Quantitative phenotyping of inflammatory bowel disease in the IL-10-deficient mouse by use of noninvasive magnetic resonance imaging.

BACKGROUND: Although magnetic resonance imaging (MRI) is an increasingly used diagnostic tool in the assessment of inflammatory bowel disease (IBD) in humans, diagnosis and quantitation of intestinal inflammation in animal models of IBD still depends on ex vivo techniques. The aim of this study was to evaluate whether high-field MRI is suitable for the quantitative phenotyping of gut inflammation in a dextran sulfate sodium (DSS)-triggered interleukin (IL)10-deficient (IL-10(-/-)) mouse model of IBD, especially in longitudinal studies. METHODS: Using colitis-susceptible and -resistant backgrounds, MRI and ex vivo analyses were applied to characterize this specific model, differentiating disease severity and time-dependent alterations. Colon wall thickness, cecum wall tissue intensity, spleen, and mesenteric lymph node (MLN) volumes were evaluated 1, 2, 4, and 12 weeks after disease onset by T2-weighted MRI. Ex vivo parameters included histology, spleen, and MLN weight and analysis of cytokine expression. RESULTS: MRI and ex vivo determined parameters correlated well, revealing a mouse strain-specific colitis development over time with characteristics typical for the DSS model in the initial and for the IL-10(-/-) model in the chronic phase. To evaluate the use of high-field MRI for monitoring therapeutic studies, mice with a profound colitis were treated with IL-10-producing Saccharomyces boulardii and monitored by MRI. CONCLUSIONS: MRI can be utilized to quantify colitis development in the IL-10(-/-) model of IBD. Therefore, this noninvasive technique might be highly advantageous for an individual follow-up of colitis development in chronic models of IBD, facilitating the reduction of animal numbers in this kind of research.

Identification of an MHC class I ligand for the single member of a killer cell lectin-like receptor family, KLRH1.

Natural killer cells are able to recognize and kill target cells according to differences in MHC class I expression. In rodents, the Ly49 receptors are primarily responsible for this MHC differentiation. We previously described the cloning of a novel C-type lectin-like receptor, KLRH1, encoded in the NK complex adjacent to the Ly49 genes and expressed by subsets of NK and NKT cells. MHC influence on selection of KLRH1(+) NK cells in congenic strains suggested that KLRH1 may have an MHC ligand, although we were unable to identify any such ligand. In this study, we have used a sensitive reporter system and Fc fusion protein to demonstrate that KLRH1 binds specifically to the classical MHC class I molecule RT1-A2 of the RT1(n) haplotype. Cytolytic activity of KLRH1-transfected RNK-16 cells was also inhibited by target cells expressing RT1-A2(n). Thus, KLRH1 represents a novel family of MHC allele-specific inhibitory receptors expressed by NK cells.

Strain-specific colitis susceptibility in IL10-deficient mice depends on complex gut microbiota-host interactions.

