RESUMEN
The life of an mRNA is dynamic within a cell. The development of quantitative fluorescent microscopy techniques to image single molecules of RNA has allowed many aspects of the mRNA lifecycle to be directly observed in living cells. Recent advances in live-cell multicolor RNA imaging, however, have now made it possible to investigate RNA metabolism in greater detail. In this chapter, we present an overview of the design and implementation of the translating RNA imaging by coat protein knockoff RNA biosensor, which allows untranslated mRNAs to be distinguished from ones that have undergone a round of translation. The methods required for establishing this system in mammalian cell lines and Drosophila melanogaster oocytes are described here, but the principles may be applied to any experimental system.
Asunto(s)
Técnicas Biosensibles/métodos , Drosophila melanogaster/citología , Microscopía Fluorescente/métodos , Oocitos/citología , ARN Mensajero/análisis , Animales , Proteínas de la Cápside/genética , Células Cultivadas , Drosophila melanogaster/genética , Levivirus/genética , Proteínas Luminiscentes/genética , Imagen Molecular/métodos , Oocitos/metabolismo , ARN Mensajero/genéticaRESUMEN
Primary torsion dystonia is an autosomal-dominant inherited movement disorder. Most cases are caused by an in-frame deletion (GAG) of the DYT1 gene encoding torsinA. Reduced penetrance and phenotypic variability suggest that alteration of torsinA amino acid sequence is necessary but not sufficient for development of clinical symptoms and that additional factors must contribute to the factual manifestation of the disease. We generated 4 independent transgenic mouse lines, two overexpressing human mutant torsinA and two overexpressing human wildtype torsinA using a strong murine prion protein promoter. Our data provide for the first time in vivo evidence that not only mutant torsinA is detrimental to neuronal cells but that also wildtype torsinA can lead to neuronal dysfunction when overexpressed at high levels. This hypothesis is supported by (i) neuropathological findings, (ii) neurochemistry, (iii) behavioral abnormalities and (iv) DTI-MRI analysis.
Asunto(s)
Encéfalo/metabolismo , Encéfalo/patología , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Neuronas/ultraestructura , Animales , Western Blotting , Química Encefálica , Distonía/genética , Distonía/metabolismo , Distonía/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Actividad Motora , Neuronas/metabolismo , Neurotransmisores/análisis , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The cellular attachment receptor for adenovirus (Ad), Coxsackie adenovirus receptor (CAR), required for delivery of Ad into primary cells, is not present on all cell types, thus restricting Ad-gene delivery systems. To circumvent this constrain, a transgenic mouse has been generated that expresses a truncated human CAR in all tissues analyzed. These mice allowed efficient in vitro infections at low multiplicities into lymphoid, myeloid, and endothelial cells. Furthermore, in vivo administration of Ad-vectors results in infection of macrophages, lymphocytes, and endothelial cells. In addition, tail vein injection resulted in targeting of virus into previously inaccessible areas, such as the lung and the capillaries of the brain. The CAR transgenic mice will be useful for rapid functional genomic analysis in vivo, for testing the efficacy of gene therapy procedures or as a source of easily transducible cells.
Asunto(s)
Infecciones por Adenoviridae/genética , Adenoviridae/genética , Regulación Viral de la Expresión Génica , Ratones Transgénicos , Animales , Modelos Animales de Enfermedad , Técnicas de Transferencia de Gen , Transferencia de Gen Horizontal , Genes Virales , Humanos , RatonesRESUMEN
To study further the role of gonadotropins in reproductive functions, we generated mice with LH receptor (LHR) knockout (LuRKO) by inactivating, through homologous recombination, exon 11 on the LHR gene. LuRKO males and females were born phenotypically normal, with testes, ovaries, and genital structures indistinguishable from their wild-type (WT) littermates. Postnatally, testicular growth and descent, and external genital and accessory sex organ maturation, were blocked in LuRKO males, and their spermatogenesis was arrested at the round spermatid stage. The number and size of Leydig cells were dramatically reduced. LuRKO females also displayed underdeveloped external genitalia and uteri postnatally, and their age of vaginal opening was delayed by 5-7 days. The (-/-) ovaries were smaller, and histological analysis revealed follicles up to the early antral stage, but no preovulatory follicles or corpora lutea. Reduced gonadal sex hormone production was found in each sex, as was also reflected by the suppressed accessory sex organ weights and elevated gonadotropin levels. Completion of meiosis of testicular germ cells in the LuRKO males differs from other hypogonadotropic/cryptorchid mouse models, suggesting a role for FSH in this process. In females, FSH appears to stimulate developing follicles from the preantral to early antral stage, and LH is the stimulus beyond this stage. Hence, in each sex, the intrauterine sex differentiation is independent of LH action, but it has a crucial role postnatally for attaining sexual maturity. The LuRKO mouse is a close phenocopy of recently characterized human patients with inactivating LHR mutations, although the lack of pseudohermaphroditism in LuRKO males suggests that the intrauterine sex differentiation in this species is not dependent on LH action.
Asunto(s)
Desarrollo Embrionario y Fetal , Genitales/embriología , Genitales/crecimiento & desarrollo , Receptores de HL/deficiencia , Maduración Sexual , Animales , Línea Celular , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Gonadotropina Coriónica/metabolismo , Embrión de Mamíferos , Exones , Femenino , Hormona Folículo Estimulante/sangre , Hormona Folículo Estimulante/farmacología , Expresión Génica , Hormona Luteinizante/sangre , Hormona Luteinizante/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovario/anatomía & histología , Ovario/crecimiento & desarrollo , Fenotipo , Receptores de HL/genética , Receptores de HL/fisiología , Células Madre , Esteroide 17-alfa-Hidroxilasa/genética , Esteroides/sangre , Testículo/anatomía & histología , Testículo/crecimiento & desarrolloRESUMEN
To determine the spatio-temporal expression in brain of the high-affinity kainate receptor subunit KA1, we generated transgenic mice expressing Cre recombinase from the KA1 gene on a chromosomally integrated 550 kb yeast artificial chromosome (YAC). Activity of the KA1 gene promoter during brain development was visualized by Cre immunohistochemistry, and by X-gal staining of beta-galactosidase induced by Cre recombinase in double transgenic KA1-Cre/lacZ indicator mice. During early brain development, expression from the YAC-carried KA1-Cre transgene was observed in all major brain areas, predicting a function for KA1 in the developing central nervous system. In the adult brain, KA1-Cre transgene expression was restricted mainly to hippocampal CA3 pyramidal and dentate gyrus granule cells, an adult expression pattern characteristic for the endogenous KA1 alleles. KA1-Cre transgenic mice may help in elucidating the role of floxed genes ablated in vivo in KA1 expressing neurons.
Asunto(s)
Envejecimiento/metabolismo , Animales Recién Nacidos/metabolismo , Integrasas/metabolismo , Receptores de Ácido Kaínico/metabolismo , Proteínas Virales , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Genes Reporteros/fisiología , Inmunohistoquímica , Integrasas/genética , Operón Lac/fisiología , Ratones , Ratones Transgénicos/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptores de Ácido Kaínico/genética , Distribución TisularRESUMEN
The differentiation potential of stem cells in tissues of the adult has been thought to be limited to cell lineages present in the organ from which they were derived, but there is evidence that some stem cells may have a broader differentiation repertoire. We show here that neural stem cells from the adult mouse brain can contribute to the formation of chimeric chick and mouse embryos and give rise to cells of all germ layers. This demonstrates that an adult neural stem cell has a very broad developmental capacity and may potentially be used to generate a variety of cell types for transplantation in different diseases.