Versatile applications of transcriptional pulsing to study mRNA turnover in mammalian cells.

Development of transcriptional pulsing approaches using the c-fos and Tet-off promoter systems greatly facilitated studies of mRNA turnover in mammalian cells. However, optimal protocols for these approaches vary for different cell types and/or physiological conditions, limiting their widespread application. In this study, we have further optimized transcriptional pulsing systems for different cell lines and developed new protocols to facilitate investigation of various aspects of mRNA turnover. We apply the Tet-off transcriptional pulsing strategy to investigate ARE-mediated mRNA decay in human erythroleukemic K562 cells arrested at various phases of the cell cycle by pharmacological inhibitors. This application facilitates studies of the role of mRNA stability in control of cell-cycle dependent gene expression. To advance the investigation of factors involved in mRNA turnover and its regulation, we have also incorporated recently developed transfection and siRNA reagents into the transcriptional pulsing approach. Using these protocols, siRNA and DNA plasmids can be effectively cotransfected into mouse NIH3T3 cells to obtain high knockdown efficiency. Moreover, we have established a tTA-harboring stable line using human bronchial epithelial BEAS-2B cells and applied the transcriptional pulsing approach to monitor mRNA deadenylation and decay kinetics in this cell system. This broadens the application of the transcriptional pulsing system to investigate the regulation of mRNA turnover related to allergic inflammation. Critical factors that need to be considered when employing these approaches are characterized and discussed.

Conditional and inducible transgene expression in endothelial and hematopoietic cells using Cre/loxP and tetracycline-off systems

In the present study, the tetracycline-off and Cre/loxP systems were combined to gain temporal and spatial control of transgene expression. Mice were generated that carried three transgenes: Tie2-tTA, tet-O-Cre and either the ZEG or ZAP reporter. Tie2-tTA directs expression of tetracycline-controlled transactivator (tTA) in endothelial and hematopoietic cells under the control of the Tie2 promoter. Tet-O-Cre produces Cre recombinase from a minimal promoter containing the tet-operator (tetO). ZEG or ZAP contains a strong promoter and a loxP-flanked stop sequence, followed by an enhanced green fluorescence protein (EGFP) or human placental alkaline phosphatase (hPLAP) reporter. In the presence of tetracycline, the tTA transactivator produced by Tie-2-tTA is disabled and Cre is not expressed. In the absence of tetracycline, the tTA binds tet-O-Cre to drive the expression of Cre, which recombines the loxP sites of the ZEG or ZAP transgene and results in reporter gene expression. In the present study, the expression of the ZEG or ZAP reporter genes in embryos and adult animals with and without tetracycline treatment was examined. In the presence of tetracycline, no reporter gene expression was observed. When tetracycline was withdrawn, Cre excision was activated and the reporter genes were detected in endothelial and hematopoietic cells. These results demonstrate that this system may be used to bypass embryonic lethality and access adult phenotypes.

Postnatal Notch1 activation induces T‑cell malignancy in conditional and inducible mouse models

The Notch1 signaling pathway is essential for hematopoietic development. However, the effects of postnatal activation of Notch1 signaling on hematopoietic system is not yet fully understood. We previously generated ZEG‑IC‑Notch1 transgenic mice that have a floxed β‑geo/stop signal between a CMV promoter and intracellular domain of Notch1 (IC‑Notch1). Constitutively active IC‑Notch1 is silent until the introduction of Cre recombinase. In this study, endothelial/hematopoietic specific expression of IC‑Notch1 in double transgenic ZEG‑IC‑Notch1/Tie2‑Cre embryos induced embryonic lethality at E9.5 with defects in vascular system but not in hematopoietic system. Inducible IC‑Notch1 expression in adult mice was achieved by using tetracycline regulated Cre system. The ZEG‑IC‑Notch1/Tie2‑tTA/tet‑O‑Cre triple transgenic mice survived embryonic development when maintained on tetracycline. Post‑natal withdrawal of tetracycline induced expression of IC‑Notch1 transgene in hematopoietic cells of adult mice. The triple transgenic mice displayed extensive T‑cell infiltration in multiple organs and T‑cell malignancy of lymph nodes. In addition, the protein levels of p53 and alternative reading frame (ARF) were decreased in lymphoma‑like neoplasms from the triple transgenic mice while their mRNA expression remained unchanged, suggesting that IC‑Notch1 might repress ARF‑p53 pathway by a post‑transcriptional mechanism. This study demonstrated that activation of constitutive Notch1 signaling after embryonic development alters adult hematopoiesis and induces T‑cell malignancy.

Determinación de la época más adecuada para la propagación ágamica de gramíneas ornamentales : : Mendoza (Argentina)

La división de matas es una técnica de propagación difundida con gran éxito entre las Gramíneas. Cuando se trata de la producción comercial de este grupo de plantas, es importante conocer la época en que se realiza esta práctica para obtener plantas de la mejor calidad en el menor tiempo posible. Según algunos autores, la estación apropiada para dividir gramíneas está relacionada con el momento de activo crecimiento: primavera tardía para las especies estivales y otoño o primavera temprana para las invernales. En este trabajo se estudió la influencia de la época del año en la división de matas de Miscanthus sinensis "Variegatus", Miscanthus sinensis "Zebrinus", Miscanthus sinensis "Morning Light", Paspalum haumanii, Leymus arenarius, Pennisetum setaceum y Trichloris crinita en diciembre y febrero. Se evaluaron las características ornamentales y la precocidad, con fines comerciales, de las plantas obtenidas en otoño y primavera tardía. Las variables utilizadas fueron altura vegetativa, diámetro de canopia y de corona, número de cañas y porcentaje de sobrevivencia. Desde el punto de vista comercial y productivo, es conveniente realizar la división de matas en otoño para obtener precocidad sólo en M. sinensis "Variegatus" y M. sinensis "Zebrinus". En el caso de M. sinensis "Morning Light", P. setaceum, T. crinita, P. haumanii y Leymus es más adecuada la división de primavera.

Mejora de la gestión del agua de riego mediante el uso de indicadores de riego

La gestión del agua de riego en la zona regable del Genil-Cabra, situada en la provincia de Córdoba, sur de España, se ha estudiado usando tres indicadores de riego: el Suministro Relativo de Agua de Riego (RIS); el Suministro Relativo de Agua (RWS) y el Suministro Relativo de Agua por Precipitaciones (RRS). Estos tres indicadores se han calculado tanto de forma global como agrupando los datos según el tipo de cultivo, el método de riego, la textura del suelo y el tamaño de la parcela. Toda la información relativa a variables agronómicas e hidráulicas se ha incluido en un Sistema de Información Geográfica (SIG) para facilitar su manejo. Los resultados muestran que los riegos son deficitarios ya que el valor del indicador RIS es relativamente bajo. No obstante, dado que el indicador RWS alcanza valores más altos, la demanda evaporativa puede ser satisfecha a lo largo del ciclo de desarrollo del cultivo. El indicador RRS oscila menos y junto al RWS permite conocer la fracción de evapotranspiración cubierta por el agua de lluvia. Los valores medios de los indicadores calculados son muy útiles para conocer el comportamiento del regante y la tendencia general, aunque la muestra usada es aún insuficiente para poder caracterizar una gran área de riego en su conjunto.