have been important in analysis of resistance to intracellular pathogens.

Animals↗
PubMed2002

A pox on thee! Manipulation of the host immune system by myxoma virus and implications for viral-host co-adaptation.

The poxviruses have evolved a diverse array of proteins which serve to subvert innate and adaptive host responses that abort or at least limit viral infections. Myxoma virus and its rabbit host are considered to represent an ideal poxvirus-host system in which to study the effects of these immunomodulatory proteins. Studies of laboratory rabbits (Oryctolagus cuniculus) infected with gene knockout variants of myxoma virus have provided compelling evidence that several myxoma virus gene products contribute to the pathogenic condition known as myxomatosis. However, myxomatosis, which is characterized by skin lesions, systemic immunosuppression, and a high mortality rate, does not occur in the virus' natural South American host, Sylvilogus brasiliensis. Moreover, in Australia where myxoma virus was willfully introduced to control populations of O. cuniculus, myxomatosis-resistant rabbits emerged within a year of myxoma virus introduction into the field. In this review I discuss the characterized immunomodulatory proteins of myxoma virus, their biochemical properties, their pathogenic effects in laboratory rabbits, the role of the host immune system in the susceptibility or resistance to myxomatosis, and the evidence that immunomodulatory genes may have been attenuated during the co-adaptation of myxoma virus and O. cuniculus in Australia.

Adaptation, Physiological↗
PubMed2000

Dual regulation of proliferation and apoptosis: c-myc in bitransgenic murine mammary tumor models.

Recent progress in the study of c-Myc has convincingly demonstrated that it possesses a dual role in regulating both proliferation and apoptosis; however, the manner in which c-Myc influences these cellular response pathways remains incompletely characterized. Deregulation of c-Myc expression, via many mechanisms, is a common feature of multiple cancers and is an especially prominent feature of many breast cancers. Of significant interest to those who study mammary gland development and neoplasia is the unresolved nature and contribution of apoptosis to breast tumorigenesis. Recently, the use of transgenic mice and gene-knockout mice has allowed investigators to evaluate the pathological mechanisms by which different genes influence tumor development and progression. In this review, we address two distinct c-myc-containing bitransgenic murine mammary tumor models and discuss the contribution and possible future directions for resolution of cancer-relevant molecular pathways influenced by c-Myc.

Animals↗
PubMed2004

Structure and regulated expression of mammalian RUNX genes.

The RUNX are key regulators of lineage-specific gene expression in major developmental pathways. The expression of RUNX genes is tightly regulated, leading to a highly specific spatio/temporal expression pattern and to distinct phenotypes of gene knockouts. This review highlights the extensive structural similarities between the three mammalian RUNX genes and delineates how regulation of their expression at the levels of transcription and translation are orchestrated into the unique RUNX expression pattern.

Animals↗
PubMed2026

Simplifying multiplex genome engineering in Saccharomyces cerevisiae with intron-mediated Random Assembly and INtegration (RAIN).

Engineering of multistep enzymatic pathways often involves extensive optimization of heterologous gene expression levels and requires cloning of promoter and open reading frames (ORFs) to generate expression cassettes. We present work on a nascent method for multiplex genome engineering in Saccharomyces cerevisiae that negates the requirement for cloning of expression cassettes. Our system, Random Assembly and INtegration (RAIN), uses intron-mediated homologous recombination (HR) for random in vivo assembly of exogenous promoter and ORF libraries, which are combined and cotransformed in a one-pot method. The libraries include consensus homology arms which target long terminal repeat regions of the Ty1 retrotransposon, providing over a hundred possible integration loci. In this way, our developmental system aims to negate the need for in vitro combinatorial cloning of promoters and ORFs to generate expression cassettes, simplifying in vitro DNA preparation before multiplex genome engineering. This paper presents findings from a series of experiments to demonstrate a proof of concept for the RAIN system. These include: the first reported use of intron-mediated assembly of promoters and ORFs for expression of a functional gene product; up to three markerless genomic integrations; and up to five integrations with antibiotic selection. We also present a number of innovations to improve integration efficiency during multiplex engineering in S. cerevisiae including: SGS1 gene knockout; disruption of heteroduplex rejection; modified Cas9 expression architecture; and overexpression of HR genes RAD52, MRE11, and RAD59. To demonstrate how our system can be used for single transformation phenotype engineering of multiple strains, we also transformed a library of methylotrophy associated genes to generate four new strains that were able to grow on a solid minimal medium with methanol as the sole additional carbon source. Our findings contribute to the ongoing efforts to improve multiplex genome engineering tools in S. cerevisiae, and provide the foundations for further development of a novel toolbox for generating useful genetic diversity for metabolic pathway engineering.

