Project description:Full-length genomic DNA of the recently identified laboratory mouse papillomavirus 1 (MusPV1) was synthesized in vitro and was used to establish and characterize a mouse model of papillomavirus pathobiology. MusPV1 DNA, whether naked or encapsidated by MusPV1 or human papillomavirus 16 (HPV 16) capsids, efficiently induced the outgrowth of papillomas as early as 3 weeks after application to abraded skin on the muzzles and tails of athymic NCr nude mice. High concentrations of virions were extracted from homogenized papillomatous tissues and were serially passaged for >10 generations. Neutralization by L1 antisera confirmed that infectious transmission was capsid mediated. Unexpectedly, the skin of the murine back was much less susceptible to virion-induced papillomas than the muzzle or tail. Although reporter pseudovirions readily transduced the skin of the back, infection with native MusPV1 resulted in less viral genome amplification and gene expression on the back, including reduced expression of the L1 protein and very low expression of the L2 protein, results that imply skin region-specific control of postentry aspects of the viral life cycle. Unexpectedly, L1 protein on the back was predominantly cytoplasmic, while on the tail the abundant L1 was cytoplasmic in the lower epithelial layers and nuclear in the upper layers. Nuclear localization of L1 occurred only in cells that coexpressed the minor capsid protein, L2. The pattern of L1 protein staining in the infected epithelium suggests that L1 expression occurs earlier in the MusPV1 life cycle than in the life cycle of high-risk HPV and that virion assembly is regulated by a previously undescribed mechanism.
Project description:Epidemiological and experimental data implicate cutaneous human papillomavirus infection as co-factor in the development of cutaneous squamous cell carcinomas (cSCCs), particularly in immunocompromised organ transplant recipients (OTRs). Herein, we established and characterized a skin cancer model, in which Mus musculus papillomavirus 1 (MmuPV1) infection caused cSCCs in cyclosporine A (CsA)-treated mice, even in the absence of UV light. Development of cSCCs and their precursors were observed in 70% of MmuPV1-infected, CsA-treated mice on back as well as on tail skin. Immunosuppression by systemic CsA, but not UV-B irradiation, was a prerequisite, as immunocompetent or UV-B-irradiated mice did not develop skin malignancies after infection. In the virus-driven cSCCs the MmuPV1-E6/E7 oncogenes were abundantly expressed, and transcriptional activity and productive infection demonstrated. MmuPV1 infection induced the expression of phosphorylated H2AX, but not degradation of proapoptotic BAK in the cSCCs. Transfer of primary cells, established from a MmuPV1-induced cSCC from back skin, into athymic nude mice gave rise to secondary cSCCs, which lacked viral DNA, demonstrating that maintenance of the malignant phenotype was virus independent. This papillomavirus-induced skin cancer model opens future investigations into viral involvement, pathogenesis, and cancer surveillance, aiming at understanding and controlling the high incidence of skin cancer in OTRs.
Project description:Individual researchers are struggling to keep up with the accelerating emergence of high-throughput biological data, and to extract information that relates to their specific questions. Integration of accumulated evidence should permit researchers to form fewer - and more accurate - hypotheses for further study through experimentation.Here a method previously used to predict Gene Ontology (GO) terms for Saccharomyces cerevisiae (Tian et al.: Combining guilt-by-association and guilt-by-profiling to predict Saccharomyces cerevisiae gene function. Genome Biol 2008, 9(Suppl 1):S7) is applied to predict GO terms and phenotypes for 21,603 Mus musculus genes, using a diverse collection of integrated data sources (including expression, interaction, and sequence-based data). This combined 'guilt-by-profiling' and 'guilt-by-association' approach optimizes the combination of two inference methodologies. Predictions at all levels of confidence are evaluated by examining genes not used in training, and top predictions are examined manually using available literature and knowledge base resources.We assigned a confidence score to each gene/term combination. The results provided high prediction performance, with nearly every GO term achieving greater than 40% precision at 1% recall. Among the 36 novel predictions for GO terms and 40 for phenotypes that were studied manually, >80% and >40%, respectively, were identified as accurate. We also illustrate that a combination of 'guilt-by-profiling' and 'guilt-by-association' outperforms either approach alone in their application to M. musculus.
