Mouse Gestation Length Is Genetically Determined

Background: Preterm birth is an enormous public health problem, affecting over 12 % of live births and costing over $26 billion in the United States alone. The causes are complex, but twin studies support the role of genetics in determining gestation length. Despite widespread use of the mouse in studies of the genetics of preterm birth, there have been few studies that actually address the precise natural gestation length of the mouse, and to what degree the timing of labor and birth is genetically determined. Methodology/Principal Findings: To further develop the mouse as a genetic model of preterm birth, we developed a highthroughput monitoring system and measured the gestation length in 15 inbred strains. Our results show an unexpectedly wide variation in overall gestation length between strains that approaches two full days, while intra-strain variation is quite low. Although litter size shows a strong inverse correlation with gestation length, genetic difference alone accounts for a significant portion of the variation. In addition, ovarian transplant experiments support a primary role of maternal genetics in the determination of gestation length. Preliminary analysis of gestation length in the C57BL/6J-Chr # A/J /NaJ chromosome substitution strain (B.A CSS) panel suggests complex genetic control of gestation length. Conclusions/Significance: Together, these data support the role of genetics in regulating gestation length and present th

Mutation discovery in mice by whole exome sequencing

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis

Mouse genomic variation and its effect on phenotypes and gene regulation

We report genome sequences of 17 inbred strains of laboratory mice and identify almost ten times more variants than previously known. We use these genomes to explore the phylogenetic history of the laboratory mouse and to examine the functional consequences of allele-specific variation on transcript abundance, revealing that at least 12% of transcripts show a significant tissue-specific expression bias. By identifying candidate functional variants at 718 quantitative trait loci we show that the molecular nature of functional variants and their position relative to genes vary according to the effect size of the locus. These sequences provide a starting point for a new era in the functional analysis of a key model organism

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Studies of OC-STAMP in Osteoclast Fusion: A New Knockout Mouse Model, Rescue of Cell Fusion, and Transmembrane Topology.

The fusion of monocyte/macrophage lineage cells into fully active, multinucleated, bone resorbing osteoclasts is a complex cell biological phenomenon that utilizes specialized proteins. OC-STAMP, a multi-pass transmembrane protein, has been shown to be required for pre-osteoclast fusion and for optimal bone resorption activity. A previously reported knockout mouse model had only mononuclear osteoclasts with markedly reduced resorption activity in vitro, but with paradoxically normal skeletal micro-CT parameters. To further explore this and related questions, we used mouse ES cells carrying a gene trap allele to generate a second OC-STAMP null mouse strain. Bone histology showed overall normal bone form with large numbers of TRAP-positive, mononuclear osteoclasts. Micro-CT parameters were not significantly different between knockout and wild type mice at 2 or 6 weeks old. At 6 weeks, metaphyseal TRAP-positive areas were lower and mean size of the areas were smaller in knockout femora, but bone turnover markers in serum were normal. Bone marrow mononuclear cells became TRAP-positive when cultured with CSF-1 and RANKL, but they did not fuse. Expression levels of other osteoclast markers, such as cathepsin K, carbonic anhydrase II, and NFATc1, were not significantly different compared to wild type. Actin rings were present, but small, and pit assays showed a 3.5-fold decrease in area resorbed. Restoring OC-STAMP in knockout cells by lentiviral transduction rescued fusion and resorption. N- and C-termini of OC-STAMP were intracellular, and a predicted glycosylation site was shown to be utilized and to lie on an extracellular loop. The site is conserved in all terrestrial vertebrates and appears to be required for protein stability, but not for fusion. Based on this and other results, we present a topological model of OC-STAMP as a 6-transmembrane domain protein. We also contrast the osteoclast-specific roles of OC- and DC-STAMP with more generalized cell fusion mechanisms

Supporting conditional mouse mutagenesis with a comprehensive cre characterization resource.