BACKGROUND: Colitis susceptibility in Il10(-/-) mice depends on genetic background and microbiota composition. A major genetic locus mediating colitis susceptibility, Cdcs1, was transferred from susceptible C3Bir-Il10(-/-) to resistant B6-Il10(-/-) mice, resulting in susceptible congenic BC-R3-Il10(-/-) mice. The aim of this study was to determine the impact of microbiota on this differential colitis susceptibility using a Helicobacter hepaticus infection model. METHODS: Parental C3Bir-Il10(-/-) , B6-Il10(-/-) , and congenic BC-R3-Il10(-/-) mice were inoculated with H. hepaticus and analyzed for inflammation. In parental Il10(-/-) mice, microbiota composition was determined by terminal restriction fragment length polymorphism (T-RFLP) and quantitative polymerase chain reaction (qPCR). RESULTS: Most severe inflammation was observed in C3Bir-Il10(-/-) in the cecum, in BC-R3-Il10(-/-) in cecum and colon, and, unexpectedly, in B6-Il10(-/-) in the colon. C3Bir-Il10(-/-) and BC-R3-Il10(-/-) secreted significantly more interferon-gamma (IFNγ) and interleukin (IL)17 than B6-Il10(-/-) . T-RFLP analyses in C3Bir-Il10(-/-) and B6-Il10(-/-) mice revealed 1) a significant impact of H. hepaticus infection on species richness and diversity, and 2) strain differences in microbiota composition only after H. hepaticus infection. qPCR revealed higher numbers of Clostridia leptum and Bacteroides spp. in the cecum of infected C3Bir-Il10(-/-) mice, and Lactobacillus spp. in B6-Il10(-/-) mice. CONCLUSIONS: Cdcs1 modifies the response to H. hepaticus infection. However, this infection alone does not reflect the original response to a complex colitogenic biota. H. hepaticus-induced inflammation altered intestinal microbiota in a mouse strain-specific manner. Bacteroides spp. became more abundant in susceptible C3Bir-Il10(-/-) , lactobacilli in B6-Il10(-/-) mice. Therefore, both host immune response and differential compositional changes of microbiota play a role in strain-specific colitis susceptibility in Il10(-/-) mice.

Therapeutic concentrations of glucagon-like peptide-1 in cerebrospinal fluid following cell-based delivery into the cerebral ventricles of cats.

BACKGROUND: Neuropeptides may have considerable potential in the treatment of acute and chronic neurological diseases. Encapsulated genetically engineered cells have been suggested as a means for sustained local delivery of such peptides to the brain. In our experiments, we studied human mesenchymal stem cells which were transfected to produce glucagon-like peptide-1 (GLP-1). METHODS: Cells were packed in a water-permeable mesh bag containing 400 polymeric microcapsules, each containing 3000 cells. The mesh bags were either transplanted into the subdural space, into the brain parenchyma or into the cerebral ventricles of the cat brain. Mesh bags were explanted after two weeks, and cell viability, as well as GLP-1 concentration in the cerebrospinal fluid (CSF), was measured. RESULTS: Viability of cells did not significantly differ between the three implantation sites. However, CSF concentration of GLP-1 was significantly elevated only after ventricular transplantation with a maximum concentration of 73 pM (binding constant = 70 pM). CONCLUSIONS: This study showed that ventricular cell-based delivery of soluble factors has the capability to achieve concentrations in the CSF which may become pharmacologically active. Despite the controversy about the pharmacokinetic limitations of ventricular drug delivery, there might be a niche in this for encapsulated cell biodelivery of soluble, highly biologically-effective neuropeptides of low molecular weight like GLP-1.

A mutation in Myo15 leads to Usher-like symptoms in LEW/Ztm-ci2 rats.

The LEW/Ztm-ci2 rat is an animal model for syndromal deafness that arose from a spontaneous mutation. Homozygous animals show locomotor abnormalities like lateralized circling behavior. Additionally, an impaired vision can be observed in some animals through behavioral studies. Syndromal deafness as well as retinal degeneration are features of the Usher syndrome in humans. In the present study, the mutation was identified as a base substitution (T->C) in exon 56 of Myo15, leading to an amino acid exchange from leucine (Leu) to proline (Pro) within the carboxy-terminal MyTH4 domain in the proteins' tail region. Myo15 mRNA was expressed in the retina as demonstrated for the first time with the help of in-situ hybridization and PCR. To characterize the visual phenotype, rats were examined by scotopic and photopic electroretinography and, additionally, histological analyses of the retinas were conducted. The complete loss of sight was detected along with a severe degeneration of photoreceptor cells. Interestingly, the manifestation of the disease does not solely depend on the mutation, but also on environmental factors. Since the LEW/Ztm-ci2 rat features the entire range of symptoms of the human Usher syndrome we think that this strain is an appropriate model for this disease. Our findings display that mutations in binding domains of myosin XV do not only cause non-syndromic hearing loss but can also lead to syndromic disorders including retinal dysfunction.

Presence of Minute virus of mice in immunocompetent mice despite the onset of host immunity.

