Efficient Generation of Germ Line Transmitting Chimeras from C57BL/6N ES Cells by Aggregation with Outbred Host Embryos

Genetically modified mouse strains derived from embryonic stem (ES) cells have become essential tools for functional genomics and biomedical research. Large scale mutagenesis projects are producing libraries of mutant C57BL/6 (B6) ES cells to enable the functional annotation of every gene of the mouse genome. To realize the utility of these resources, efficient and accessible methods of generating mutant mice from these ES cells are necessary. Here, we describe a combination of ICR morula aggregation and a chemically-defined culture medium with widely available and accessible components for the high efficiency generation of germline transmitting chimeras from C57BL/6N ES cells. Together these methods will ease the access of the broader biomedical research community to the publicly available B6 ES cell resources

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

Efficient differentiation and function of human macrophages in humanized CSF-1 mice

Humanized mouse models are useful tools to understand pathophysiology and to develop therapies for human diseases. While significant progress has been made in generating immunocompromised mice with a human hematopoietic system, there are still several shortcomings, one of which is poor human myelopoiesis. Here, we report that human CSF-1 knockin mice show augmented frequencies and functions of human myeloid cells. Insertion of human CSF1 into the corresponding mouse locus of Balb/c Rag2(-/-) γc(-/-) mice through VELOCIGENE technology resulted in faithful expression of human CSF-1 in these mice both qualitatively and quantitatively. Intra-hepatic transfer of human fetal liver derived hematopoietic stem and progenitor cells (CD34(+)) in humanized CSF-1 (CSF1(h/h)) newborn mice resulted in more efficient differentiation and enhanced frequencies of human monocytes/macrophages in the bone marrow, spleens, peripheral blood, lungs, liver and peritoneal cavity. Human monocytes/macrophages obtained from the humanized CSF-1 mice show augmented functional properties including migration, phagocytosis, activation and responses to LPS. Thus, humanized mice engineered to express human cytokines will significantly help to overcome the current technical challenges in the field. In addition, humanized CSF-1 mice will be a valuable experimental model to study human myeloid cell biology

Conditional Activation of Akt in Adult Skeletal Muscle Induces Rapid Hypertrophy

Skeletal muscle atrophy is a severe morbidity caused by a variety of conditions, including cachexia, cancer, AIDS, prolonged bedrest, and diabetes. One strategy in the treatment of atrophy is to induce the pathways normally leading to skeletal muscle hypertrophy. The pathways that are sufficient to induce hypertrophy in skeletal muscle have been the subject of some controversy. We describe here the use of a novel method to produce a transgenic mouse in which a constitutively active form of Akt can be inducibly expressed in adult skeletal muscle and thereby demonstrate that acute activation of Akt is sufficient to induce rapid and significant skeletal muscle hypertrophy in vivo, accompanied by activation of the downstream Akt/p70S6 kinase protein synthesis pathway. Upon induction of Akt in skeletal muscle, there was also a significant decrease in adipose tissue. These findings suggest that pharmacologic approaches directed toward activating Akt will be useful in inducing skeletal muscle hypertrophy and that an increase in lean muscle mass is sufficient to decrease fat storage

Diverse Phenotypes and Specific Transcription Patterns in Twenty Mouse Lines with Ablated LincRNAs

<div><p>In a survey of 20 knockout mouse lines designed to examine the biological functions of large intergenic non-coding RNAs (lincRNAs), we have found a variety of phenotypes, ranging from perinatal lethality to defects associated with premature aging and morphological and functional abnormalities in the lungs, skeleton, and muscle. Each mutant allele carried a <i>lacZ</i> reporter whose expression profile highlighted a wide spectrum of spatiotemporal and tissue-specific transcription patterns in embryos and adults that informed our phenotypic analyses and will serve as a guide for future investigations of these genes. Our study shows that lincRNAs are a new class of encoded molecules that, like proteins, serve essential and important functional roles in embryonic development, physiology, and homeostasis of a broad array of tissues and organs in mammals.</p></div

Monoclonal antibodies against GFRα3 are efficacious against evoked hyperalgesic and allodynic responses in mouse join pain models but, one of these, REGN5069, was not effective against pain in a randomized, placebo-controlled clinical trial in patients with osteoarthritis pain

