Why serology just is not enough: Strategic parvovirus risk assessment using a novel qPCR assay.

Health monitoring of laboratory rodents not only improves animal health but also enhances the validity of animal experiments. In particular, infections of laboratory animals with murine parvoviruses influence biomedical research data. Despite strict barrier housing, prevalence remains high in animal facilities, leading to increased risk of parvovirus introduction after the import of contaminated mice. Unfortunately, hygienic rederivation can be challenging, since gametes often contain residual virus material. Consequently, the process has to be closely monitored with highly sensitive diagnostic methods to verify parvovirus decontamination of the rederived progeny. However, diagnostic sensitivity of traditional methods is often low and requires testing of large animal cohorts. Therefore, we aimed to develop a powerful quantitative real-time polymerase chain reaction (qPCR) assay for the fast and reliable detection of murine parvoviruses in different sample materials. We validated the assay within an infection experiment and systematically analysed various animal-derived and environmental sample materials. We further developed a strategic risk assessment procedure for parvovirus monitoring after embryo transfer. Our novel qPCR assay reliably detected parvovirus DNA in a broad variety of sample materials, with environmental samples dominating in the acute phase of infection, whereas animal-derived samples were more suitable to detect low virus loads in the chronic phase. Here, the assay served as a highly sensitive screening method for parvovirus contamination in mouse colonies, requiring significantly lower sample sizes than traditional methods like conventional PCR and serology. Thus, the use of our novel qPCR assay substantially improves parvovirus diagnostics, enhancing research validity according to the 6Rs.

A combination of genetics and microbiota influences the severity of the obesity phenotype in diet-induced obesity.

Obesity has emerged as a major global health problem and is associated with various diseases, such as metabolic syndrome, type 2 diabetes mellitus, and cardiovascular diseases. The inbred C57BL/6 mouse strain is often used for various experimental investigations, such as metabolic research. However, over time, genetically distinguishable C57BL/6 substrains have evolved. The manifestation of genetic alterations has resulted in behavioral and metabolic differences. In this study, a comparison of diet-induced obesity in C57BL/6JHanZtm, C57BL/6NCrl and C57BL/6 J mice revealed several metabolic and immunological differences such as blood glucose level and cytokine expression, respectively, among these C57BL/6 substrains. For example, C57BL/6NCrl mice developed the most pronounced adiposity, whereas C57BL/6 J mice showed the highest impairment in glucose tolerance. Moreover, our results indicated that the immunological phenotype depends on the intestinal microbiota, as the cell subset composition of the colon was similar in obese ex-GF B6NRjB6JHanZtm and obese B6JHanZtm mice. Phenotypic differences between C57BL/6 substrains are caused by a complex combination of genetic and microbial alterations. Therefore, in performing metabolic research, considering substrain-specific characteristics, which can influence the course of study, is important. Moreover, for unbiased comparison of data, the entire strain name should be shared with the scientific community.

One for two: A novel and highly sensitive virulence factor-based quantitative polymerase chain reaction assay for the simultaneous detection of Rodentibacter pneumotropicus and Rodentibacter heylii in environmental sample material.

Hygienic monitoring of laboratory rodents has focused more and more on the analysis of environmental sample material by quantitative polymerase chain reaction (qPCR) assays. This approach requires profound knowledge of specific genetic sequences of the agents to be monitored and the assays need to be permanently adapted to take the latest research into account. [Pasteurella] pneumotropica was recently reclassified into the new genus Rodentibacter, with Rodentibacter (R.) pneumotropicus and R. heylii as the most commonly detected species in laboratory mouse colonies. This study aimed at the development of a specific qPCR assay for the simultaneous detection of both agents. A novel primer probe set, based on detection of the specific virulence factor' 'inclusion body protein A' gene (ibpA), was confirmed by testing the assay on currently described Rodentibacter type species and other Pasteurellaceae. Furthermore, it was validated within four different barrier units and results were compared with the cultural analysis of sentinel mice. The assay was suitable to specifically detect R. pneumotropicus and R. heylii and discriminate them from other murine Rodentibacter spp. In addition, it revealed high sensitivity for the detection of both agents in environmental sampling material including exhaust air dust in individually ventilated cage systems. Altogether, higher pathogen prevalence was detected via qPCR of environmental samples compared with cultural diagnostics of sentinel mice. This study describes a qPCR assay for the simultaneous detection of R. pneumotropicus and R. heylii. This assay was demonstrated to be beneficial during routine health monitoring, especially with regard to environmental sampling strategies.