Saccharomyces cerevisiae↗
PubMed1992

The Src family of tyrosine protein kinases in hemopoietic signal transduction.

The Src family of tyrosine protein kinases represent an expanding class of closely related intracellular enzymes that participate in the signal transduction pathways of a variety of surface receptors. One of the more surprising aspects of the information relating Src protein kinases to receptor signaling is the apparent diversity of receptor types with which the Src-related enzymes are reported to interact physically or functionally. Traditional biochemical and genetic approaches have yielded much information regarding the interactions between the Src tyrosine protein kinases and other cellular proteins in defined cell types, and emerging technologies, most notably homologous recombination in embryonal stem cells to achieve gene "knockouts," are providing new insights into the participation of the Src-related gene products in signal transduction and development.

Animals↗
PubMed2005

Mechanisms of drug resistance in cancer chemotherapy.

The management of cancer involves procedures, which include surgery, radiotherapy and chemotherapy. Development of chemoresistance is a persistent problem during the treatment of local and disseminated disease. A plethora of cytotoxic drugs that selectively, but not exclusively, target actively proliferating cells include such diverse groups as DNA alkylating agents, antimetabolites, intercalating agents and mitotic inhibitors. Resistance constitutes a lack of response to drug-induced tumour growth inhibition; it may be inherent in a subpopulation of heterogeneous cancer cells or be acquired as a cellular response to drug exposure. Resistance varies. Although regulatory approval may require efficacy in as few as 20% of trial cohorts, a drug may subsequently be used in unselected patients displaying resistance to the treatment. Principal mechanisms may include altered membrane transport involving the P-glycoprotein product of the multidrug resistance (MDR) gene as well as other associated proteins, altered target enzyme (e.g. mutated topoisomerase II), decreased drug activation, increased drug degradation due to altered expression of drug-metabolising enzymes, drug inactivation due to conjugation with increased glutathione, subcellular redistribution, drug interaction, enhanced DNA repair and failure to apoptose as a result of mutated cell cycle proteins such as p53. Attempts to overcome resistance mainly involve the use of combination drug therapy using different classes of drugs with minimally overlapping toxicities to allow maximal dosages and with narrowest cycle intervals, necessary for bone marrow recovery. Adjuvant therapy with P-glycoprotein inhibitors and, in specific instances, the use of growth factor and protein kinase C inhibitors are newer experimental approaches that may also prove effective in abrogating or delaying onset of resistance. Gene knockout using antisense molecules may be another effective way of blocking drug resistance genes. Conversely, drug resistance may also be used to good purpose by transplanting retrovirally transformed CD34 cells expressing the MDR gene to protect the bone marrow during high-dose chemotherapy.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Mosaic analysis with double markers in mice.

We describe a method termed MADM (mosaic analysis with double markers) in mice that allows simultaneous labeling and gene knockout in clones of somatic cells or isolated single cells in vivo. Two reciprocally chimeric genes, each containing the N terminus of one marker and the C terminus of the other marker interrupted by a loxP-containing intron, are knocked in at identical locations on homologous chromosomes. Functional expression of markers requires Cre-mediated interchromosomal recombination. MADM reveals that interchromosomal recombination can be induced efficiently in vivo in both mitotic and postmitotic cells in all tissues examined. It can be used to create conditional knockouts in small populations of labeled cells, to determine cell lineage, and to trace neuronal connections. To illustrate the utility of MADM, we show that cerebellar granule cell progenitors are fated at an early stage to produce granule cells with axonal projections limited to specific sublayers of the cerebellar cortex.

Animals↗
PubMed1997

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

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

Animals↗
PubMed2006

Characterization of AcMNPV with a deletion of me53 gene.