Infections with the autonomous parvovirus Minute virus of mice (MVM) are generally characterized as acute and self-limiting. However, MVM remains with considerably high prevalence rates in laboratory mouse colonies impeding rodent based research. The objective of this study was to assess whether the immunosuppressive variant of MVM (MVMi) establishes a persistent infection in immunocompetent adult mice. Therefore, we approached the question whether replicating and/or infectious virus is present in mice after the decline of viral shedding and whether immunosuppression might modify the infection. Dissection or induction of immunosuppression of individually housed mice was performed at 8 weeks post inoculation after fecal samples tested negative for viral DNA for at least 2 subsequent weeks as determined by weekly PCR analyses. MVMi mRNA was detected by both, RT-PCR and in situ RT-PCR in spleens at 8 weeks post inoculation with positive cells resembling lymphocytes and macrophages. These findings and the use of explant cultures strongly indicated the presence of replicating virus in spleens at 8 weeks post inoculation. Following immunosuppression (by irradiation), an induction of viral shedding was observed. Additionally, an increase in the amount of viral DNA was detected by real-time qPCR in mesenteric lymph nodes after irradiation. In summary, our data support the notion that MVMi persists in lymphoid tissue of immunocompetent adult mice despite the onset of host immunity.

Cdcs1 a major colitis susceptibility locus in mice; subcongenic analysis reveals genetic complexity.

BACKGROUND: The cytokine-deficiency-induced colitis susceptibility (Cdcs)1 locus is a major modifier of murine inflammatory bowel disease (IBD) and was originally identified in experimental crosses of interleukin-10-deficient (Il10(-/-)) mice. Congenic mice, in which this locus was reciprocally transferred between IBD-susceptible C3H/HeJBir-Il10(-/-) and resistant C57BL/6J-Il10(-/-) mice, revealed that this locus likely acts by inducing innate hypo- and adaptive hyperresponsiveness, associated with impaired NF-kappaB responses of macrophages. The aim of the present study was to dissect the complexity of Cdcs1 by further development and characterization of reciprocal Cdcs1 congenic strains and to identify potential candidate genes in the congenic interval. METHODS: In total, 15 reciprocal congenic strains were generated from Il10(-/-) mice of either C3H/HeJBir or C57BL/6J genetic backgrounds by 10 cycles of backcrossing. Colitis activity was monitored by histological grading. Candidate genes were identified by fine mapping of congenic intervals, sequencing, microarray analysis, and a high-throughput real-time reverse-transcription polymerase chain reaction (RT-PCR) approach using bone marrow-derived macrophages. RESULTS: Within the originally identified Cdcs1-interval, 3 independent regions were detected that likely contain susceptibility-determining genetic factors (Cdcs1.1, Cdcs1.2, and Cdcs1.3). Combining results of candidate gene approaches revealed Fcgr1, Cnn3, Larp7, and Alpk1 as highly attractive candidate genes with polymorphisms in coding or regulatory regions and expression differences between susceptible and resistant mouse strains. CONCLUSIONS: Subcongenic analysis of the major susceptibility locus Cdcs1 on mouse chromosome 3 revealed a complex genetic structure. Candidate gene approaches revealed attractive genes within the identified regions.

Analysis of Cd14 as a genetic modifier of experimental inflammatory bowel disease (IBD) in mice.

