RESUMEN
Acute promyelocytic leukemia (APML) is characterized by abnormal myeloid development, resulting an accumulation of leukemic promyelocytes that are often highly sensitive to retinoic acid. A balanced t(15;17) (q22;q21) reciprocal chromosomal translocation is found in approximately 90% of APML patients; this translocation fuses the PML gene on chromosome 15 to the retinoic acid receptor alpha (RAR alpha) gene on chromosome 17, creating two novel fusion genes, PML-RAR alpha and RAR alpha-PML. The PML-RAR alpha fusion gene product, which is expressed in virtually all patients with t(15;17), is thought to play a direct role in the pathogenesis of APML. To determine whether PML-RAR alpha is sufficient to cause APML in an animal model, we used the promyelocyte-specific targeting sequences of the human cathepsin G (hCG) gene to direct the expression of a PML-RAR alpha cDNA to the early myeloid cells of transgenic mice. Mice expressing the hCG-PML-RAR alpha transgene were found to have altered myeloid development that was characterized by increased percentages of immature and mature myeloid cells in the peripheral blood, bone marrow, and spleen. In addition, approximately 30% of transgene-expressing mice eventually developed acute myeloid leukemia after a long latent period. The splenic promyelocytes of mice with both the nonleukemic and leukemic phenotypes responded to all-trans retinoic acid (ATRA) treatment, which caused apoptosis of myeloid precursors. Although low-level expression of the hCG-PML-RAR alpha transgene is not sufficient to directly cause acute myeloid leukemia in mice, its expression alters myeloid development, resulting in an accumulation of myeloid precursors that may be susceptible to cooperative transforming events.
Asunto(s)
Catepsinas/genética , Regulación Neoplásica de la Expresión Génica , Hematopoyesis/genética , Leucemia Promielocítica Aguda/genética , Ratones Transgénicos , Proteínas de Neoplasias/genética , Proteínas de Fusión Oncogénica/genética , Animales , Catepsina G , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Técnicas de Transferencia de Gen , Humanos , Leucemia Promielocítica Aguda/patología , Ratones , Serina EndopeptidasasRESUMEN
To examine the function of murine beta-globin locus region (LCR) 5' hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5' HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human gamma-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5' HS3 reduces expression of the linked embryonic epsilon y- and beta H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult beta (beta major plus beta minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5' HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult erythrocytes.
Asunto(s)
Genes Reguladores , Globinas/genética , Animales , Línea Celular , Eritrocitos/metabolismo , Muerte Fetal/genética , Eliminación de Gen , Expresión Génica , Marcación de Gen , Genes de Cambio , Homocigoto , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Familia de Multigenes , Recombinación Genética , Talasemia/genéticaRESUMEN
Macrophages secrete a variety of proteinases that are thought to participate in remodeling of the extracellular matrix associated with inflammatory processes. We have eliminated expression of the macrophage metalloelastase (MME) gene by targeted disruption to assess the role of this protein in macrophage-mediated proteolysis. We found that the macrophages of MME-deficient (MME-/-) mice have a markedly diminished capacity to degrade extracellular matrix components. In addition, MME-/- macrophages are essentially unable to penetrate reconstituted basement membranes in vitro and in vivo. MME is therefore required for macrophage-mediated extracellular matrix proteolysis and tissue invasion.
Asunto(s)
Elastina/metabolismo , Matriz Extracelular/fisiología , Macrófagos Peritoneales/fisiología , Metaloendopeptidasas/metabolismo , Animales , Secuencia de Bases , Membrana Basal/fisiología , Clonación Molecular , Cartilla de ADN , Biblioteca de Genes , Inflamación , Metaloproteinasa 12 de la Matriz , Metaloendopeptidasas/biosíntesis , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
T cell hybridomas require the immediate-early gene NGFI-B (nur77) for T cell receptor (TCR)-mediated apoptosis, a model for negative selection of self-reactive T cells. TCR-mediated death was examined in mice bearing an NGFI-B loss-of-function mutation, either by administration of antibodies to CD3 (anti-CD3) or in two well-characterized transgenic models expressing self-reactive TCRs. Both the extent and the rate of thymocyte death were unimpaired. Anti-CD3-induced death was normal in CD4+ peripheral T cells, in which death is mediated predominantly by the Fas signaling pathway. Thus, no unique requirement for NGFI-B is observed for thymic or peripheral T cell death.
