Genetische Analyse von Conductin durch zielgerichtete Mutagenese in der Maus

Die Signalübertragung durch Wnt/beta-Catenin stellt einen der wichtigsten Signalwege während der Embryogenese sowie im adulten Organismus dar. Die homologen Gerüstproteine Conductin und Axin stehen im Mittelpunkt eines zentralen Multiproteinkomplexes, der im Zytoplasma für die Regulation des Wnt/beta-Catenin-Signalwegs verantwortlich ist. In der vorliegenden Arbeit habe ich eine kombinierte genetische Analyse von Conductin und Axin in der Maus durchgeführt. Dazu habe ich mit Hilfe der Technik der homologen Rekombination das Conductin-Gen deletiert und ein Reportergen unter die Kontrolle des endogenen Conductin-Promotors gebracht. Im Weiteren habe ich festgestellt, dass der gemeinsame Verlust von Conductin und einem Axin-Allel (Con-/-;Ax+/-) zu Holoprosenzephalie (HPE) führt, die durch schwere kraniofazialen und Vorderhirndefekten in der Maus charakterisiert ist. Dabei zeigte die detaillierte Analyse eine genetische Interaktion des Wnt-Signalwegs mit dem Shh-Signalweg. Störungen im Shh-Signalweg sind auch beim Menschen für die Ausbildung von HPE verantwortlich gemacht worden. Daneben führt die gleichzeitige Abwesenheit von Conductin und Axin (Con-/-;Ax-/-) zum Verlust der anterior-posterioren Achse in einem frühen Entwicklungsstadium und zum Absterben der Embryonen nach dem Tag 6,5 der Entwicklung. Die vorliegende Arbeit zeigt, wie Unterschiede in der biologischen Bedeutung zweier funktionell redundanter Faktoren mit genetischen Methoden in der Maus aufgeklärt werden können.The Wnt/beta-Catenin pathway represents one of the most important signaling cascades during development as well as in the adult organism. The homologous scaffolding proteins Conductin and Axin are the backbone of a central multi protein complex that is responsible for the tight regulation of the Wnt/beta-Catenin pathway in the cytoplasm. In the present study I have performed a combined genetic analysis of Conductin and Axin in the mouse. To this end I have deleted the Conductin gene by homologous recombination in embryonic stem cells and inserted a reporter gene under the control of the endogenous Conductin promoter. I could show that the simultaneous loss of Conductin and one Axin allele (Con-/-;Ax+/-) causes Holoprosencephaly (HPE), which is characterized by severe craniofacial and forebrain defects in the mouse. The detailed analysis of the mutant mice reveals a genetic interaction of Wnt and Shh signaling and defective Shh signaling has previously been implicated in the formation of HPE in human patients. Moreover, complete absence of both Conductin and Axin (Con-/-;Ax-/-) leads to loss of the anterior-posterior axis early in development and death of the embryos after E6.5. The present study exemplifies how differences in the biological function of two mechanistically redundant factors can be studied by genetic means in the mouse

3R measures in facilities for the production of genetically modified rodents

Sociocultural changes in the human–animal relationship have led to increasing demands for animal welfare in biomedical research. The 3R concept is the basis for bringing this demand into practice: Replace animal experiments with alternatives where possible, Reduce the number of animals used to a scientifically justified minimum and Refine the procedure to minimize animal harm. The generation of gene-modified sentient animals such as mice and rats involves many steps that include various forms of manipulation. So far, no coherent analysis of the application of the 3Rs to gene manipulation has been performed. Here we provide guidelines from the Committee on Genetics and Breeding of Laboratory Animals of the German Society for Laboratory Animal Science to implement the 3Rs in every step during the generation of genetically modified animals. We provide recommendations for applying the 3Rs as well as success/intervention parameters for each step of the process, from experiment planning to choice of technology, harm–benefit analysis, husbandry conditions, management of genetically modified lines and actual procedures. We also discuss future challenges for animal welfare in the context of developing technologies. Taken together, we expect that our comprehensive analysis and our recommendations for the appropriate implementation of the 3Rs to technologies for genetic modifications of rodents will benefit scientists from a wide range of disciplines and will help to improve the welfare of a large number of laboratory animals worldwide

Practical Application of the 3Rs in Rodent Transgenesis

The principles of the 3Rs (replace, reduce, refine), as originally published by Russell and Burch, are internationally acclaimed guidelines for meeting ethical and welfare standards in animal experimentation. Genome manipulation is a standard technique in biomedical research and beyond. The goal of this chapter is to give practical advice on the implementation of the 3Rs in laboratories generating genetically modified rodents. We cover 3R aspects from the planning phase through operations of the transgenic unit to the final genome-manipulated animals. The focus of our chapter is on an easy-to-use, concise protocol that is close to a checklist. While we focus on mice, the proposed methodological concepts can be easily adapted for the manipulation of other sentient animals