Project description:The immunocytes that regulate papillomavirus infection and lesion development in humans and animals remain largely undefined. We found that immunocompetent mice with varying H-2 haplotypes displayed asymptomatic skin infection that produced L1 when challenged with 6×1010 MusPV1 virions, the recently identified domestic mouse papillomavirus (also designated "MmuPV1"), but were uniformly resistant to MusPV1-induced papillomatosis. Broad immunosuppression with cyclosporin A resulted in variable induction of papillomas after experimental infection with a similar dose, from robust in Cr:ORL SENCAR to none in C57BL/6 mice, with lesional outgrowth correlating with early viral gene expression and partly with reported strain-specific susceptibility to chemical carcinogens, but not with H-2 haplotype. Challenge with 1×1012 virions in the absence of immunosuppression induced small transient papillomas in Cr:ORL SENCAR but not in C57BL/6 mice. Antibody-induced depletion of CD3+ T cells permitted efficient virus replication and papilloma formation in both strains, providing experimental proof for the crucial role of T cells in controlling papillomavirus infection and associated disease. In Cr:ORL SENCAR mice, immunodepletion of either CD4+ or CD8+ T cells was sufficient for efficient infection and papillomatosis, although deletion of one subset did not inhibit the recruitment of the other subset to the infected epithelium. Thus, the functional cooperation of CD4+ and CD8+ T cells is required to protect this strain. In contrast, C57BL/6 mice required depletion of both CD4+ and CD8+ T cells for infection and papillomatosis, and separate CD4 knock-out and CD8 knock-out C57BL/6 were also resistant. Thus, in C57BL/6 mice, either CD4+ or CD8+ T cell-independent mechanisms exist that can protect this particular strain from MusPV1-associated disease. These findings may help to explain the diversity of pathological outcomes in immunocompetent humans after infection with a specific human papillomavirus genotype.
Project description:This experiment is one of a series of experiments on interspecific recombinant congenic strain (IRCS) mice that aimed to identify novel genes involved in male or female hyporfertility by comparing characteristics of the sperm, number of offspring, quality ofÂ implantation etc. in C57B6/J and IRCS mice. <br>The goal of this experiment was to understand the basis of female hypofertility/embryonicÂ resorption in a mouse model of congenic strains. The IRCS strain used in this experiment is the 66H Ch13 mouse. This strain was derived by introgression of a ~6 Mb fragment of mus spretus originÂ inside the genome of Mus musculus (C57B6/J) (L'hÃ´te etÂ al, Bioessays, 2010. PMID:20091755 ) Previous ultrasonographic analysis of this line revealed an increased rate of embryonic resorption compared to the C57B6/J parent (Laissue et al, Int. J . Dev. Biol, 2009 PMID: 19488966 ). <br>In this experiment we measured gene expression in the tissues that are relevant for implantation and early development, i.e. the uterus and the placenta, in C57B6/J and 66H Chr13 mice at 12 days post-coÃ¯tus with C57B6/J males. Pools of RNA from four mice per sample were obtained and analysed using a Nimblegen mouse expression array.
Project description:Wound-induced hair follicle neogenesis (WIHN) has been demonstrated in laboratory mice (Mus musculus) after large (>1.5 × 1.5 cm2 ) full-thickness wounds. WIHN occurs more robustly in African spiny mice (Acomys cahirinus), which undergo autotomy to escape predation. Yet, the non-WIHN regenerative ability of the spiny mouse skin has not been explored. To understand the regenerative ability of the spiny mouse, we characterized skin features such as hair types, hair cycling, and the response to small and large wounds. We found that spiny mouse skin contains a large portion of adipose tissue. The spiny mouse hair bulge is larger and shows high expression of stem cell markers, K15 and CD34. All hair types cycle synchronously. To our surprise, the hair cycle is longer and less frequent than in laboratory mice. Newborn hair follicles in anagen are more mature than C57Bl/6 and demonstrate molecular features similar to C57Bl/6 adult hairs. The second hair cycling wave begins at week 4 and lasts for 5 weeks, then telogen lasts for 30 weeks. The third wave has a 6-week anagen, and even longer telogen. After plucking, spiny mouse hairs regenerate in about 5 days, similar to that of C57Bl/6. After large full-thickness excisional wounding, there is more de novo hair formation than C57Bl/6. Also, all hair types are present and pigmented, in contrast to the unpigmented zigzag hairs in C57Bl/6 WIHN. These findings shed new light on the regenerative biology of WIHN and may help us understand the control of skin repair vs regeneration.
Project description:During the process of embryonic development in mammals, epigenetic modifications must be erased and reconstructed. In particular, the trimethylation of histone 3 lysine 27 (H3K27me3) is associated with gene-specific transcriptional repression and contributes to the maintenance of the pluripotent embryos. In this study, we determined that the global levels of the H3K27me3 marker were elevated in MII oocyte chromatin and decrease to minimal levels at the 8-cell and morula stages. When the blastocyst hatched, H3K27me3 was re-established in the inner cell mass. We also determined that H3K27me3-specific demethylases, UTX and JMJD3, were observed at high transcript and protein levels in mouse preimplantation embryos. In the activated oocytes, when the H3K27me3 disappeared at the 8-cell stage, the UTX (but not JMJD3) protein levels were undetectable. Using RNA interference, we suppressed UTX and JMJD3 gene expression in the embryos and determined that the functions of UTX and JMJD3 were complementary. When JMJD3 levels were decreased by RNA interference, the embryo development rate and quality were improved, but the knockdown of UTX produced the opposite results. Understanding the epigenetic mechanisms controlling preimplantation development is critical to comprehending the basis of embryonic development and to devise methods and approaches to treat infertility.
