TY - JOUR A1 - Yan, Yan A1 - Hong, Ni A1 - Chen, Tiansheng A1 - Li, Mingyou A1 - Wang, Tiansu A1 - Guan, Guijun A1 - Qiao, Yongkang A1 - Chen, Songlin A1 - Schartl, Manfred A1 - Li, Chang-Ming A1 - Hong, Yunhan T1 - p53 Gene Targeting by Homologous Recombination in Fish ES Cells JF - PLoS One N2 - Background: Gene targeting (GT) provides a powerful tool for the generation of precise genetic alterations in embryonic stem (ES) cells to elucidate gene function and create animal models for human diseases. This technology has, however, been limited to mouse and rat. We have previously established ES cell lines and procedures for gene transfer and selection for homologous recombination (HR) events in the fish medaka (Oryzias latipes). Methodology and Principal Findings: Here we report HR-mediated GT in this organism. We designed a GT vector to disrupt the tumor suppressor gene p53 (also known as tp53). We show that all the three medaka ES cell lines, MES1 similar to MES3, are highly proficient for HR, as they produced detectable HR without drug selection. Furthermore, the positive-negative selection (PNS) procedure enhanced HR by similar to 12 folds. Out of 39 PNS-resistant colonies analyzed, 19 (48.7%) were positive for GT by PCR genotyping. When 11 of the PCR-positive colonies were further analyzed, 6 (54.5%) were found to be bona fide homologous recombinants by Southern blot analysis, sequencing and fluorescent in situ hybridization. This produces a high efficiency of up to 26.6% for p53 GT under PNS conditions. We show that p53 disruption and long-term propagation under drug selection conditions do not compromise the pluripotency, as p53-targeted ES cells retained stable growth, undifferentiated phenotype, pluripotency gene expression profile and differentiation potential in vitro and in vivo. Conclusions: Our results demonstrate that medaka ES cells are proficient for HR-mediated GT, offering a first model organism of lower vertebrates towards the development of full ES cell-based GT technology. KW - mouse KW - in-vitro KW - drug selection KW - chimera formation KW - medakafish oryzias latipes KW - embryonic stem-cells KW - zebrafish KW - differentiation KW - cultures KW - pluripotency Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-133416 VL - 8 IS - 3 ER - TY - JOUR A1 - Degenkolbe, Elisa A1 - König, Jana A1 - Zimmer, Julia A1 - Walther, Maria A1 - Reißner, Carsten A1 - Nickel, Joachim A1 - Plöger, Frank A1 - Raspopovic, Jelena A1 - Sharpe, James A1 - Dathe, Katharina A1 - Hecht, Jacqueline T. A1 - Mundlos, Stefan A1 - Doelken, Sandra C. A1 - Seemann, Petra T1 - A GDF5 Point Mutation Strikes Twice - Causing BDA1 and SYNS2 JF - PLOS Genetics N2 - Growth and Differentiation Factor 5 (GDF5) is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP) family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA) and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2). Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1) caused by a single point mutation in GDF5 (p.W414R). Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5 W-414R variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C) or SYNS2 (p.E491K) revealed a dual pathomechanism characterized by a gain-and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG) leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A), is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA. KW - dominant-negative mutatio KW - morphogenetic protein receptors KW - brachtydacyly type A2 KW - BMP KW - gene encoding noggin KW - growth factor beta KW - signal tranduction KW - molecular mechanism KW - crystal-structure KW - differentiation Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127556 SN - 1553-7404 VL - 9 IS - 10 ER - TY - JOUR A1 - Nolte, Thomas A1 - Zadeh-Khorasani, Maryam A1 - Safarov, Orkhan A1 - Rueff, Franziska A1 - Varga, Rita A1 - Herbach, Nadja A1 - Wanke, Rüdiger A1 - Wollenberg, Andreas A1 - Mueller, Thomas A1 - Gropp, Roswitha A1 - Wolf, Eckhard A1 - Siebeck, Matthias T1 - Induction of oxazolone-mediated features of atopic dermatitis in NOD-scid IL2R \(γ^{null}\) mice engrafted with human peripheral blood mononuclear cells JF - Disease