Asunto(s)
Apoptosis , Proteínas de Unión al ADN/fisiología , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología , Receptores de Esteroides/fisiología , Linfocitos T/citología , Factores de Transcripción/fisiología , Animales , Anticuerpos , Complejo CD3/inmunología , Complejo CD3/fisiología , Células Cultivadas , Supresión Clonal , Cruzamientos Genéticos , Proteínas de Unión al ADN/genética , Femenino , Marcación de Gen , Hibridomas , Masculino , Ratones , Ratones Transgénicos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Receptores Citoplasmáticos y Nucleares , Receptores de Esteroides/genética , Células Madre , Subgrupos de Linfocitos T/citología , Linfocitos T/inmunología , Timo/citología , Factores de Transcripción/genéticaRESUMEN
NGFI-A (also known as EGR-1, zif/268, and Krox-24) is a zinc finger transcription factor induced in many cell types by a variety of growth and differentiation stimuli. To determine if NGFI-A plays a requisite role in these processes, we used homologous recombination to mutate both alleles of NGFI-A in embryonic stem (ES) cells and examined its effect on growth and differentiation. We find that ES cells lacking NGFI-A exhibit similar growth rates and serum-induced gene expression profiles compared to wild-type parental cells. They are capable of differentiating into neurons, cardiac myocytes, chondrocytes, and squamous epithelium. Chimeric mice were generated from targeted ES cells, and their progeny were crossed to produce homozygous mutant mice. Growth and histological analyses of mice lacking NGFI-A confirm the finding in ES cells that NGFI-A is not required for many of the processes associated with its expression and suggest that the function of NGFI-A is either more subtle in vivo or masked by redundant expression provided by other gene family members such as NGFI-C, Krox-20, or EGR3.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes Inmediatos-Precoces , Proteínas Inmediatas-Precoces , Factores de Transcripción/genética , Animales , Diferenciación Celular/genética , División Celular/genética , Línea Celular , Proteínas de Unión al ADN/fisiología , Proteína 1 de la Respuesta de Crecimiento Precoz , Homocigoto , Ratones , Ratones Endogámicos BALB C , Mutación , Recombinación Genética , Factores de Transcripción/fisiologíaRESUMEN
We have generated H-2b mice with a homozygous null mutation in the granzyme (gzm) B gene. Gzm B is a neutral serine protease with Aspase activity that is found only in the granules of activated cytolytic T cells, natural killer cells, and lymphokine-activated killer cells. Gzm B-/- mice develop normally and have normal hematopoiesis and lymphopoiesis. In vitro, cytotoxic T lymphocytes (CTL) derived from gzm B-/- animals are able to induce 51Cr release from allotarget cells, but with reduced efficiency. However, gzm B-/- CTL have a profound defect in their ability to induce rapid DNA fragmentation and apoptosis in allogeneic target cells. This defect is kinetic since DNA fragmentation is partially compensated and 51Cr release is completely rescued with long incubation times. We conclude that gzm B serves a critical and nonredundant role for the rapid induction of target cell DNA fragmentation and apoptosis by alloreactive cytotoxic T lymphocytes.
Asunto(s)
Apoptosis/fisiología , ADN/metabolismo , Células Asesinas Naturales/fisiología , Serina Endopeptidasas/fisiología , Linfocitos T Citotóxicos/fisiología , Animales , Embrión de Mamíferos/citología , Granzimas , Hematopoyesis/fisiología , Células Asesinas Naturales/enzimología , Activación de Linfocitos/fisiología , Prueba de Cultivo Mixto de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Familia de Multigenes , Mutación , Serina Endopeptidasas/genética , Células Madre , Linfocitos T Citotóxicos/enzimologíaRESUMEN
Lipoprotein-associated coagulation inhibitor (LACI) is a multivalent, Kunitz-type proteinase inhibitor which appears to play an important role in the regulation of hemostasis. LACI directly inhibits factor Xa, and, in a Xa-dependent fashion, also inhibits the factor VIIa-tissue factor catalytic complex. Hybridization of a LACI cDNA probe to DNA isolated from a panel of human-mouse somatic cell hybrids containing different human chromosomes localized the human LACI gene to chromosome 2. In situ hybridization to metaphase chromosomes further mapped the gene to the region 2q31----2q32.1. Exons of the human LACI gene were cloned from genomic or chromosome 2-specific phage libraries and sequenced, including approximately 500 base pairs of 5' upstream DNA. The 5' DNA did not contain a prototypical TATAA box or CCAAT sequence, and attempts to identify a unique site for the initiation of transcription were unsuccessful in that primer extension and S1 nuclease protection analysis indicate multiple transcription initiation sites for LACI messages. Comparing the gene sequence with LACI cDNA sequences indicates that the gene contains nine exons and that alternative splicing can occur, resulting in the absence of exon 2 in the 5' untranslated region of some messages. The three Kunitz domains in LACI are encoded on separate exons. Introns which interrupt coding sequences all occur in the same codon phase interrupting the first and second bases of the codon triplets. The data are consistent with LACI evolving by a combination of gene segment duplications and exon shuffling.