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Saragusty J.,The Leibniz Institute for Zoo and Wildlife ResearchBerlinGermany | Diecke S.,Max Delbruck Center for Molecular Medicine | Drukker M.,Institute of Stem Cell Research | Durrant B.,San Diego Zoo Institute for Conservation ResearchEscondido | And 11 more authors.
Zoo Biology | Year: 2016

With only three living individuals left on this planet, the northern white rhinoceros (Ceratotherium simum cottoni) could be considered doomed for extinction. It might still be possible, however, to rescue the (sub)species by combining novel stem cell and assisted reproductive technologies. To discuss the various practical options available to us, we convened a multidisciplinary meeting under the name "Conservation by Cellular Technologies." The outcome of this meeting and the proposed road map that, if successfully implemented, would ultimately lead to a self-sustaining population of an extremely endangered species are outlined here. The ideas discussed here, while centered on the northern white rhinoceros, are equally applicable, after proper adjustments, to other mammals on the brink of extinction. Through implementation of these ideas we hope to establish the foundation for reversal of some of the effects of what has been termed the sixth mass extinction event in the history of Earth, and the first anthropogenic one. Zoo Biol. XX:XX-XX, 2016. © 2016 Wiley Periodicals, Inc.

Chau Y.-Y.,Western Research Institute | Brownstein D.,Queens Medical Research Institute | Mjoseng H.,University of Edinburgh | Lee W.-C.,Chang Gung University | And 13 more authors.
PLoS Genetics | Year: 2011

There is much interest in the mechanisms that regulate adult tissue homeostasis and their relationship to processes governing foetal development. Mice deleted for the Wilms' tumour gene, Wt1, lack kidneys, gonads, and spleen and die at mid-gestation due to defective coronary vasculature. Wt1 is vital for maintaining the mesenchymal-epithelial balance in these tissues and is required for the epithelial-to-mesenchyme transition (EMT) that generates coronary vascular progenitors. Although Wt1 is only expressed in rare cell populations in adults including glomerular podocytes, 1% of bone marrow cells, and mesothelium, we hypothesised that this might be important for homeostasis of adult tissues; hence, we deleted the gene ubiquitously in young and adult mice. Within just a few days, the mice suffered glomerulosclerosis, atrophy of the exocrine pancreas and spleen, severe reduction in bone and fat, and failure of erythropoiesis. FACS and culture experiments showed that Wt1 has an intrinsic role in both haematopoietic and mesenchymal stem cell lineages and suggest that defects within these contribute to the phenotypes we observe. We propose that glomerulosclerosis arises in part through down regulation of nephrin, a known Wt1 target gene. Protein profiling in mutant serum showed that there was no systemic inflammatory or nutritional response in the mutant mice. However, there was a dramatic reduction in circulating IGF-1 levels, which is likely to contribute to the bone and fat phenotypes. The reduction of IGF-1 did not result from a decrease in circulating GH, and there is no apparent pathology of the pituitary and adrenal glands. These findings 1) suggest that Wt1 is a major regulator of the homeostasis of some adult tissues, through both local and systemic actions; 2) highlight the differences between foetal and adult tissue regulation; 3) point to the importance of adult mesenchyme in tissue turnover. © 2011 Chau et al.

Hesse M.,University of Bonn | Raulf A.,University of Bonn | Pilz G.-A.,Institute of Stem Cell Research | Haberlandt C.,University of Bonn | And 14 more authors.
Nature Communications | Year: 2012

Current approaches to monitor and quantify cell division in live cells, and reliably distinguish between acytokinesis and endoreduplication, are limited and complicate determination of stem cell pool identities. Here we overcome these limitations by generating an in vivo reporter system using the scaffolding protein anillin fused to enhanced green fluorescent protein, to provide high spatiotemporal resolution of mitotic phase. This approach visualizes cytokinesis and midbody formation as hallmarks of expansion of stem and somatic cells, and enables distinction from cell cycle variations. High-resolution microscopy in embryonic heart and brain tissues of enhanced green fluorescent protein-anillin transgenic mice allows live monitoring of cell division and quantitation of cell cycle kinetics. Analysis of cell division in hearts post injury shows that border zone cardiomyocytes in the infarct respond with increasing ploidy, but not cell division. Thus, the enhanced green fluorescent protein-anillin system enables monitoring and measurement of cell division in vivo and markedly simplifies in vitro analysis in fixed cells. © 2012 Macmillan Publishers Limited. All rights reserved.

Petrezselyova S.,Academy of Sciences of the Czech Republic | Kinsky S.,Academy of Sciences of the Czech Republic | Truban D.,Academy of Sciences of the Czech Republic | Sedlacek R.,Academy of Sciences of the Czech Republic | And 2 more authors.
Cellular and Molecular Biology Letters | Year: 2015

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusion, our approach facilitates programmed integration of exogenous DNA sequences at a target locus and thus could serve as a basis for more sophisticated genome engineering approaches, such as generation of reporters and conditional knock-out alleles. © 2016 University of Wroclaw, Poland.

Burtscher I.,Helmholtz Center Munich | Burtscher I.,Institute of Stem Cell Research | Barkey W.,Helmholtz Center Munich | Barkey W.,Institute of Stem Cell Research | And 2 more authors.
Genesis | Year: 2013

The Foxa2-winged helix/forkhead box transcription factor (TF) is absolutely required for endoderm formation and organogenesis. Foxa2 plays essential roles during lung, liver, pancreas, and gastrointestinal tract development and regulates cell-type specific programs in the adult organism. To specifically address Foxa2 function during organ development and homeostasis, we generated a Foxa2-Venus fusion (FVF) reporter protein by gene targeting in embryonic stem (ES) cells. The FVF knock-in reporter is expressed under endogenous Foxa2 control and the fluorescent protein fusion does not interfere with TF function, as homozygous mice are viable and fertile. Moreover, the FVF protein localizes to the nucleus, associates with chromatin during mitosis, and reflects the endogenous Foxa2 protein distribution pattern in several tissues in heterozygous animals. Importantly, live-cell imaging on single-cell level of the FVF and Sox17-Cherry fusion double knock-in reporter ES cell line reveals the dynamics of endoderm TF accumulation during ES cell differentiation. The FVF reporter also allowed us to identify the endoderm progenitors during gastrulation and to visualize the different branching morphogenesis modes of the lung and pancreas epithelium in ex vivo embryo and organ cultures. In summary, the generation of the FVF reporter line adds an important new tool to visualize and analyse endoderm-derived organ development and homeostasis on the cellular and molecular level. © 2013 Wiley Periodicals, Inc.

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