The artemin-GFRα3 signaling pathway has been implicated in various painful conditions including migraine, cold allodynia, hyperalgesia, inflammatory bone pain, and mouse knees contain GFRα3-immunoreactive nerve endings. We developed high affinity mouse (REGN1967) and human (REGN5069) GFRα3-blocking monoclonal antibodies and, following in vivo evaluations in mouse models of chronic joint pain (osteoarthritic-like and inflammatory), conducted a first-in-human phase 1 pharmacokinetics (PK) and safety trial of REGN5069 (NCT03645746) in healthy volunteers, and a phase 2 randomized placebo-controlled efficacy and safety trial of REGN5069 (NCT03956550) in patients with knee osteoarthritis (OA) pain. In three commonly used mouse models of chronic joint pain (destabilization of the medial meniscus, intra-articular monoiodoacetate, or Complete Freund’s Adjuvant), REGN1967 and REGN5069 attenuated evoked behaviors including tactile allodynia and thermal hyperalgesia without discernably impacting joint pathology or inflammation, prompting us to further evaluate REGN5069 in humans. In the phase 1 study in healthy subjects, the safety profiles of single doses of REGN5069 up to 3000 mg (intravenous) or 600 mg (subcutaneous) were comparable to placebo; PK were consistent with a monoclonal antibody exhibiting target-mediated disposition. In the phase 2 study in patients with OA knee pain, two doses of REGN5069 (100 mg or 1000 mg intravenous every 4 weeks) for 8 weeks failed to achieve the 12-week primary and secondary efficacy endpoints relative to placebo. In addition to possible differences in GFRα3 biology between mice and humans, we highlight here differences in experimental parameters that could have contributed to a different profile of efficacy in mouse models versus human OA pain. Additional research is required to more fully evaluate any potential role of GFRα3 in human pain

Abnormal Lung Morphology in <i>Fendrr</i> Knockout Mice at E13.5.

<p>(A) <i>LacZ</i> reporter gene expression at E12.5 in <i>Fendrr</i>^{<i>−⁄−</i>} embryos in the frontonasal region (FN) of the face, the aorta gonad mesonephros (AGM) region, and the respiratory tract, including the lungs (L) and trachea (T). (B) Dissection of lungs at E13.5 revealed an abnormal, disorganized, globular phenotype in the lobes of <i>Fendrr</i>^{<i>−⁄−</i>} embryos compared with <i>Fendrr</i>^{<i>+⁄−</i>}.</p

<i>LacZ</i> Reporter Expression in Brains of 6–8 Week Old lincRNA Gene-Targeted F0 Generation Heterozygotes.

<p>The brain <i>lacZ</i> expression pattern (blue) for each lincRNA gene is as follows: (A<b>)</b><i>Crnde</i>, the colliculi (dorsal view, arrow); (B) <i>Pantr1</i>, neocortex, olfactory bulb, basal forebrain, and hypothalamus; (C<b>)</b><i>Pantr2</i>, neocortex, olfactory bulb, cerebellum, hypothalamus, and basal forebrain; (D<b>)</b><i>Lincenc1</i>, neocortex, parts of the cerebellum and medial hypothalamus, especially strong patterning in the olfactory projection and olfactory projection areas of the temporal cortex (ventral view, red arrow); (E<b>)</b><i>Celrr</i>, broadly in gray matter with the exception of the lateral cerebellum and ventral pons; (F) <i>Kantr</i>, possibly in deep cerebellar layers (dorsal view, star); (G<b>)</b><i>Lincpint</i>, ubiquitously in gray matter, especially intense in the hypothalamus; (H<b>)</b><i>Lincppara</i>, ubiquitously in gray matter, especially dense in the hypothalamus; (I<b>)</b><i>Peril</i>, midline of the hypothalamus (ventral view, arrowhead); and (J) <i>Tug1</i>, spinal cord gray matter and light gray matter in most structures except for the neocortex. n = 2, genotype confirmed male mice per lincRNA knockout project.</p

Aging-associated Phenotypes in <i>Lincpint</i> Knockout Mice.

