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

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

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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Abstract C9: MACC1 enhances intestinal tumorigenesis in ApcMin mice.

Abstract Colorectal cancer is one of the most frequently occurring malignancies worldwide. Although about 50% of the patients with colorectal cancer can be cured by surgery and multimodal treatment, the successful outcome of CRC patients is seriously affected by the metastatic dissemination of primary tumors. We have previously identified a novel gene termed MACC1 (metastasis-associated in colon cancer 1), which has a strong prognostic value for colon cancer metastasis, enhancing the metastatic potential of colon cancer cells both in vitro and in vivo. To further understand the role of MACC1 on colorectal carcinogenesis, we generated a transgenic mouse model with intestine-specific overexpression of MACC1 (vil-MACC1). Additionally, we crossed these animals with ApcMin mice to create vil-MACC1/ApcMin mice. Four vil-MACC1 founder lines showed MACC1 transgene mRNA and protein expression in the small intestine and colon, but not in other organs including liver, lung, and kidney. Although the villin promoter drove MACC1 expression through the whole vertical (crypt-villus) and horizontal (duodenum-colon) intestinal axes, higher levels of transgenic MACC1 were detected in the small intestine as compared with the colon, and in the villi as compared with the crypts. Histopathological analysis of the intestine of 3-months old vil-MACC1 mice showed no abnormalities as compared with age-matched wild-type littermate controls. Similar analysis is currently being performed in older animals (≥ 1 year old). In line with what we observed in the vil-MACC1 animals, the vil-MACC1/ApcMin mice displayed a similar pattern of MACC1 mRNA and protein expression throughout the whole small intestine and colon. Remarkably, transgenic overexpression of MACC1 in the intestine of vil-MACC1/ApcMin mice significantly increased the total number of adenomas, as compared with ApcMin littermate controls (P = 0.0146). Moreover, the small intestine of vil-MACC1/ApcMin mice displayed an increased number of large-sized adenomas (diameter ≥ 3 mm; P = 0.0033). Additionally, we also observed a slight increase in spleen size in vil-MACC1/ApcMin mice, as compared with ApcMin controls. Despite the increased tumor burden on vil-MACC1/ApcMin mice, we didn't observe a significant difference on survival between the two animal groups. We report here for the first time the generation of a mouse model with genetically engineered MACC1 expression. More importantly, our current findings demonstrate that MACC1 is instrumental for intestinal adenoma formation and development in vivo. These results further strengthen the importance of MACC1 for intestinal tumorigenesis and metastasis, underlying its potential as a new target for colorectal cancer treatment. Funded by the Alexander von Humboldt Foundation, the Wilhelm Sander Foundation, the Preclinical Comprehensive Cancer Center and the German Cancer Consortium Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C9. Citation Format: Clara Lemos, Cynthia Voss, Boris Jerchow, Wolfram Haider, Ulrike Stein. MACC1 enhances intestinal tumorigenesis in ApcMin mice. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C9.</jats:p