BACKGROUND AND AIM: By combining QTL and gene expression analyses, we have previously identified Cd14 as a potential candidate gene contributing to the differential IBD susceptibility of C3H/HeJBir (C3/J)-Il10(-/-) mice [carrying IBD-resistance alleles at this QTL (Cdcs6)] and C57BL/6J (B6)-Il10(-/-) mice, corroborating studies that showed an association of a CD14-promoter polymorphism with Crohn's disease and ulcerative colitis. The aim of the present study was to analyze the molecular mechanisms leading to differential intestinal expression of Cd14 and its contribution to IBD development. METHODS: Intestinal CD14 expression was assessed by FACS, immunohistochemistry, and ELISA on supernatants of primary epithelial cell and tissue cultures. RAW264.7 cells were stimulated with LPS and PGN in the presence or absence of CD14. Cd14 alleles were sequenced and promoters cloned for luciferase assays in transfected RAW264.7 cells. The severity of typhlocolitis between Cd14(-/-) and wild-type mice was compared in 2 distinct mouse models of IBD (acute DSS and Il10(-/-) ). RESULTS: In the gut, CD14 was detected mainly in its soluble form (sCD14), with higher expression in C3/J-Il10(-/-) mice. Polymorphisms in C3/J mice caused higher activity of the Cd14 promoter (luciferase assays). Intestinal sCD14 concentrations influenced the LPS and PGN responses of RAW264.7 cells. In vivo, genetic deletion of Cd14 aggravated colitis in both mouse models of IBD. CONCLUSIONS: Our study shows that Cd14-promoter polymorphisms affect CD14 expression and confirms the protective effect of CD14 against experimental IBD, potentially mediated by TLR2- and TLR4-dependent effects on intestinal barrier function. These findings support the concept that human CD14-promoter polymorphisms contribute to disease development.

Risk assessment of minute virus of mice transmission during rederivation: detection in reproductive organs, gametes, and embryos of mice after in vivo infection.

Murine parvoviruses, including minute virus of mice (MVM), represent major infectious disease problems encountered in contemporary laboratory animal research facilities with embryo transfer (ET), one of the most widely used techniques for rederivation. Using an in vivo approach, the objectives of this study were to assess the risk of MVM transmission during rederivation and to provide data that allow recommendation of preventive measures. Therefore, we determined whether immunosuppressive variant MVMi viral DNA is detectable in reproductive organs, gametes (oocytes and spermatozoa), and embryos collected from experimentally infected mice and whether washing as recommended before ET eliminates MVMi sufficiently from gametes and embryos. Fractions of reproductive organs tested positive from Day 5 to Day 30 postinoculation, demonstrating a risk for a minimum period of 4 wk; the highest incidence of positive organs was found between Day 9 and Day 13 postinoculation. Real-time PCR detected viral DNA to a lesser extent in male than in female reproductive organs. MVMi DNA was detected in oocytes and sperm cells derived after in vivo infection but not in two-cell embryos. In vitro contamination studies revealed that the virus firmly adheres to the zona pellucida after 10 wash steps, indicating that even extensive washing might not eliminate MVMi completely from embryos. According to this systematic in vivo approach, recommended measures to prevent transmission of MVM during rederivation include sufficient washing of embryos, accompanying testing using adequate (PCR) methods, and using embryos rather than in vitro fertilization techniques; furthermore, the exchange of gametes should be considered a risk factor.

CpG motifs of bacterial DNA exert protective effects in mouse models of IBD by antigen-independent tolerance induction.

BACKGROUND & AIMS: Prophylactic treatment of mice with CpG motifs of bacterial DNA protects from experimental inflammatory bowel disease, at least partly via induction of inhibitory T-cells. The aim of this study was to elucidate whether these CpG-dependent protective effects require presence of bacterial flora suggesting antigen-specific regulatory activity. METHODS: Germ-free BALB/c and IL-10(-/-) mice were treated with CpG-oligodeoxynucleotides (ODN), control-ODN, or PBS. CD4(+)CD62L(+) cells of these mice were transferred into SCID recipients. CpG-ODN-treated germ-free IL-10(-/-) mice were transferred into colitogenic environment. Monoclonal antibodies were used to neutralize TGF-beta and IFN-alpha/beta during CpG-ODN treatment. CD4(+)CD62L(+) cells of donors were evaluated for cytokine secretion and FOXP3, PD-1, and CD25 expression. RESULTS: Compared to PBS or control-ODN treatment, CpG-ODN application to germ-free donors led to decreased intestinal inflammation as indicated by histology, decreased proinflammatory cytokines, and increased IL-10 secretion. Protection was also observed after cotransfer of cells from PBS and CpG-ODN treated donors. Anti-TGF-beta and anti-INF-alpha/beta partly reversed the protective CpG-ODN effect. CpG-ODN-treated germ-free IL-10(-/-) mice transferred into colitogenic environment developed significantly less colitis than controls but not recipients of IL-10(-/-)CD4(+)CD62L(+)cells. CD4(+)CD62L(+)cells of CpG-treated germ-free animals displayed increased expression of regulatory markers. CONCLUSIONS: Even without pre-existence of bacterial flora CpG-ODN exposition induces tolerance, indicating that CpG-ODN-induced regulatory T-cells are not bacterial antigen specific. TGF-beta and IFN-alpha/beta play major roles in induction of regulatory cells, and although IL10-independent mechanisms play a role in CpG-ODN protection, this cytokine likely is important for the effector mechanism of CpG-ODN-induced regulatory T-cells.

