A new generation of pPRIG-based retroviral vectors

<p>Abstract</p> <p>Background</p> <p>Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c).</p> <p>Results</p> <p>The convenience of pPRIG makes it a good basic vector to generate additional derivatives for an extended range of use. Here we present several novel pPRIG-based vectors (collectively referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames 3' of the MCS (3'HA PRIGs); ii) a functional domain (ER, VP16 or KRAB) was inserted either 5' or 3' of the MCS (« modular » PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puro-R (« single color/resistance » PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS of the IRES-eGFP, IRES-eCFP or IRES-Puro-R containing PRIGs, respectively (« dual color/selection » PRIGs). Additionally, some of these PRIGs were also constructed in a pMigR MSCV background which has been widely used in pluripotent cells.</p> <p>Conclusion</p> <p>These novel vectors allow for straightforward detection of any expressed protein (3'HA PRIGs), for functional studies of chimeric proteins (« modular » PRIGs), for multiple transductions and fluorescence analyses of transduced cells (« single color/resistance » PRIGs), or for quantitative detection of studied proteins in independently identified/selected transduced cells (« dual color/selection » PRIGs). They maintain the original advantages of pPRIG and provide suitable tools for either transient or stable expression and functional studies in a large range of experimental settings.</p

Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse Yolk Sac: a comparative study between in situ electroporation and retroviral transduction

<p>Abstract</p> <p>Background</p> <p>Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.</p> <p>Results</p> <p>One transduction protocol involves transient modification of gene expression through <it>in situ </it>electroporation of the prospective blood islands, which allows the evolution of transfected mesodermal cells in their "normal" environment, upon organ culture. Following <it>in situ </it>electroporation of a GFP reporter construct into the YS cavity of embryos at post-streak (mesodermal/pre-hematopoietic precursors) or early somite (hematopoietic precursors) stages, high GFP expression levels as well as a good preservation of cell viability is observed in YS explants. Moreover, the erythro-myeloid progeny typical of the YS arises from GFP⁺mesodermal cells or hematopoietic precursors, even if the number of targeted precursors is low. The second approach, based on retroviral transduction allows a very efficient transduction of large precursor numbers, but may only be used to target 8 dpc YS hematopoietic precursors. Again, transduced cells generate a progeny quantitatively and qualitatively similar to that of control YS.</p> <p>Conclusion</p> <p>We thus provide two protocols whose combination may allow a thorough study of both early and late events of hematopoietic development in the murine YS. <it>In situ </it>electroporation constitutes the only possible gene transfer method to transduce mesodermal/pre-hematopoietic precursors and analyze the earliest steps of hematopoietic development. Both <it>in situ </it>electroporation and retroviral transduction may be used to target early hematopoietic precursors, but the latter appears more convenient if a large pool of stably transduced cells is required. We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.</p

Making the Earth: Combining Dynamics and Chemistry in the Solar System

No terrestrial planet formation simulation completed to date has considered the detailed chemical composition of the planets produced. While many have considered possible water contents and late veneer compositions, none have examined the bulk elemental abundances of the planets produced as an important check of formation models. Here we report on the first study of this type. Bulk elemental abundances based on disk equilibrium studies have been determined for the simulated terrestrial planets of O'Brien et al. (2006). These abundances are in excellent agreement with observed planetary values, indicating that the models of O'Brien et al. (2006) are successfully producing planets comparable to those of the Solar System in terms of both their dynamical and chemical properties. Significant amounts of water are accreted in the present simulations, implying that the terrestrial planets form "wet" and do not need significant water delivery from other sources. Under the assumption of equilibrium controlled chemistry, the biogenic species N and C still need to be delivered to the Earth as they are not accreted in significant proportions during the formation process. Negligible solar photospheric pollution is produced by the planetary formation process. Assuming similar levels of pollution in other planetary systems, this in turn implies that the high metallicity trend observed in extrasolar planetary systems is in fact primordial.Comment: 61 pages (including online material), 12 figures (7 in paper, 5 online). Accepted to Icaru