Models & Mechanisms N2 - Animal models mimicking human diseases have been used extensively to study the pathogenesis of autoimmune diseases and the efficacy of potential therapeutics. They are, however, limited with regard to their similarity to the human disease and cannot be used if the antagonist and its cognate receptor require high similarity in structure or binding. Here, we examine the induction of oxazolone-mediated features of atopic dermatitis (AD) in NOD-scid IL2R \(γ^{null}\) mice engrafted with human peripheral blood mononuclear cells (PBMC). The mice developed the same symptoms as immunocompetent BALB/c mice. Histological alterations induced by oxazolone were characterized by keratosis, epithelial hyperplasia and influx of inflammatory cells into the dermis and epidermis. The cellular infiltrate was identified as human leukocytes, with T cells being the major constituent. In addition, oxazolone increased human serum IgE levels. The response, however, required the engraftment of PBMC derived from patients suffering from AD, which suggests that this model reflects the immunological status of the donor. Taken together, the model described here has the potential to evaluate the efficacy of therapeutics targeting human lymphocytes in vivo and, in addition, might be developed further to elucidate molecular mechanisms inducing and sustaining flares of the disease. KW - expression KW - model KW - pbl KW - differentiation KW - mechanisms KW - antagonists KW - gamma KW - human interleukin-4 KW - rheumatoid-arthritis KW - T-cells Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122189 VL - 6 ER - TY - THES A1 - Raaijmakers, Nadja T1 - Osteoporoseprophylaxe mit pflanzlichen Wirkstoffen T1 - Osteoporosis prevention with plant ingredients N2 - Osteoporose ist einer der häufigsten Knochenerkrankungen im fortschreitenden Alter und zählt, aufgrund der damit verbundenen hohen direkten und indirekten Behandlungskosten, zu einer der zehn wichtigsten volkswirtschaftlichen Krankheiten. Die Behandlung der Osteoporose ist langjährig und umfasst eine medikamentöse Therapie auf der Basis einer „knochengesunden“ Lebensweise hinsichtlich Ernährung und Bewegung. Im Rahmen von Untersuchungen zur Linderung von postmenopausalen Beschwerden, zeigte ein Extrakt der Pflanze Cimicifuga racemosa Potential zu osteoprotektiver Wirksamkeit und rückte somit in den Fokus für eine mögliche Anwendung in der Therapie und Prophylaxe von Osteoporose. Das Ziel der vorliegenden Arbeit war es daher, in enger Zusammenarbeit mit der Bionorica SE, welche für die Aufreinigung und Fraktionierung des Pflanzenextraktes zuständig war, und mit der Arbeitsgruppe um Prof. Wuttke, welche parallele Rattenstudien durchführte, Methoden anzuwenden, mit denen osteoprotektive Wirksamkeiten nachgewiesen und auf einzelne Fraktionen des Extraktes limitiert werden können. ... N2 - Osteoporosis is one of the most common bone diseases in advancing age and is, due to the associated high direct and indirect costs of treatment, one of the ten most important economic diseases. The treatment of osteoporosis lasts for many years and covers a drug therapy based on a "bone-healthy" lifestyle regarding diet and exercise. As part of investigations for the relief of postmenopausal symptoms Cimicifuga racemosa showed potential to osteoprotective effectiveness and thus the focus places special emphasis to the potential application in the treatment and prevention of osteoporosis. The aim of the present study was therefore, in close cooperation with the Bionorica SE, which was responsible for the purification and fractionation of the plant extract and the research group of Professor Wuttke, which conduct parallel rat studies, to investigate methods which demonstrate osteoprotective efficacies and may be limited to individual fractions of the extract. ... KW - Osteoporose KW - Prävention KW - Traubensilberkerze KW - humane mesenchymale Stammzellen KW - Differenzierung KW - osteoprosis KW - prevention KW - black cohosh KW - human mesenchymal stem cells KW - differentiation KW - Arzneimittelforschung KW - Phytopharmakon Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-83341 ER -