Conditional brain-specific knockdown of MAPK using Cre/loxP regulated RNA interference

In the last years, RNA interference (RNAi)-mediated gene knockdown has developed into a routine method to assess gene function in cultured mammalian cells in a fast and easy manner. For the use of this technique in developing or adult mice, short hairpin (sh)RNA vectors expressed stably from the genome are a faster alternative to conventional knockout approaches. Here we describe an advanced strategy for conditional gene knockdown in mice, where we used the Cre/loxP system to activate RNAi in a time and tissue dependent manner in the adult mouse brain. By placing conditional RNAi constructs into the defined genomic Rosa26 locus and by using recombinase mediated cassette exchange (RMCE) instead of laborious homologous recombination, we developed a fast, easy and reproducible approach to assess gene function in adult mice. We applied this technique to three genes of the MAPK signaling pathway—Braf, Mek1 and Mek2—and demonstrate here the potential of this new tool in mouse mutagenesis

Using integrated knowledge acquisition to prepare sophisticated expert plans for their re-use in novel situations

Plans which were constructed by human experts and have been repeatedly executed to the complete satisfaction of some customer in a complex real world domain contain very valuable planning knowledge. In order to make this compiled knowledge re-usable for novel situations, a specific integrated knowledge acquisition method has been developed: First, a domain theory is established from documentation materials or texts, which is then used as the foundation for explaining how the plan achieves the planning goal. Secondly, hierarchically structured problem class definitions are obtained from the practitioners\u27 highlevel problem conceptualizations. The descriptions of these problem classes also provide operationality criteria for the various levels in the hierarchy. A skeletal plan is then constructed for each problem class with an explanation-based learning procedure. These skeletal plans consist of a sequence of general plan elements, so that each plan element can be independently refined. The skeletal plan thus accounts for the interactions between the various concrete operations of the plan at a general level. The complexity of the planning problem is thereby factored in a domain-specific way and the compiled knowledge of sophisticated expert plans can be re-used in novel situations

Uncertainty in coupled models of cyber-physical systems

The development of cyber-physical systems typically involves the association between multiple coupled models that capture different aspects of the system and the environment where it operates. Due to the dynamic aspect of the environment, unexpected conditions and uncertainty may impact the system. In this work, we tackle this problem and propose a taxonomy for characterizing uncertainty in coupled models. Our taxonomy extends existing proposals to cope with the particularities of coupled models in cyber-physical systems. In addition, our taxonomy discusses the notion of uncertainty propagation to other parts of the system. This allows for studying and (in some cases) quantifying the effects of uncertainty on other models in a system even at design time. We show the applicability of our uncertainty taxonomy in real use cases motivated by our envisioned scenario of automotive development

Pulses of enhanced North Pacific Intermediate Water ventilation from the Okhotsk Sea and Bering Sea during the last deglaciation

Under modern conditions only North Pacific Intermediate Water is formed in the Northwest Pacific Ocean. This situation might have changed in the past. Recent studies with General Circulation Models indicate a switch to deep-water formation in the Northwest Pacific during Heinrich Stadial 1 (17.5–15.0 kyr) of the last glacial termination. Reconstructions of past ventilation changes based on paleoceanographic proxy records are still insufficient to test whether a deglacial mode of deep-water formation in the North Pacific Ocean existed. Here we present deglacial ventilation records based on radiocarbon-derived ventilation ages in combination with epibenthic stable carbon isotopes from the Northwest Pacific including the Okhotsk Sea and Bering Sea, the two potential source regions for past North Pacific ventilation changes. Evidence for most rigorous ventilation of the mid-depth North Pacific occurred during Heinrich Stadial 1 and the Younger Dryas, simultaneous to significant reductions in Atlantic Meridional Overturning Circulation. Concurrent changes in δ13C and ventilation ages point to the Okhotsk Sea as driver of millennial-scale changes in North Pacific Intermediate Water ventilation during the last deglaciation. Our records additionally indicate that changes in the δ13C intermediate water (700–1750 m water depth) signature and radiocarbon-derived ventilation ages are in antiphase to those of the deep North Pacific Ocean (>2100 m water depth) during the last glacial termination. Thus, intermediate and deep-water masses of the Northwest Pacific have a differing ventilation history during the last deglaciation

Development of a species-specific RNA polymerase I-based shRNA expression vector

RNA interference (RNAi) can be induced in vitro either by application of synthetic short interfering RNAs (siRNAs), or by intracellular expression of siRNAs or short hairpin RNAs (shRNAs) from transfected vectors. The most widely used promoters for siRNA/shRNA expression are based on polymerase III (Pol III)-dependent transcription. We developed an alternative vector for siRNA/shRNA expression, using a mouse RNA polymerase I (Pol I) promoter. Pol I-dependent transcription serves in cells for production of ribosomal RNA (rRNA), and as such, is ubiquitously and stably active in different cell types. As Pol I-dependent transcription is highly species-specific, Pol I-based system provides an important biosafety advantage with respect to silencing of genes with unknown functions

Enhancement of Precise Gene Editing by the Association of Cas9 With Homologous Recombination Factors

The CRISPR-Cas9 system is used for genome editing in mammalian cells by introducing double-strand breaks (DSBs) which are predominantly repaired via non-homologous end joining (NHEJ) or to lesser extent by homology-directed repair (HDR). To enhance HDR for improving the introduction of precise genetic modifications, we tested fusion proteins of Cas9 nuclease with HDR effectors to enforce their localization at DSBs. Using a traffic-light DSB repair reporter (TLR) system for the quantitative detection of HDR and NHEJ events in human HEK cells we found that Cas9 fusions with CtIP, Rad52, and Mre11, but not Rad51C promote HDR up to twofold in human cells and significantly reduce NHEJ events. We further compared, as an alternative to the direct fusion with Cas9, two components configurations that associate CtIP fusion proteins with a Cas9-SunTag fusion or with guide RNA that includes MS2 binding loops. We found that the Cas9-CtIP fusion and the MS2-CtIP system, but not the SunTag approach increase the ratio of HDR/NHEJ 4.5–6-fold. Optimal results are obtained by the combined use of Cas9-CtIP and MS2-CtIP, shifting the HDR/NHEJ ratio by a factor of 14.9. Thus, our findings provide a simple and effective tool to promote precise gene modifications in mammalian cells

A global view of gene expression in lithium and zinc treated sea urchin embryos: new components of gene regulatory networks

Novel territory-specific markers from the sea urchin Strongylocentrotus purpuratus have been identified using screens for genes that are differentially expressed in lithium-treated embryos, which form an excess of endomesoderm, and in zinc-treated embryos, in which endomesoderm specification is blocked