A high specificity and affinity interaction with serum albumin stimulates an anion conductance in malaria-infected erythrocytes.

The intraerythrocytic development of P. falciparum induces New Permeability Pathways (NPP) in the membrane of the parasitized erythrocyte which provide the parasite with nutrients, adjust the erythrocyte electrolyte composition to the needs of the parasite, and dispose of metabolic waste products and osmolytes. Patch-clamp recordings identified inwardly and outwardly rectifying (OR) anion conductances in the host erythrocyte membrane as electrophysiological correlate of the NPP. The OR conductance is regulated by serum. Here we show that serum albumin (SA) stimulated OR-generated Cl(-) and lactate outward currents with an EC(50) of approximately 100 nM while other proteins such as ovalbumin or casein did not. The stimulatory efficacy did not differ between fatty acid free bovine SA and recombinant human SA and disruption of the SA tertiary structure abolished the effect suggesting that intact SA protein and not other bound factors interact with the erythrocyte membrane. Taken together, the data indicate a high affinity and specificity interaction of native SA with the parasitized erythrocytes which might underlie the observed dependence of P. falciparum growth in vitro on SA.

Minute virus of mice: antibody response, viral shedding, and persistence of viral DNA in multiple strains of mice.

Minute virus of mice (MVM) is a major concern for laboratory animal facilities because it remains with considerably high prevalence despite strict barrier systems. The aim of this study was to elucidate potential risks associated with MVM infection by investigating the role of the genetic background on antibody production and persistence as well as viral shedding. Mice of various strains and stocks were inoculated oronasally with the immunosuppressive strain MVMi; in addition, natural infection was modeled through contact exposure. As determined by serology, seroconversion and serum levels of IgG differed considerably among strains and stocks, especially in the contact-exposed group. For example, C57BL/6J mice responded well to exposure in contrast to FVB/N, NMRI, ICR, and C3H/HeN mice. Titration studies indicated that the viral dose necessary to induce seroconversion was strain-dependent. Experiments to dissect the role of the major histocompatibility complex haplotype in the response to MVMi gave inconclusive results. To detect viral persistence, spleens and feces were analyzed by PCR at 16 wk after exposure, and the infectivity of PCR-positive spleens was investigated by IP and oronasal inoculation of naive mice. Although DNA was detected in the spleens of some mice, feces remained negative, and naive mice were not infected by inoculation. In addition, viral shedding declined rapidly after day 20 postinoculation. In summary, the data show that seroconversion and antibody response to MVMi infection depend on the genetic background of mice, with the infective dose being a critical factor. The role of viral DNA in chronically infected mice will require further elucidation.

Establishing an in vivo model of canine prostate carcinoma using the new cell line CT1258.