3R Measures in facilities for the production of genetically modified rodents

Sociocultural changes in the human-animal relationship have led to increasing demands for animal welfare in biomedical research. The 3R concept is the basis for bringing this demand into practice: Replace animal experiments with alternatives where possible, Reduce the number of animals used to a scientifically justified minimum and Refine the procedure to minimize animal harm. The generation of gene-modified sentient animals such as mice and rats involves many steps that include various forms of manipulation. So far, no coherent analysis of the application of the 3Rs to gene manipulation has been performed. Here we provide guidelines from the Committee on Genetics and Breeding of Laboratory Animals of the German Society for Laboratory Animal Science to implement the 3Rs in every step during the generation of genetically modified animals. We provide recommendations for applying the 3Rs as well as success/intervention parameters for each step of the process, from experiment planning to choice of technology, harm-benefit analysis, husbandry conditions, management of genetically modified lines and actual procedures. We also discuss future challenges for animal welfare in the context of developing technologies. Taken together, we expect that our comprehensive analysis and our recommendations for the appropriate implementation of the 3Rs to technologies for genetic modifications of rodents will benefit scientists from a wide range of disciplines and will help to improve the welfare of a large number of laboratory animals worldwide

Kennzeichnung und Genotypisierung von Nagern

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TT2016 meeting report on the 13th Transgenic Technology meeting in Prague, Czech Republic

International audienc

Negative Feedback Loop of Wnt Signaling through Upregulation of Conductin/Axin2 in Colorectal and Liver Tumors

Activation of Wnt signaling through β-catenin/TCF complexes is a key event in the development of various tumors, in particular colorectal and liver tumors. Wnt signaling is controlled by the negative regulator conductin/axin2/axil, which induces degradation of β-catenin by functional interaction with the tumor suppressor APC and the serine/threonine kinase GSK3β. Here we show that conductin is upregulated in human tumors that are induced by β-catenin/Wnt signaling, i.e., high levels of conductin protein and mRNA were found in colorectal and liver tumors but not in the corresponding normal tissues. In various other tumor types, conductin levels did not differ between tumor and normal tissue. Upregulation of conductin was also observed in the APC-deficient intestinal tumors of Min mice. Inhibition of Wnt signaling by a dominant-negative mutant of TCF downregulated conductin but not the related protein, axin, in DLD1 colorectal tumor cells. Conversely, activation of Wnt signaling by Wnt-1 or dishevelled increased conductin levels in MDA MB 231 and Neuro2A cells, respectively. In time course experiments, stabilization of β-catenin preceded the upregulation of conductin by Wnt-1. These results demonstrate that conductin is a target of the Wnt signaling pathway. Upregulation of conductin may constitute a negative feedback loop that controls Wnt signaling activity

Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits.

Germline transgenesis is an important procedure for functional investigation of biological pathways, as well as for animal biotechnology. We have established a simple, nonviral protocol in three important biomedical model organisms frequently used in physiological studies. The protocol is based on the hyperactive Sleeping Beauty transposon system, SB100X, which reproducibly promoted generation of transgenic founders at frequencies of 50-64, 14-72, and 15% in mice, rats, and rabbits, respectively. The SB100X-mediated transgene integrations are less prone to genetic mosaicism and gene silencing as compared to either the classical pronuclear injection or to lentivirus-mediated transgenesis. The method was successfully applied to a variety of transgenes and animal models, and can be used to generate founders with single-copy integrations. The transposon vector also allows the generation of transgenic lines with tissue-specific expression patterns specified by promoter elements of choice, exemplified by a rat reporter strain useful for tracking serotonergic neurons. As a proof of principle, we rescued an inborn genetic defect in the fawn-hooded hypertensive rat by SB100X transgenesis. A side-by-side comparison of the SB100X- and piggyBac-based protocols revealed that the two systems are complementary, offering new opportunities in genome manipulation.-Katter, K., Geurts, A. M., Hoffmann, O., Mates, L., Landa,V., Hiripi, L., Moreno, C., Lazar, J., Bashir, S., Zidek, V., Popova, E., Jerchow, B., Becker, K., Devaraj, A., Walter, I., Grzybowksi, M., Corbett, M., Rangel Filho, A., Hodges, M. R., Bader, M., Ivics, Z., Jacob, H. J., Pravenec, M., Bosze, Z., Rulicke, T., Izsvak, Z. Transposon-mediated transgenesis, transgenic rescue, and tissue-specific gene expression in rodents and rabbits