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.

Guidelines on severity assessment and classification of genetically altered mouse and rat lines.

Genetic alterations can unpredictably compromise the wellbeing of animals. Thus, more or less harmful phenotypes might appear in the animals used in research projects even when they are not subjected to experimental treatments. The severity classification of suffering has become an important issue since the implementation of Directive 2010/63/EU on the protection of animals used for scientific purposes. Accordingly, the breeding and maintenance of genetically altered (GA) animals which are likely to develop a harmful phenotype has to be authorized. However, a determination of the degree of severity is rather challenging due to the large variety of phenotypes. Here, the Working Group of Berlin Animal Welfare Officers (WG Berlin AWO) provides field-tested guidelines on severity assessment and classification of GA rodents. With a focus on basic welfare assessment and severity classification we provide a list of symptoms that have been classified as non-harmful, mild, moderate or severe burdens. Corresponding monitoring and refinement strategies as well as specific housing requirements have been compiled and are strongly recommended to improve hitherto applied breeding procedures and conditions. The document serves as a guide to determine the degree of severity for an observed phenotype. The aim is to support scientists, animal care takers, animal welfare bodies and competent authorities with this task, and thereby make an important contribution to a European harmonization of severity assessments for the continually increasing number of GA rodents.

MACC1 Induces Tumor Progression in Transgenic Mice and Colorectal Cancer Patients via Increased Pluripotency Markers Nanog and Oct4.

PURPOSE: We have previously identified the gene MACC1 as a strong prognostic biomarker for colorectal cancer metastasis and patient survival. Here, we report for the first time the generation of transgenic mouse models for MACC1. EXPERIMENTAL DESIGN: We generated mice with transgenic overexpression of MACC1 in the intestine driven by the villin promoter (vil-MACC1) and crossed them with Apc(Min) mice (vil-MACC1/Apc(Min)). RESULTS: vil-MACC1/Apc(Min) mice significantly increased the total number of tumors (P = 0.0056). This was particularly apparent in large tumors (≥3-mm diameter; P = 0.0024). A detailed histopathologic analysis of these lesions demonstrated that the tumors from the vil-MACC1/Apc(Min) mice had a more invasive phenotype and, consequently, showed a significantly reduced survival time than Apc(Min) mice (P = 0.03). Molecular analysis revealed an increased Wnt and pluripotency signaling in the tumors of vil-MACC1/Apc(Min) mice. Specifically, we observed a prominent upregulation of the pluripotency markers Oct4 and Nanog in these tumors compared with Apc(Min) controls. Finally, we could also validate that Oct4 and Nanog are regulated by MACC1 in vitro and strongly correlate with MACC1 levels in a cohort of 60 tumors of colorectal cancer patients (r = 0.7005 and r = 0.6808, respectively; P > 0.0001 and P > 0.0002, respectively). CONCLUSIONS: We provide proof of principle that MACC1-induced tumor progression in colorectal cancer acts, at least in part, via the newly discovered MACC1/Nanog/Oct4 axis. These findings might have important implications for the design of novel therapeutic intervention strategies to restrict tumor progression. Clin Cancer Res; 22(11); 2812-24. ©2016 AACR.

Global increase of p16INK4a in APC-deficient mouse liver drives clonal growth of p16INK4a-negative tumors.