<p>(A) <i>Lincpint</i>^{<i>−⁄−</i>} and <i>Lincpint</i>^{<i>+⁄−</i>} male mice exhibit a significantly slower growth rate than their wild type (WT) littermates and begin to show significant weight loss near 6 months of age. Data are plotted as the mean +/− SEM, n > 9 mice for each group. Significance was assessed by a one-way ANOVA (*, <i>P</i> < 0.05; **, <i>P</i> < 0.005; ***, <i>P</i> < 0.001). (B) Kaplan-Meier analysis of homozygous with heterozygous and WT mice. <i>Lincpint</i>^{<i>−⁄−</i>} male mice exhibit a significant reduction in survival compare to <i>Lincpint</i>^{<i>+⁄−</i>} and wild type littermates. Data are plotted as percent survival over 1 year observation. (C) Ventral and dorsal skin sections in <i>Lincpint</i>^{<i>−⁄−</i>} mice compared with <i>Lincpint</i>^{<i>+⁄−</i>} and WT littermates. (D, E, F, and G) MicroCT evaluation of body composition at 12-, 26- and 52-weeks of age. (D, E) Male <i>Lincpint</i>^{<i>−⁄−</i>} and <i>Lincpint</i>^{<i>+⁄−</i>} mice exhibit a significant reduction in body fat as early as 26-week of age. Female <i>Lincpint</i>^{<i>−⁄−</i>} mice have reduced body fat at an older age noticeably at 52-week of age (***, <i>P</i> < 0.001, one-way ANOVA). (F, G) A significant reduction in femur bone mineral density (BMD) observed in both males and females <i>Lincpint</i>^{<i>−⁄−</i>} compared with their <i>Lincpint</i>^{<i>+⁄−</i>} and WT littermates (*, <i>P</i> < 0.05; ***, <i>P</i> < 0.001, one-way ANOVA). (H) MicroCT images depict pronounced lordokyphosis (curvature of the spinal column) seen in older male and female <i>Lincpint</i>^{<i>−⁄−</i>} mice compared with WT littermates. (I) Approximately 70% (6/9 males and 7/10 females) of <i>Lincpint</i>^{<i>−⁄−</i>} mice have lordokyphosis by 12 weeks of age, compared with 0–20% of <i>Lincpint</i>^{<i>+⁄−</i>} (1/12 males and 2/10 females) and WT (1/10 males and 0/11 females) littermates. By 26 weeks of age the proportion of <i>Lincpint</i>^{<i>−⁄−</i>} mice with lordokyphosis increased to nearly 90% (7/8 males and 8/9 females) and appeared in approximately 60% (8/12 males and 6/10 females) of <i>Lincpint</i>^{<i>+⁄−</i>} mice, compared with less than 20% (2/10 males and 2/11 females) of WT littermates. n ≥ 9 mice per group for all observations reported.</p

Abnormal Hindlimb Posture, Reduced Grip Strength, and Muscle Wasting in <i>Hottip</i>^{<i>−⁄−</i>} Mice.

<p>(A) <i>Hottip</i>^{<i>−⁄−</i>} mice demonstrated unusual hindlimb clasping posture when suspended by the tail. (B) Cage endurance testing revealed that <i>Hottip</i>^{<i>−⁄−</i>} mice have a reduced ability to remain suspended from an inverted wire cage top, n = 5 mice for each group. (C) The right and left TA (tibialis anterior), GA (gastrocnemius) and Quad (quadriceps) muscles from WT, <i>Hottip</i>^{<i>+⁄−</i>} and <i>Hottip</i>^{<i>−⁄−</i>} mice were weighed. Muscle weights are normalized to body weight and calculated to include both right/left muscle weights. Data are means +/−SEM, n = 6 mice for each group. A significant decrease in muscle weight was observed only in the GA of <i>Hottip</i>^{<i>−⁄−</i>} animal in both males and females (male data not shown). Asterisks indicate a significant difference in the <i>Hottip</i>^{<i>−⁄−</i>} GA muscle weights compared to all other control groups (P< 0.01). (D) Comparison of GA muscle fiber numbers in WT, <i>Hottip</i>^{<i>+⁄−</i>} and <i>Hottip</i>^{<i>−⁄−</i>}. A significant reduction of fiber count was observed in <i>Hottip</i>^{<i>−⁄−</i>}. Significance assessed by one-way ANOVA (P < .0001 (E) Comparison of mean cross-sectional area of muscle fibers. Cross sections taken from the GA muscle were stained with an antibody against laminin and measured. There is no noticeable size difference between <i>Hottip</i>^{<i>−⁄−</i>} and control skeletal muscles, n = 6 mice per group for all muscle analyses.</p