BACKGROUND: Prostate cancer is a frequent finding in man. In dogs, malignant disease of the prostate is also of clinical relevance, although it is a less common diagnosis. Even though there are numerous differences in origin and development of the disease, man and dog share many similarities in the pathological presentation. For this reason, the dog might be a useful animal model for prostate malignancies in man.Although prostate cancer is of great importance in veterinary medicine as well as in comparative medicine, there are only few cell lines available. Thus, it was the aim of the present study to determine whether the formerly established prostate carcinoma cell line CT1258 is a suitable tool for in vivo testing, and to distinguish the growth pattern of the induced tumours. METHODS: For characterisation of the in vivo behaviour of the in vitro established canine prostate carcinoma cell line CT1258, cells were inoculated in 19 NOD.CB17-PrkdcScid/J (in the following: NOD-Scid) mice, either subcutaneously or intraperitoneally. After sacrifice, the obtained specimens were examined histologically and compared to the pattern of the original tumour in the donor. Cytogenetic investigation was performed. RESULTS: The cell line CT 1258 not only showed to be highly tumourigenic after subcutaneous as well as intraperitoneal inoculation, but also mimicked the behaviour of the original tumour. CONCLUSION: Tumours induced by inoculation of the cell line CT1258 resemble the situation in naturally occurring prostate carcinoma in the dog, and thus could be used as in vivo model for future studies.

Role of intestinal microbiota in transformation of bismuth and other metals and metalloids into volatile methyl and hydride derivatives in humans and mice.

The present study shows that feces samples of 14 human volunteers and isolated gut segments of mice (small intestine, cecum, and large intestine) are able to transform metals and metalloids into volatile derivatives ex situ during anaerobic incubation at 37 degrees C and neutral pH. Human feces and the gut of mice exhibit highly productive mechanisms for the formation of the toxic volatile derivative trimethylbismuth [(CH(3))(3)Bi] at rather low concentrations of bismuth (0.2 to 1 mumol kg(-1) [dry weight]). An increase of bismuth up to 2 to 14 mmol kg(-1) (dry weight) upon a single (human volunteers) or continuous (mouse study) administration of colloidal bismuth subcitrate resulted in an average increase of the derivatization rate from approximately 4 pmol h(-1) kg(-1) (dry weight) to 2,100 pmol h(-1) kg(-1) (dry weight) in human feces samples and from approximately 5 pmol h(-1) kg(-1) (dry weight) to 120 pmol h(-1) kg(-1) (dry weight) in mouse gut samples, respectively. The upshift of the bismuth content also led to an increase of derivatives of other elements (such as arsenic, antimony, and lead in human feces or tellurium and lead in the murine large intestine). The assumption that the gut microbiota plays a dominant role for these transformation processes, as indicated by the production of volatile derivatives of various elements in feces samples, is supported by the observation that the gut segments of germfree mice are unable to transform administered bismuth to (CH(3))(3)Bi.

The mutation of the LEW.1AR1-iddm rat maps to the telomeric end of rat chromosome 1.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes mellitus (T1DM) with an autosomal recessive mode of inheritance. T1DM susceptibility loci could be localized on chromosome (RNO) 20 in the major histocompatibility complex region (Iddm1) and on RNO1 (Iddm8, Iddm9) in a BN backcross cohort. In this study the impact of the different susceptibility regions on diabetes development was investigated in a backcross population of the diabetes-resistant PAR strain. A cohort of 130 [(PAR x LEW.1AR1-iddm) x LEW.1AR1-iddm] N2 rats was monitored for blood glucose and analyzed by linkage analysis. Sixteen percent of the PAR backcross animals developed T1DM. Genetic analysis revealed significant linkage to T1DM in the MHC region on RNO20p12. In contrast to the linkage analysis of the BN backcross cohort, only one susceptibility locus for T1DM could be identified on RNO1. This susceptibility region on RNO1 mapped to the telomeric end corresponding to Iddm8. Eighty-nine percent of diabetic PAR backcross animals were homozygous for Iddm8. The Iddm9 diabetes susceptibility region showed no linkage to diabetes in the PAR backcross cohort. The data of this study provide evidence that the mutation leading to T1DM in the LEW.1AR1-iddm rat is located at the telomeric end of RNO1 corresponding to Iddm8.

Sensitivity to Escherichia coli Nissle 1917 in mice is dependent on environment and genetic background.