UNLABELLED: Reduction of ß-catenin (CTNNB1) destroying complex components, for example, adenomatous polyposis coli (APC), induces ß-catenin signaling and subsequently triggers activation of genes involved in proliferation and tumorigenesis. Though diminished expression of APC has organ-specific and threshold-dependent influence on the development of liver tumors in mice, the molecular basis is poorly understood. Therefore, a detailed investigation was conducted to determine the underlying mechanism in the development of liver tumors under reduced APC levels. Mouse liver at different developmental stages was analyzed in terms of ß-catenin target genes including Cyp2e1, Glul, and Ihh using real-time RT-PCR, reporter gene assays, and immunohistologic methods with consideration of liver zonation. Data from human livers with mutations in APC derived from patients with familial adenomatous polyposis (FAP) were also included. Hepatocyte senescence was investigated by determining p16(INK4a) expression level, presence of senescence-associated ß-galactosidase activity, and assessing ploidy. A ß-catenin activation of hepatocytes does not always result in ß-catenin positive but unexpectedly also in mixed and ß-catenin-negative tumors. In summary, a senescence-inducing program was found in hepatocytes with increased ß-catenin levels and a positive selection of hepatocytes lacking p16(INK4a), by epigenetic silencing, drives the development of liver tumors in mice with reduced APC expression (Apc(580S) mice). The lack of p16(INK4a) was also detected in liver tumors of mice with triggers other than APC reduction. IMPLICATIONS: Epigenetic silencing of p16(Ink4a) in selected liver cells bypassing senescence is a general principle for development of liver tumors with ß-catenin involvement in mice independent of the initial stimulus.

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.

Reciprocal requirements for EDA/EDAR/NF-kappaB and Wnt/beta-catenin signaling pathways in hair follicle induction.

Wnt/beta-catenin and NF-kappaB signaling mechanisms provide central controls in development and disease, but how these pathways intersect is unclear. Using hair follicle induction as a model system, we show that patterning of dermal Wnt/beta-catenin signaling requires epithelial beta-catenin activity. We find that Wnt/beta-catenin signaling is absolutely required for NF-kappaB activation, and that Edar is a direct Wnt target gene. Wnt/beta-catenin signaling is initially activated independently of EDA/EDAR/NF-kappaB activity in primary hair follicle primordia. However, Eda/Edar/NF-kappaB signaling is required to refine the pattern of Wnt/beta-catenin activity, and to maintain this activity at later stages of placode development. We show that maintenance of localized expression of Wnt10b and Wnt10a requires NF-kappaB signaling, providing a molecular explanation for the latter observation, and identify Wnt10b as a direct NF-kappaB target. These data reveal a complex interplay and interdependence of Wnt/beta-catenin and EDA/EDAR/NF-kappaB signaling pathways in initiation and maintenance of primary hair follicle placodes.

Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates.

The Sleeping Beauty (SB) transposon is a promising technology platform for gene transfer in vertebrates; however, its efficiency of gene insertion can be a bottleneck in primary cell types. A large-scale genetic screen in mammalian cells yielded a hyperactive transposase (SB100X) with approximately 100-fold enhancement in efficiency when compared to the first-generation transposase. SB100X supported 35-50% stable gene transfer in human CD34(+) cells enriched in hematopoietic stem or progenitor cells. Transplantation of gene-marked CD34(+) cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution. In addition, SB100X supported sustained (>1 year) expression of physiological levels of factor IX upon transposition in the mouse liver in vivo. Finally, SB100X reproducibly resulted in 45% stable transgenesis frequencies by pronuclear microinjection into mouse zygotes. The newly developed transposase yields unprecedented stable gene transfer efficiencies following nonviral gene delivery that compare favorably to stable transduction efficiencies with integrating viral vectors and is expected to facilitate widespread applications in functional genomics and gene therapy.

Transgenic Technology meeting 2008 in Toronto, Canada: a meeting report.

In a history that goes back to 1999, the Transgenic Technology meetings started out in Sweden and over the years began to attract a growing community of technicians and researchers mainly from Europe. As the meetings started to attract an expanding worldwide audience, the community decided to found the International Society for Transgenic Technologies at the Barcelona meeting in 2005. The 2007 convention was held at Brisbane, Australia, and in 2008, the 8th Transgenic Technology meeting was held for the second time on behalf of ISTT and for the second time outside of Europe in Toronto, Canada. Due to its excellent program with over 400 participants the meeting was able to attract the highest number of delegates of all past TT meetings. With extended times for plenary discussions about technical and organizational aspects, as well as top level scientific presentations, both technicians and scientists enjoyed this as an extremely fruitful meeting from which they could take home solutions for daily routines as well as new insights and ideas for coming projects.