The Autographa Californica multiple nucleopolyhedrovirus (AcMNPV) me53 gene, which was previously reported as one of the major early-transcribed genes, was deleted through homologous recombination from an AcMNPV genome propagated as a bacmid DNA in E. coli, generating a me53 gene knockout bacmid. Green fluorescent protein (GFP) expression analysis and supernatant passage assay revealed that the me53 knockout bacmid was unable to replicate in cell culture, while me53 repair bacmid, which was generated by reinsertion of the me53 gene into the mutant at a different locus (the gentamicin locus) with ET-recombination technique, resumed viral replication ability at wild-type levels, indicating that the defective phenotype of the me53 knockout virus was directly due to the deletion of the me53-ORF. Subsequent electron microscopy revealed that the me53 knockout bacmid failed to form nucleocapsid in the nuclei of the transfected cells, though viral infection seemed to be initiated. Meanwhile, real-time PCR analysis based on SYBR Green fluorescence indicated abolishment of the viral DNA replication by me53 gene inactivation. Thus, it is demonstrated for the first time that me53 knockout blocked viral DNA replication, nucleocapsid formation, and consequent BV and ODV production.

Animals↗
PubMed1999

Signal transduction pathways controlling the switch between keratinocyte growth and differentiation.

Self-renewing epithelia are characterized by a high turnover rate and a fine balance between growth and differentiation. Such a balance is influenced by many exogenous factors, including gradients of diffusible molecules, cell/substrate adhesion contacts, and direct cell-cell communication. The inter-connection between these various extracellular signals and underlying intracellular pathways is clearly of great interest. Primary keratinocytes of either human or murine origin provide an ideal experimental system to elucidate early signaling events involved in the control of epithelial differentiation. Relative to established cell lines, use of a primary system eliminates the possibility of alterations in critical regulatory events which may occur during prolonged propagation in culture. Primary keratinocytes are easily grown in large numbers, and their differentiation can be induced under well-defined culture conditions. The ensuing rapid and homogeneous response is amenable to careful biochemical analysis. Gene transfer technology (transient transfections, adenoviral and retroviral vectors), together with the use of keratinocytes derived from gene knockout and transgenic mice, makes it possible to assess the specific contribution of individual genes to the control of the differentiation process. This review focuses on the significant progress that has been made over the last few years in our understanding of the specific signals that trigger keratinocyte differentiation, the underlying signaling pathways, and how they impinge on specific transcription and cell-cycle control mechanisms associated with the onset of keratinocyte differentiation. Recent developments and future directions in this important area of research will be highlighted.

Animals↗
PubMed2005

Review: complement receptor 1 therapeutics for prevention of immune hemolysis.

The complement system plays a crucial role in fighting infections and is an important link between the innate and adaptive immune responses. However, inappropriate complement activation can cause tissue damage, and it underlies the pathology of many diseases. In the transfusion medicine setting, complement sensitization of RBCs can lead to both intravascular and extravascular destruction. Moreover, complement deficiencies are associated with autoimmune disorders, including autoimmune hemolytic anemia (AIHA). Complement receptor 1 (CR1) is a large single-pass glycoprotein that is expressed on a variety of cell types in blood, including RBCs and immune cells. Among its multiple functions is its ability to inhibit complement activation. Furthermore, gene knockout studies in mice implicate a role for CR1 (along with the alternatively spliced gene product CR2) in prevention of autoimmunity. This review discusses the possibility that the CR1 protein may be manipulated to prevent and treat AIHA. In addition, it will be shown in an in vivo mouse model of transfusion reaction that recombinant soluble forms of CR1 can reduce complement-mediated RBC destruction, thereby prolonging survival of transfused RBCs. It is proposed that CR1-based therapeutics have potential for effective and safe prophylactic short-term use and for treatment of hemolytic transfusion reactions.

Anemia, Hemolytic, Autoimmune↗
PubMed1999

Effects of Npt2 gene ablation and low-phosphate diet on renal Na(+)/phosphate cotransport and cotransporter gene expression.

