Bypass of lethality with mosaic mice generated by Cre–loxP-mediated recombination

AbstractBackground The analysis of gene function based on the generation of mutant mice by homologous recombination in embryonic stem cells is limited if gene disruption results in embryonic lethality. Mosaic mice, which contain a certain proportion of mutant cells in all organs, allow lethality to be circumvented and the potential of mutant cells to contribute to different cell lineages to be analyzed. To generate mosaic animals, we used the bacteriophage P1-derived Cre–loxP recombination system, which allows gene alteration by Cre-mediated deletion of loxP-flanked gene segments.Results We generated nestin–cre transgenic mouse lines, which expressed the Cre recombinase under the control of the rat nestin promoter and its second intron enhancer. In crosses to animals carrying a loxP-flanked target gene, partial deletion of the loxP-flanked allele occurred before day 10.5 post coitum and was detectable in all adult organs examined, including germ-line cells. Using this approach, we generated mosaic mice containing cells deficient in the γ-chain of the interleukin-2 receptor (IL-2Rγ); in these animals, the IL-2Rγ-deficient cells were underrepresented in the thymus and spleen. Because mice deficient in DNA polymerase β die perinatally, we studied the effects of DNA polymerase β deficiency in mosaic animals. We found that some of the mosaic polymerase β-deficient animals were viable, but were often reduced in size and weight. The fraction of DNA polymerase β-deficient cells in mosaic embryos decreased during embryonic development, presumably because wild-type cells had a competitive advantage.Conclusions The nestin–cre transgenic mice can be used to generate mosaic animals in which target genes are mutated by Cre-mediated recombination of loxP-flanked target genes. By using mosaic animals, embryonic lethality can be bypassed and cell lineages for whose development a given target gene is critical can be identified. In the case of DNA polymerase β, deficient cells are already selected against during embryonic development, demonstrating the general importance of this protein in multiple cell types

Maintenance of the Specification of the Anterior Definitive Endoderm and Forebrain Depends on the Axial Mesendoderm: A Study Using HNF3β/Foxa2 Conditional Mutants

AbstractIn mouse embryo, the early induction of the head region depends on signals from the anterior visceral endoderm (AVE) and the anterior primitive streak. Subsequently, node derivatives, including anterior definitive endoderm and axial mesendoderm, are thought to play a role in the maintenance and elaboration of anterior neural character. Foxa2 encodes a winged-helix transcription factor expressed in signaling centers required for head development, including the AVE, anterior primitive streak, anterior definitive endoderm, and axial mesendoderm. To address Foxa2 function during formation of the head, we used conditional mutants in which Foxa2 function is preserved in extraembryonic tissues during early embryonic stages and inactivated in embryonic tissues after the onset of gastrulation. In Foxa2 conditional mutants, the anterior neural plate and anterior definitive endoderm were initially specified. In contrast, the axial mesendoderm failed to differentiate. At later stages, specification of the anterior neural plate and anterior definitive endoderm was shown to be labile. As a result, head truncations were observed in Foxa2 conditional mutants. Our results therefore indicate that anterior definitive endoderm alone is not sufficient to maintain anterior head specification and that an interaction between the axial mesendoderm and the anterior definitive endoderm is required for proper specification of the endoderm. Foxa2 therefore plays an integral role in the formation of axial mesendoderm, which is required to maintain the specification of the forebrain and the anterior definitive endoderm

Game changers in science and technology - now and beyond

The recent devastating pandemic has drastically reminded humanity of the importance of constant scientific and technological progress. A strong interdisciplinary dialogue between academic and industrial scientists of various specialties, entrepreneurs, managers and the public is paramount in triggering new breakthrough ideas which often emerge at the interface of disciplines. The following sections, compiled by a highly diverse group of authors, are summarizing recently achieved game-changing leaps in science and technology. The game-changers range from paradigm shifts in scientific theories to make impact over several decades to game-changers that have the potential to change our everyday lives tomorrow. The paper is an interdisciplinary dialogue of relevance for academic interdisciplinary thinkers, large corporations' strategic planners, and top executives alike; it provides a glimpse into what further breakthroughs the future may hold and thereby intends to spark new ideas with its readers

CACHE (Critical Assessment of Computational Hit-finding Experiments): A public-private partnership benchmarking initiative to enable the development of computational methods for hit-finding

Computational approaches in drug discovery and development hold great promise, with artificial intelligence methods undergoing widespread contemporary use, but the experimental validation of these new approaches is frequently inadequate. We are initiating Critical Assessment of Computational Hit-finding Experiments (CACHE) as a public benchmarking project that aims to accelerate the development of small molecule hit-finding algorithms by competitive assessment. Compounds will be identified by participants using a wide range of computational methods for dozens of protein targets selected for different types of prediction scenarios, as well as for their potential biological or pharmaceutical relevance. Community-generated predictions will be tested centrally and rigorously in an experimental hub(s), and all data, including the chemical structures of experimentally tested compounds, will be made publicly available without restrictions. The ability of a range of computational approaches to find novel compounds will be evaluated, compared, and published. The overarching goal of CACHE is to accelerate the development of computational chemistry methods by providing rapid and unbiased feedback to those developing methods, with an ancillary and valuable benefit of identifying new compound-protein binding pairs for biologically interesting targets. The initiative builds on the power of crowd sourcing and expands the open science paradigm for drug discovery