Aberrant Wnt/beta-catenin signaling can induce chromosomal instability in colon cancer.

Chromosomal instability (CIN), a hallmark of most colon tumors, may promote tumor progression by increasing the rate of genetic aberrations. CIN is thought to arise as a consequence of improper mitosis and spindle checkpoint activity, but its molecular basis remains largely elusive. The majority of colon tumors develop because of mutations in the tumor suppressor APC that lead to Wnt/beta-catenin signaling activation and subsequent transcription of target genes, including conductin/AXIN2. Here we demonstrate that Wnt/beta-catenin signaling causes CIN via up-regulation of conductin. Human colon tumor samples with CIN show significantly higher expression of conductin than those without. Conductin is up-regulated during mitosis, localizes along the mitotic spindles of colon cancer cells, and binds to polo-like kinase 1. Ectopic expression of conductin or its up-regulation through small interfering RNA-mediated knock-down of APC leads to CIN in chromosomally stable colon cancer cells. High conductin expression compromises the spindle checkpoint, and this requires localized polo-like kinase 1 activity. Knock-down of conductin by small interfering RNA in colon carcinoma cells or gene ablation in mouse embryo fibroblasts enforces the checkpoint.

The role of Axin2 in calvarial morphogenesis and craniosynostosis.

Axin1 and its homolog Axin2/conductin/Axil are negative regulators of the canonical Wnt pathway that suppress signal transduction by promoting degradation of beta-catenin. Mice with deletion of Axin1 exhibit defects in axis determination and brain patterning during early embryonic development. We show that Axin2 is expressed in the osteogenic fronts and periosteum of developing sutures during skull morphogenesis. Targeted disruption of Axin2 in mice induces malformations of skull structures, a phenotype resembling craniosynostosis in humans. In the mutants, premature fusion of cranial sutures occurs at early postnatal stages. To elucidate the mechanism of craniosynostosis, we studied intramembranous ossification in Axin2-null mice. The calvarial osteoblast development is significantly affected by the Axin2 mutation. The Axin2 mutant displays enhanced expansion of osteoprogenitors, accelerated ossification, stimulated expression of osteogenic markers and increases in mineralization. Inactivation of Axin2 promotes osteoblast proliferation and differentiation in vivo and in vitro. Furthermore, as the mammalian skull is formed from cranial skeletogenic mesenchyme, which is derived from mesoderm and neural crest, our data argue for a region-specific effect of Axin2 on neural crest dependent skeletogenesis. The craniofacial anomalies caused by the Axin2 mutation are mediated through activation of beta-catenin signaling, suggesting a novel role for the Wnt pathway in skull morphogenesis.

LRP2/megalin is required for patterning of the ventral telencephalon.

Megalin is a low-density lipoprotein receptor-related protein (LRP2) expressed in the neuroepithelium and the yolk sac of the early embryo. Absence of megalin expression in knockout mice results in holoprosencephaly, indicating an essential yet unidentified function in forebrain development. We used mice with complete or conditional megalin gene inactivation in the embryo to demonstrate that expression of megalin in the neuroepithelium but not in the yolk sac is crucial for brain development. During early forebrain development, megalin deficiency leads to an increase in bone morphogenic protein (Bmp) 4 expression and signaling in the rostral dorsal neuroepithelium, and a subsequent loss of sonic hedgehog (Shh) expression in the ventral forebrain. As a consequence of absent SHH activity, ventrally derived oligodendroglial and interneuronal cell populations are lost in the forebrain of megalin-/- embryos. Similar defects are seen in models with enhanced signaling through BMPs, central regulators of neural tube patterning. Because megalin mediates endocytic uptake and degradation of BMP4, these findings indicate a role for megalin in neural tube specification, possibly by acting as BMP4 clearance receptor in the neuroepithelium.

Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors.

Activation of Wnt signaling through beta-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 beta-catenin by functional interaction with the tumor suppressor APC and the serine/threonine kinase GSK3beta. Here we show that conductin is upregulated in human tumors that are induced by beta-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 beta-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.