Transcription Factor SP2 Enhanced the Expression of Cd14 in Colitis-Susceptible C3H/HeJBir.

Genetic analysis in the IL10-deficient mouse model revealed a modifier locus of experimental inflammatory bowel disease (IBD) on chromosome 18, with the allele of the strain C3H/HeJBir (C3Bir) conferring resistance and the allele of C57BL/6J (B6) conferring susceptibility. Differential Cd14 expression was associated with this background specific susceptibility to intestinal inflammation. Polymorphisms of the Cd14 promoter were found to be likely causative for strain specific expression, and Cd14-knockout mice revealed a protective role of this gene-product in experimental IBD. In this study, luciferase reporter assays confirmed an increased activity of the C3Bir derived Cd14 promoter compared to the one of B6. Promoter truncation experiments and site-directed mutagenesis in both strains resulted in reduced Cd14 promoter activity and confirmed that a central AP1 and the proximal SP1 transcription factor binding sites mediated the basal activity of the Cd14 promoter in the mouse. Moreover, a T to C exchange at position -259 replaced putative STAT1 and CDX1 sites in the Cd14 promoter from B6 by a SP2 site in C3Bir. Ablation of the Sp2 site through truncation was associated with a decreased promoter activity. Site-directed mutagenesis also demonstrated that the inactivation of SP2 led to a substantial loss of promoter activity in C3Bir. Performing electrophoretic mobility shift and supershift assays demonstrated interaction of SP2 with its potential binding site. In addition, retroviral-mediated overexpression of the SP2 transcription factor in primary bone marrow macrophages derived from C3Bir mice caused a significant increase in Cd14 transcription. These data characterized SP2 as important factor responsible for higher Cd14 expression and reduced IBD susceptibility mediated by the C3Bir allele.

Immortalized tumor derived rat fibroblasts as feeder cells facilitate the cultivation of male embryonic stem cells from the rat strain WKY/Ztm.

Feeder cells are essential for the establishment and culture of pluripotent rat embryonic stem cells (ESC) in vitro. Therefore, we tested several fibroblast and epithelial cell lines derived from the female genital tract as feeder cells to further improve ESC culture conditions. The immortalized tumor derived rat fibroblast TRF-O3 cells isolated from a Dnd1-deficient teratoma were identified as optimal feeder cells supporting stemness and proliferation of rat ESC. The TRF-O3 cells were characterized as myofibroblasts by expression of fibroblast specific genes alpha-2 type I collagen, collagen prolyl 4-hydroxylase alpha (II), vimentin, S100A4, and smooth muscle α-actin. Culture of inner cell masses (ICM) derived from WKY/Ztm rat blastocysts in 2i-LIF medium on TRF-O3 feeder cells lacking LIF, SCF and FGF2 expression resulted in pluripotent and germ-line competent rat ESC lines. Therein, genotyping confirmed up to 26% male ESC lines. On the other hand the TRF-O3 specific BMP4 expression was correlated with transcriptional activity of the mesodermal marker T-brachyury and the ectoderm specific nestin in the ESC line ES21 demonstrating mesodermal or ectodermal cell lineage differentiation processes within the ESC population. Substitution of 2i-LIF by serum-containing YPAC medium supplemented with TGF-ß and rho kinase inhibitors or by 4i medium in combination with TRF-O3 feeder cells led to enhanced differentiation of ES21 cells and freshly isolated ICMs. These results suggest that the ESC culture conditions using TRF-O3 feeder cells and 2i-LIF medium supported the establishment of male ESC lines from WKY/Ztm rats, which represent a favored, permissive genetic background for rat ESC culture.

Zinc-finger nuclease mediated disruption of Rag1 in the LEW/Ztm rat.

BACKGROUND: Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain. RESULTS: After microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαß+ as well as CD8+/CD3+/TCRαß+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat. CONCLUSION: The Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.