Project description:BACKGROUND: Mus spretus diverged from Mus musculus over one million years ago. These mice are genetically and phenotypically divergent. Despite the value of utilizing M. musculus and M. spretus for quantitative trait locus (QTL) mapping, relatively little genomic information on M. spretus exists, and most of the available sequence and polymorphic data is for one strain of M. spretus, Spret/Ei. In previous work, we mapped fifteen loci for skin cancer susceptibility using four different M. spretus by M. musculus F1 backcrosses. One locus, skin tumor susceptibility 5 (Skts5) on chromosome 12, shows strong linkage in one cross. RESULTS: To identify potential candidate genes for Skts5, we sequenced 65 named and unnamed genes and coding elements mapping to the peak linkage area in outbred spretus, Spret/EiJ, FVB/NJ, and NIH/Ola. We identified polymorphisms in 62 of 65 genes including 122 amino acid substitutions. To look for polymorphisms consistent with the linkage data, we sequenced exons with amino acid polymorphisms in two additional M. spretus strains and one additional M. musculus strain generating 40.1 kb of sequence data. Eight candidate variants were identified that fit with the linkage data. To determine the degree of variation across M. spretus, we conducted phylogenetic analyses. The relatedness of the M. spretus strains at this locus is consistent with the proximity of region of ascertainment of the ancestral mice. CONCLUSION: Our analyses suggest that, if Skts5 on chromosome 12 is representative of other regions in the genome, then published genomic data for Spret/EiJ are likely to be of high utility for genomic studies in other M. spretus strains.
Project description:Human papillomaviruses (HPVs) are the most common sexually transmitted infectious agents. Because of the species specificity of HPVs, study of their natural transmission in laboratory animals is not possible. The papillomavirus, MmuPV1, which infects laboratory mice (Mus musculus), can cause infections in the female cervicovaginal epithelium of immunocompetent mice that progress to cancer. Here, we provide evidence that MmuPV1 is sexually transmitted in unmanipulated, immunocompetent male and female mice. Female 'donor' mice experimentally infected with MmuPV1 in their lower reproductive tract were housed with unmanipulated male mice. The male mice were then transferred to cages holding 'recipient' female mice. One third of the female recipient mice acquired cervicovaginal infections. Prolonged infections were verified by histopathology and in situ hybridization analyses of both male and recipient female mice at the study endpoint. These findings indicate that MmuPV1 is a new model animal papillomavirus with which to study sexually transmission of papillomaviruses.
Project description:BACKGROUND:The molecular pathways involved in the transition from uterine quiescence to overt labour are mapped and form the currently established pharmacological targets for both the induction and inhibition of human labour. However, both spontaneous premature labour and functional dystocia occur and are difficult to treat adequately. The identification of upstream regulators involved in the onset and orchestration of labour pathways is essential to develop additional therapies that will contribute to the regulation of the timing of birth. OBJECTIVES:To define uterine biological processes and their upstream activators involved in the transition from uterine quiescence to overt labour. STUDY DESIGN:The uterus of non-pregnant and pregnant FVB M. musculus is collected at embryonic days (E) 6.5, 8.5, 10.5, 12.5, 15.5 and 17.5 and the uterine transcriptome is determined using the Illumina mouse Ref8v2 micro-array platform. K-means clustering and Ingenuity Pathway Analysis are applied to further dissect the transcriptome data. RESULTS:From E6.5 to E17.5, 5405 genes are significantly differentially expressed and they segregate into 7 unique clusters. Five of the 7 clusters are enriched for genes involved in specific biological processes that include regulation of gene-expression, T-cell receptor activation, Toll-like receptor signalling and steroid metabolism. The identification of upstream activators for differentially expressed genes between consecutive time points highlights the E10.5 to E12.5 window during which the role from progesterone switches from an activated state to the inhibited state reflecting the process of functional progesterone withdrawal essential for the transgression from myometrial quiescence to synchronized contractions. For this time window in which 189 genes are differentially expressed we define 22 putative upstream activators of which NUPR1 and TBX2 are the most significant with respectively an activated and an inhibited status. CONCLUSIONS:Gene expression profiling of mice uterus from E6.5 to E17.5 results in 7 unique gene expression clusters from early to late pregnancy that define the landscape of molecular events in ongoing pregnancy. In the current dataset progesterone is predicted as an activated upstream regulator and maintainer of myometrial quiescence and is active till E10.5. Progesterone is predicted as an inhibited upstream regulator at E12.5. We identify 22 upstream regulators in the E10.5 to E12.5 time window where the switch to progesterone withdrawal occurs. They are putative relevant upstream activators of labour.