Escherichia coli Nissle 1917 (EcN) is a well-characterized probiotic bacterium. Although genomic comparisons of EcN with the uropathogenic E. coli strain CFT073 revealed high degrees of similarity, EcN is generally considered a non-pathogenic organism. However, as recent evidence suggests that EcN is capable of inducing inflammatory responses in host intestinal epithelial cells, we aimed to investigate potential pathogenic properties of EcN in an in vivo model using various germ-free (GF) mouse strains. With the exception of C3H/HeJZtm mice, which carry a defective toll-like receptor (TLR)4-allele, no lesions were obvious in mice of different strains orally inoculated with EcN for 1 week, although organ cultures (blood, lung, mesenteric lymph node, pancreas, spleen, liver and kidney) tested positive to various degrees. C3H/HeJZtm mice inoculated with EcN became clinically ill and the majority died or had to be euthanized. Organs of all gnotobiotic C3H/HeJZtm mice were positive for EcN by culture; major histological findings were moderate to severe pyogranulomatous serositis, typhlitis and pancreatitis. Histological findings were corroborated by highly elevated tumour necrosis factor (TNF) serum levels. Lesions were not detected in specified pathogen free maintained C3H/HeJZtm mice, GF C3H/HeJ mice lacking the interleukin-10 gene, or GF C3H/HeJZtm mice that were inoculated with E. coli K12 strain MG1655 as a control. In addition, mild histological lesions were detected in Ztm:NMRI mice 3 months after oral inoculation with EcN. This study shows that EcN is capable of displaying a virulent phenotype in GF C3H/HeJZtm mice. Whether this phenotype is linked to the bacterium's probiotic nature should be the focus of further studies.

Cryopreservation and orthotopic transplantation of rat ovaries as a means of gamete banking.

Besides the exponentially increasing number of mouse strains, the rising number of rat strains, due to the establishment of transgenic and coisogenic strains in this species, surpasses the capacity of most animal houses. Cryopreservation of gametes may be a means of solving these problems. Here we describe an easy and fast method for the cryopreservation and transplantation of frozen-thawed ovaries of the rat. Ovaries of the rat inbred strain WKY/Ztm were frozen with dimethylsulphoxide as cryoprotectant and stored at -196 degrees C. Orthotopical transplantation was performed into ovariectomized syngenic recipients. Re-establishment of the reproductive cycle in the recipients was confirmed by vaginal cytology. The morphological integrity of frozen and unfrozen ovaries was compared by histological means after staining with haematoxylin and eosin. The number of litters and offspring was recorded. Reproductive cycle was re-established in all recipients of unfrozen ovaries and in more than 50% of recipients that received frozen-thawed ovaries. One-third of the former and more than 25% of the latter became pregnant and delivered at least one litter. Cyropreservation of ovaries can thus be considered as a reliable method of preserving scientifically and economically important mutant stock, as well as congenic rat strains that are currently not required.

Cd14, Gbp1, and Pla2g2a: three major candidate genes for experimental IBD identified by combining QTL and microarray analyses.

Induction of inflammatory bowel (IBD)-like disease in mice by a targeted mutation in the Il10 gene (Il10(-/-)) is inbred strain dependent. C3H/HeJBir (C3) mice are colitis susceptible, whereas C57BL/6J (B6) mice are resistant. Genetic dissection of this susceptibility revealed 10 colitogenic quantitative trait loci (QTL). The aim of this study was to identify valuable candidate genes by a combination of QTL mapping and microarray analyses. Sixteen genes were differentially expressed between B6- and C3-Il10(-/-) mice and were located within the QTL intervals. Three major candidate genes (Cd14, Gbp1, Pla2g2a) showed prominent expression differences between B6- and C3-Il10(-/-) as well as between B6 and C3 wild-type mice, which was confirmed by semiquantitative or real-time RT-PCR. Because strain differences are known for Gbp1 and Pla2g2a, further analyses focused on Cd14. Western blot analysis revealed strain differences also on the protein level. Cd14 expression in animals with defective and intact Toll-like receptor (TLR)4 signaling (C3, C3H/HeN, B6, B6-Tlr4(tm1Aki)) make the TLR4 defect of C3 mice unlikely to be the reason for higher Cd14 expression. Less Cd14 expression in germ-free mice indicates a contribution of the microflora on Cd14 expression. Stimulation of naive peritoneal macrophages with bacterial antigens showed lower CD14 surface expression in B6 than in C3 mice. In conclusion, the large number of candidate genes was reduced to three major candidates that play an important role in inflammatory processes and immune response. Strain differences for them are already known or are shown in this study.

