Pooled RNAi Screens - Technical and Biological Aspects

RNA interference (RNAi) screens have recently emerged as an exciting new tool for studying gene function in mammalian cells. In order to facilitate those studies, short hairpin RNA (shRNA) expression libraries covering the entire human transcriptome have become commercially available. To make use of the full potential of such large-scale shRNA libraries, microarray-based methods have been developed to analyze complex pooled RNAi screens. In terms of microarray analysis, different strategies have been pursued by different research groups, largely influenced by the employed shRNA library. In this review, we compare the three major shRNA expression libraries with a focus on their suitability for a microarray-based analysis of pooled screens. We analyze and compare approaches previously used to perform pooled RNAi screens and point out their advantages as well as limitations

Test of structural models for the (4 4) phase formed by oxygen adsorption on the Pt3Sn(1 1 1) surface

Exposure of the Pt3Sn(1 1 1) surface to oxygen at a pressure in the 10-6 mbar range at 800 K leads to the formation of a chemisorbed phase with a (4 × 4) periodicity. In previous works we investigated the structure of the (4 × 4) phase by means of scanning tunneling microscopy (STM) and XPD. STM images show that the (4 × 4) periodicity results from an array of protrusions and holes. XPD data suggest that oxygen adsorption induces the reconstruction of the substrate with the formation of a Sn-O overlayer. In the present work, several structural models compatible with the STM and XPD results were tested by tensor LEED analysis. The best agreement with the experimental I-V curves was obtained for a model similar to that proposed to interpret the STM images of the Ag(1 1 1)(4 × 4)-O phase. According to this structure, the protrusions observed in STM would correspond to tin atoms occupying on-top positions

Six years of continuous carbon isotope composition measurements of methane in Heidelberg (Germany) – a study of source contributions and comparison to emission inventories

Mitigation of greenhouse gases requires a precise knowledge of their sources at both global and regional scales. With improving measurement techniques, in situ δ(13C,CH4) records are analysed in a growing number of studies to characterise methane emissions and to evaluate inventories at regional and local scales. However, most of these studies cover short time periods of a few months, and the results show a large regional variability. In this study, a 6-year time record of in situ δ(13C,CH4), measured with a cavity ring-down spectroscopy (CRDS) analyser in Heidelberg, Germany, is analysed to obtain information about seasonal variations and trends of CH4 emissions. The Keeling plot method is applied to atmospheric measurements on different timescales, and the resulting source contributions are used to evaluate the CH4 emissions reported by two emission inventories: the Emissions Database for Global Atmospheric Research (EDGAR v6.0) and the inventory of the State Institute for the Environment Baden-Württemberg (LUBW). The mean isotopic carbon source signature for the Heidelberg catchment area derived from atmospheric measurements is (-52.3±0.4) ‰ and shows an annual cycle with 5.8 ‰ more depleted values in summer than in winter. This annual cycle can only be partly explained by seasonal variations in the 13C-enriched emissions from heating and reveals strong seasonal variations in biogenic CH4 emissions in the Heidelberg catchment area, which are not included in EDGAR v6.0. The comparison with emission inventories also shows that EDGAR v6.0 overestimates the CH4 emissions from less depleted sources. In situ CH4 isotope analysers at continental and urban monitoring stations can make an important contribution to the verification and improvement of emission inventories.</p

Mutation of a gene for a Drosophila kinesin-like protein, Klp38B, leads to failure of cytokinesis

Mutations in a gene (Klp38B) encoding a novel kinesin-like protein in Drosophila melanogaster lead to the formation of polyploid cells in the larval central nervous system and in the follicle cells of adult egg chambers. Some homozygous mutants survive to adulthood and also exhibit morphological defects indicative of abnormal cell cycle progression, including rough eyes, missing bristles, and abnormal abdominal cuticles. In larval brains, there is no accumulation of mitotic cells and the frequency of anaphase figures is comparable to wild type, suggesting that nuclear division is not affected. Such brains contain polyploid cells with metaphase and anaphase chromosomes associated with bipolar spindles. Such spindles have a number of unseparated centrosomes at their poles reflecting the degree of polyploidy of the cell. Follicle cells frequently contain two nuclei of roughly equal size. Taken together, we conclude that these Klp38B mutations lead to a failure of cytokinesis resulting in polyploidy, and discuss whether or not this is a direct effect of the mutation

Effects of three-body interactions on the structure and thermodynamics of liquid krypton

Large-scale molecular dynamics simulations are performed to predict the structural and thermodynamic properties of liquid krypton using a potential energy function based on the two-body potential of Aziz and Slaman plus the triple-dipole Axilrod-Teller (AT) potential. By varying the strength of the AT potential we study the influence of three-body contribution beyond the triple-dipole dispersion. It is seen that the AT potential gives an overall good description of liquid Kr, though other contributions such as higher order three-body dispersion and exchange terms cannot be ignored.Comment: 11 pages, 3 figures, LaTeX, to appear in J. Chem. Phy

An integrated gene annotation and transcriptional profiling approach towards the full gene content of the Drosophila genome

BACKGROUND: While the genome sequences for a variety of organisms are now available, the precise number of the genes encoded is still a matter of debate. For the human genome several stringent annotation approaches have resulted in the same number of potential genes, but a careful comparison revealed only limited overlap. This indicates that only the combination of different computational prediction methods and experimental evaluation of such in silico data will provide more complete genome annotations. In order to get a more complete gene content of the Drosophila melanogaster genome, we based our new D. melanogaster whole-transcriptome microarray, the Heidelberg FlyArray, on the combination of the Berkeley Drosophila Genome Project (BDGP) annotation and a novel ab initio gene prediction of lower stringency using the Fgenesh software. RESULTS: Here we provide evidence for the transcription of approximately 2,600 additional genes predicted by Fgenesh. Validation of the developmental profiling data by RT-PCR and in situ hybridization indicates a lower limit of 2,000 novel annotations, thus substantially raising the number of genes that make a fly. CONCLUSIONS: The successful design and application of this novel Drosophila microarray on the basis of our integrated in silico/wet biology approach confirms our expectation that in silico approaches alone will always tend to be incomplete. The identification of at least 2,000 novel genes highlights the importance of gathering experimental evidence to discover all genes within a genome. Moreover, as such an approach is independent of homology criteria, it will allow the discovery of novel genes unrelated to known protein families or those that have not been strictly conserved between species