The ter mutation in the rat Dnd1 gene initiates gonadal teratomas and infertility in both genders.

A spontaneous mutation leading to the formation of congenital ovarian and testicular tumors was detected in the WKY/Ztm rat strain. The histological evaluation revealed derivatives from all three germ layers, thereby identifying these tumors as teratomas. Teratocarcinogenesis was accompanied by infertility and the underlying mutation was termed ter. Linkage analysis of 58 (WKY-ter×SPRD-Cu3) F2 rats associated the ter mutation with RNO18 (LOD = 3.25). Sequencing of candidate genes detected a point mutation in exon 4 of the dead-end homolog 1 gene (Dnd1), which introduces a premature stop codon assumed to cause a truncation of the Dnd1 protein. Genotyping of the recessive ter mutation revealed a complete penetrance of teratocarcinogenesis and infertility in homozygous ter rats of both genders. Morphologically non-tumorous testes of homozygous ter males were reduced in both size and weight. This testicular malformation was linked to a lack of spermatogenesis using immunohistochemical and histological staining. Our WKY-Dnd1(ter)/Ztm rat is a novel animal model to investigate gonadal teratocarcinogenesis and the molecular mechanisms involved in germ cell development of both genders.

Loss of Dnd1 facilitates the cultivation of genital ridge-derived rat embryonic germ cells.

Pluripotent cells referred to as embryonic germ cells (EGCs) can be derived from the embryonic precursors of the mature gametes: the primordial germ cells (PGCs). A homozygous mutation (ter) of the dead-end homolog 1 gene (Dnd1) in the rat causes gonadal teratocarcinogenesis and sterility due to neoplastic transformation and loss of germ cells. We mated heterozygous ter/+ WKY-Dnd1(ter)/Ztm rats and were able to cultivate the first genital ridge-derived EGCs of the rat embryo at day 14.5 post coitum (pc). Genotyping revealed that ten EGC lines were Dnd1 deficient, while only one wild type cell line had survived in culture. This suggests that the inactivation of the putative tumor suppressor gene Dnd1 facilitates the immortalization of late EGCs in vitro. Injection of the wild type EGCs into blastocysts resulted in the first germ-line competent chimeras. These new pluripotent rat EGCs offer an innovative approach for studies on germ cell tumor development as well as a new tool for genetic manipulations in rats.

Observations on the actual structural conditions in the stratum superficiale dermidis of porcine ear skin, with special reference to its use as model for human skin.

The results obtained from the outer ear skin of female pigs (German Landrace) by light microscopy (LM), transmission electron microscopy (TEM), and cryo scanning electron microscopy (cryo SEM) methods, in particular relying on careful and artefact-free tissue processing, exhibited that the stratum superficiale dermidis of the auricle had a very homogeneous and compact construction, especially in one area (central dorsum auriculae). Based upon the important measurements made [average thickness of stratum superficiale dermidis: 94 (+/-16) microm, region A: 81 (+/-10); average thickness of collagen fibre bundles: 12 (+/-2) microm, region A: 13 (+/-0.5); average density of subepidermal capillaries: 3134 (+/-459) loops/cm2, region A: 3497 (+/-247)], this impression was confirmed by low standard deviations for all parameters, in comparison to marginal locations studied. The capillary system present was analysed by LM and TEM for specific structural features, whereby it generally compared to the microvasculature in human skin. Moreover, a regular pattern of diffusion-relevant punctiform contacts of the capillary loop apex with the epidermal basement membrane became obvious. Cryo SEM, particularly offering the advantages of dispensation of chemical fixation, dehydration and solvents during processing, highlighted delicate structures without shrinkage and without loss of soluble sample components. Thus rather real spatial conditions in the region of the epidermo-dermal junction and the upper dermis were visualized, whereby very regular arrangements of the structures present became obvious. This pertained also to a correct demonstration of all components of the epidermal basement membrane, in particular the lamina lucida. In addition, the water-based stable character of the entire stratum superficiale dermidis could be emphasized as a basic feature for controlled diffusion processes.

Expression of a cyclin E1 isoform in mice is correlated with the quiescent cell cycle status of hepatocytes in vivo.