Spontaneous rescue from cystic fibrosis in a mouse model.

BACKGROUND: From the original CftrTgH(neoim)Hgu mutant mouse model with a divergent genetic background (129P2, C57BL/6, MF1) we have generated two inbred CftrTgH(neoim)Hgu mutant strains named CF/1-CftrTgH(neoim)Hgu and CF/3-CftrTgH(neoim)Hgu, which are fertile and show normal growth and lifespan. Initial genome wide scan analysis with microsatellite markers indicated that the two inbred strains differed on the genetic level. In order to further investigate whether these genetic differences have an impact on the disease phenotype of cystic fibrosis we characterised the phenotype of the two inbred strains. RESULTS: Reduced amounts, compared to wild type control animals, of correctly spliced Cftr mRNA were detected in the nasal epithelia, lungs and the intestine of both inbred CftrTgH(neoim)Hgu strains, with higher residual amount observed for CF/1-CftrTgH(neoim)Hgu than CF/3-CftrTgH(neoim)Hgu for every investigated tissue. Accordingly the amounts of wild type Cftr protein in the intestine were 9% for CF/1-CftrTgH(neoim)Hgu and 4% for CF/3-CftrTgH(neoim)Hgu. Unlike the apparent strain and/or tissue specific regulation of Cftr mRNA splicing, short circuit current measurements in the respiratory and intestinal epithelium revealed that both strains have ameliorated the basic defect of cystic fibrosis with a presentation of a normal electrophysiology in both tissues. CONCLUSION: Unlike the outbred CftrTgH(neoim)Hgu insertional mouse model, which displayed the electrophysiological defect in the gastrointestinal and respiratory tracts characteristic of cystic fibrosis, both inbred CftrTgH(neoim)Hgu strains have ameliorated the electrophysiological defect. On the basis of these findings both CF/1-CftrTgH(neoim)Hgu and CF/3-CftrTgH(neoim)Hgu offer an excellent model whereby determination of the minimal levels of protein required for the restoration of the basic defect of cystic fibrosis can be studied, along with the modulating factors which may affect this outcome.

Efficient single nucleotide polymorphism discovery in laboratory rat strains using wild rat-derived SNP candidates.

BACKGROUND: The laboratory rat (Rattus norvegicus) is an important model for studying many aspects of human health and disease. Detailed knowledge on genetic variation between strains is important from a biomedical, particularly pharmacogenetic point of view and useful for marker selection for genetic cloning and association studies. RESULTS: We show that Single Nucleotide Polymorphisms (SNPs) in commonly used rat strains are surprisingly well represented in wild rat isolates. Shotgun sequencing of 814 Kbp in one wild rat resulted in the identification of 485 SNPs as compared with the Brown Norway genome sequence. Genotyping 36 commonly used inbred rat strains showed that 84% of these alleles are also polymorphic in a representative set of laboratory rat strains. CONCLUSION: We postulate that shotgun sequencing in a wild rat sample and subsequent genotyping in multiple laboratory or domesticated strains rather than direct shotgun sequencing of multiple strains, could be the most efficient SNP discovery approach. For the rat, laboratory strains still harbor a large portion of the haplotypes present in wild isolates, suggesting a relatively recent common origin and supporting the idea that rat inbred strains, in contrast to mouse inbred strains, originate from a single species, R. norvegicus.