Full realization of the value of the loxP-flanked alleles generated by the International Knockout Mouse Consortium will require a large set of well-characterized cre-driver lines. However, many cre driver lines display excision activity beyond the intended tissue or cell type, and these data are frequently unavailable to the potential user. Here we describe a high-throughput pipeline to extend characterization of cre driver lines to document excision activity in a wide range of tissues at multiple time points and disseminate these data to the scientific community. Our results show that the majority of cre strains exhibit some degree of unreported recombinase activity. In addition, we observe frequent mosaicism, inconsistent activity and parent-of-origin effects. Together, these results highlight the importance of deep characterization of cre strains, and provide the scientific community with a critical resource for cre strain information

Disheveled hair and ear (Dhe), a spontaneous mouse Lmna mutation modeling human laminopathies.

Investigations of naturally-occurring mutations in animal models provide important insights and valuable disease models. Lamins A and C, along with lamin B, are type V intermediate filament proteins which constitute the proteinaceous boundary of the nucleus. LMNA mutations in humans cause a wide range of phenotypes, collectively termed laminopathies. To identify the mutation and investigate the phenotype of a spontaneous, semi-dominant mutation that we have named Disheveled hair and ear (Dhe), which causes a sparse coat and small external ears in heterozygotes and lethality in homozygotes by postnatal day 10.Genetic mapping identified a point mutation in the Lmna gene, causing a single amino acid change, L52R, in the coiled coil rod domain of lamin A and C proteins. Cranial sutures in Dhe/+ mice failed to close. Gene expression for collagen types I and III in sutures was deficient. Skulls were small and disproportionate. Skeletons of Dhe/+ mice were hypomineralized and total body fat was deficient in males. In homozygotes, skin and oral mucosae were dysplastic and ulcerated. Nuclear morphometry of cultured cells revealed gene dose-dependent blebbing and wrinkling.Dhe mice should provide a useful new model for investigations of the pathogenesis of laminopathies

Generating embryonic stem cells from the inbred mouse strain DBA/2J, a model of glaucoma and other complex diseases.

Mouse embryonic stem (ES) cells are derived from the inner cell mass of blastocyst stage embryos and are used primarily for the creation of genetically engineered strains through gene targeting. While some inbred strains of mice are permissive to the derivation of embryonic stem cell lines and are therefore easily engineered, others are nonpermissive or recalcitrant. Genetic engineering of recalcitrant strain backgrounds requires gene targeting in a permissive background followed by extensive backcrossing of the engineered allele into the desired strain background. The inbred mouse strain DBA/2J is a recalcitrant strain that is used as a model of many human diseases, including glaucoma, deafness and schizophrenia. Here, we describe the generation of germ-line competent ES cell lines derived from DBA/2J mice. We also demonstrate the utility of DBA/2J ES cells with the creation of conditional knockout allele for Endothelin-2 (Edn2) directly on the DBA/2J strain background

Genetic evidence that thyroid hormone is indispensable for prepubertal insulin-like growth factor-I expression and bone acquisition in mice.

Understanding how bone growth is regulated by hormonal and mechanical factors during early growth periods is important for optimizing the attainment of peak bone mass to prevent or postpone the occurrence of fragility fractures later in life. Using genetic mouse models that are deficient in thyroid hormone (TH) (Tshr(-/-) and Duox2(-/-)), growth hormone (GH) (Ghrhr(lit/lit)), or both (Tshr(-/-); Ghrhr(lit/lit)), we demonstrate that there is an important period prior to puberty when the effects of GH are surprisingly small and TH plays a critical role in the regulation of skeletal growth. Daily administration of T3/T4 during days 5 to 14, the time when serum levels of T3 increase rapidly in mice, rescued the skeletal deficit in TH-deficient mice but not in mice lacking both TH and GH. However, treatment of double-mutant mice with both GH and T3/T4 rescued the bone density deficit. Increased body fat in the TH-deficient as well as TH/GH double-mutant mice was rescued by T3/T4 treatment during days 5 to 14. In vitro studies in osteoblasts revealed that T3 in the presence of TH receptor (TR) α1 bound to a TH response element in intron 1 of the IGF-I gene to stimulate transcription. In vivo studies using TRα and TRβ knockout mice revealed evidence for differential regulation of insulin-like growth factor (IGF)-I expression by the two receptors. Furthermore, blockade of IGF-I action partially inhibited the biological effects of TH, thus suggesting that both IGF-I-dependent and IGF-I-independent mechanisms contribute to TH effects on prepubertal bone acquisition

Disruption

of muscle membrane and phenotype divergence in two novel mouse models of dysferlin-deficienc