Cyclin E1 controls G1/S phase transition of the eukaryotic cell cycle. We report the impact of alternative spliced cyclin E1 isoforms on cell cycle regulation in hepatocytes. We show that expression of new cyclin E1 mRNA variants IN3, Delta4, and Delta5 is associated with retarded proliferation in murine hepatocellular carcinoma. Additionally, we demonstrate that a new cyclin E1 isoform Delta3/8 lacking the central part of wild-type mRNA is expressed predominantly in nonproliferating murine hepatocytes. Following partial hepatectomy, Delta3/8 is downregulated when hepatocytes enter the cell cycle from quiescence. The Delta3/8 protein does not exhibit any cyclin box motif but binds cyclin-dependent kinase 2 without stimulating kinase activity. We demonstrate that Delta3/8 lacks any nuclear localization signal and is exclusively located in the cytoplasm. Overexpression of Delta3/8 in cultured cells leads to a delayed G0-G1 transition, indicating that this splice variant helps to maintain a quiescent state of hepatocytes. In conclusion, we identified an isoform of cyclin E1 involved in G0 maintenance and suggest an additional mechanism for cell cycle control.

Silencing of caspase-8 in murine hepatocellular carcinomas is mediated via methylation of an essential promoter element.

BACKGROUND & AIMS: Caspase-8 is the apical caspase essential for triggering Fas-induced apoptosis. In this study, we investigated caspase-8 expression in hepatocellular carcinomas (HCCs) using recently described HCC mouse models (c-myc and IgEGF transgenes). METHODS: HCCs were isolated from c-myc and IgEGF transgenic animals. Expression of caspase-8 was monitored by reverse-transcription polymerase chain reaction. The murine caspase-8 promoter was characterized by luciferase-reporter analysis and the analysis of promoter methylation was performed by bisulfite genomic sequencing. RESULTS: In HCCs investigated, we frequently found a lack of caspase-8 messenger RNA expression. Genomic deletions at the caspase-8 locus did not contribute to caspase-8 silencing. We examined tumor-derived promoter sequences and found significant hypermethylation at distinct CpG sites. In parallel, we characterized the murine caspase-8 promoter and identified a 30-bp promoter element that is indispensable for basal promoter activity. This minimal promoter element contained SP1 binding motifs that are colocalized with CpG sites and were methylated in tumor-derived promoter sequences. Electrophoretic mobility shift assay analysis showed that methylation of these SP1 sites is sufficient to prevent SP1 complex formation. To support our data, we mimicked the methylation pattern of a tumor-derived caspase-8 promoter in vitro using CpG methylase and found a strong reduction of promoter activity. CONCLUSIONS: We show that HCCs are correlated frequently with silencing of caspase-8 expression and provide data suggesting that caspase-8 silencing is a direct consequence of inhibiting SP1-dependent transactivation caused by CpG methylation at its essential binding sites in the promoter region. Our data support the hypothesis that inhibition of apoptosis triggers hepatocarcinogenesis.

[Skin layer thickness at the ear of the domesticated pig , with special reference to the use of the ear integument for human dermatological research]. / Die Hautschichtdicken am Ohr des Hausschweins, mit besonderer Berücksichtigung der Nutzung des Ohrinteguments in der humandermatologischen Forschung.

Based on a shrinkage-free methodical approach (special plastic resin embedding, frozen section technique) and histological routine staining, the thickness of the different skin layers was measured from 15 regions of the outer and the inner side of the porcine auricle. Mean thickness values were for the str. corneum: 19 microns outside/20 microns inside, vital epidermis without ridges: 52 microns outside/56 microns inside, dermis: 1175 microns outside/1112 microns inside, hypodermis: 1024 microns outside/741 microns inside, perichondrium: 295 microns outside/220 microns inside. When both sides of the auricle were compared, it became obvious that the outer side generally had a thicker dermis (1140-1290 microns) and hypodermis (780-1150 microns), whereas the inner side had a thicker vital epidermis (50-60 microns) and deeper epidermal ridges (145-165 microns). The results are discussed with regard to corresponding findings from the human skin, and one region of the outer side of the porcine auricle is recommended as suitable for human dermatological research.