The renal Na(+)/phosphate (Pi) cotransporter Npt2 is expressed in the brush border membrane (BBM) of proximal tubular cells. We examined the effect of Npt2 gene knockout on age-dependent BBM Na(+)/Pi cotransport, expression of Na(+)/Pi cotransporter genes Npt1, Glvr-1, and Ram-1, and the adaptive response to chronic Pi deprivation. Na(+)/Pi cotransport declines with age in wild-type mice (Npt2(+/+)), but not in mice homozygous for the disrupted Npt2 allele (Npt2(-/-)). At all ages, Na(+)/Pi cotransport in Npt2(-/-) mice is approximately 15% of that in Npt2(+/+) littermates. Only Npt1 mRNA abundance increases with age in Npt2(+/+) mice, whereas Npt1, Glvr-1, and Ram-1 mRNAs show an age-dependent increase in Npt2(-/-) mice. Pi deprivation significantly increases Na(+)/Pi cotransport, Npt2 protein, and mRNA in Npt2(+/+) mice. In contrast, Pi-deprived Npt2(-/-) mice fail to show the adaptive increase in transport despite exhibiting a fall in serum Pi. We conclude that (a) Npt2 is a major determinant of BBM Na(+)/Pi cotransport; (b) the age-dependent increase in Npt1, Glvr-1, and Ram-1 mRNAs in Npt2(-/-) mice is insufficient to compensate for loss of Npt2; and (c) Npt2 is essential for the adaptive BBM Na(+)/Pi cotransport response to Pi deprivation.

Adaptation, Physiological↗
PubMed2025

Development and Characterization of an Inducible Bacterial Artificial Chromosome System for Studying Lytic Replication and Pathogenesis of Kaposi's Sarcoma-Associated Herpesvirus.

Bacterial artificial chromosome (BAC) is widely used to manipulate herpesvirus genome and generate recombinant virus. Here, we developed a new KSHV BACmid, namely as iBAC, by replacing the EGFP with TET3G transactivator under EF1α promoter and inserted Tet response elements in the promoter of RTA in the original KSHV BAC16 clone and characterized KSHV lytic replication in SLK-iBAC cells. SLK-iBAC cells developed more efficient lytic replication and generated more progeny virus than iSLK-BAC16 cells upon the same conditions of doxycycline treatment. Since SLK-iBAC cells only occupied hygromycin selection marker, it is convenient to generate cellular gene knockout via lentivirus-mediated CRISPR-Cas9 or stably express viral or cellular gene via lentivirus followed by antibiotic selection, making iBAC system a better tool to identify cellular targets of viral proteins in the context of virus infection or study the role of viral or cellular genes for KSHV lytic replication and pathogenesis. In addition, iBAC is color-free and can be utilized to track subcellular localization of viral proteins or colocalization between different viral proteins by introducing fusing fluorescent proteins into the BAC backbone. Therefore, the new KSHV iBAC is a powerful inducible tool to study KSHV lytic replication and pathogenesis in cell model.

Chromosomes, Artificial, Bacterial↗
PubMed1994

Transgenic animals in toxicology.

Recent advances have been made in the characterization of a number of transgenic animal models. These animal models have provided a powerful toxicological tool for studying in vivo chemical effects and have increased our understanding of the role of specific genetic alterations as predisposing factors for chemical carcinogenesis. The goal of this symposium was to introduce the development of transgenic animals and the utilization of transgenics in toxicology research focusing on understanding tissue-specific mutation, chemical effects, and cancer. The production of transgenic animals, including gene insertions and gene knockouts, and the utilization of transgenic technology for studying multistage carcinogenesis and tumor suppressor genes are described. Data on the application and implications of transgenics as a genetic endpoint are also discussed. The use of transgenic animals in toxicology should improve our understanding of the role of specific genetic alterations in the carcinogenic process and lead to improved estimations of human health risks.

Animals↗
PubMed1994

Atherosclerosis in mice: getting to the heart of a polygenic disorder.

The mouse is being increasingly used as a model system for understanding the common and complex polygenic condition of atherosclerosis. Mice have been created in which genes involved in this condition have been overexpressed or have been altered by gene targeting. Here, we concentrate on recent experiments that use transgenic and gene knockout mice as an in vivo assay system to unravel the interactions between various genes involved in atherogenesis. We focus in particular on examples in which unique insights into the human condition have been derived from studies in mice.

Animals↗