Origin of the Ocean on the Earth: Early Evolution of Water D/H in a Hydrogen-rich Atmosphere

The origin of the Earth's ocean has been discussed on the basis of deuterium/hydrogen ratios (D/H) of several sources of water in the solar system. The average D/H of carbonaceous chondrites (CC's) is known to be close to the current D/H of the Earth's ocean, while those of comets and the solar nebula are larger by about a factor of two and smaller by about a factor of seven, respectively, than that of the Earth's ocean. Thus, the main source of the Earth's ocean has been thought to be CC's or adequate mixing of comets and the solar nebula. However, those conclusions are correct only if D/H of water on the Earth has remained unchanged for the past 4.5 Gyr. In this paper, we investigate evolution of D/H in the ocean in the case that the early Earth had a hydrogen-rich atmosphere, the existence of which is predicted by recent theories of planet formation no matter whether the nebula remains or not. Then we show that D/H in the ocean increases by a factor of 2-9, which is caused by the mass fractionation during atmospheric hydrogen loss, followed by deuterium exchange between hydrogen gas and water vapor during ocean formation. This result suggests that the apparent similarity in D/H of water between CC's and the current Earth's ocean does not necessarily support the CC's origin of water and that the apparent discrepancy in D/H is not a good reason for excluding the nebular origin of water.Comment: Accepted to Icaru

A deuterium-poor water reservoir in the asteroid 4 Vesta and the inner solar system

Recent investigations of meteorites thought to originate from the asteroid 4 Vesta have suggested an early accretion of water on rocky bodies in the inner Solar System from a carbonaceous chondrite-like source. However, these studies have been based on the hydrogen isotope compositions (δD) of late-crystallizing apatite grains in eucrites that likely do not record the primary magmatic composition. We have determined the δD and H2O concentrations in some of the earliest-formed silicates (clinopyroxenes) from several eucrites with the goal of constraining the hydrogen isotope composition of their source reservoir on their parent body. The H2O concentrations in clinopyroxenes from eucrites Juvinas, Stannern and Tirhert range from 5 to 18 μg/g, with a weighted average δD of –263 ± 70‰. Their apatites and whitlockites exhibit a higher weighted average δD of –165 ± 73‰, possibly as a result of H2 degassing during or after phosphate crystallization. Thermal metamorphism of these eucrites has most probably resulted in the loss of H, and an increase in their original δD values. While the weighted average δD value for the eucrite clinopyroxenes reported here is inferred to reflect an upper limit for the isotopic composition of the silicate mantle reservoir on their parent asteroid 4 Vesta, the average δD value of Stannern clinopyroxenes is considered to be closest to the initial δD of the source mantle (i.e., –373 ± 127‰), which is lighter than that of Earth’s depleted upper mantle and most carbonaceous chondrites. We suggest that at least some of the water in 4 Vesta (and possibly other rocky bodies in the inner Solar System) was derived from a relatively deuterium-poor reservoir in the protosolar nebula, which was incorporated into planetesimals formed early in Solar System history

Dépolarisation magnétique des niveaux de configuration 6s6d de l'atome de mercure excité optiquement par échelons

By using very bright mercury lamps to induce coherence into stepwise excited mercury levels, we have determined by Hanle effect measurements (zero field level crossing) the lifetimes of the levels of the 6s6d configuration of mercury. For these levels, the experimental results are in reasonable agreement with the theoretical predictions.A l'aide de lampes à vapeur de mercure très brillantes, on a pu induire de la cohérence dans divers niveaux du mercure excités optiquement. On a pu ainsi déterminer au moyen de l'effet Hanle (dépolarisation magnétique) les durées de vie radiatives des niveaux de la configuration 6s6d du mercure. Pour ces derniers, les résultats obtenus sont en accord raisonnable avec les prévisions théoriques

Manuel de la langue basque.

Tít. de la anteportada : Manuel de la langue basqueDigitalización Vitoria-Gasteiz Archivos y Bibliotecas Septiembre 1994 18-7