Human IL-3/GM-CSF knock-in mice support human alveolar macrophage development and human immune responses in the lung

Mice with a functional human immune system have the potential to allow in vivo studies of human infectious diseases and to enable vaccine testing. To this end, mice need to fully support the development of human immune cells, allow infection with human pathogens, and be capable of mounting effective human immune responses. A major limitation of humanized mice is the poor development and function of human myeloid cells and the absence of human immune responses at mucosal surfaces, such as the lung. To overcome this, we generated human IL-3/GM-CSF knock-in (hIL-3/GM- CSF KI) mice. These mice faithfully expressed human GM-CSF and IL-3 and developed pulmonary alveolar proteinosis because of elimination of mouse GM-CSF. We demonstrate that hIL-3/GM-CSF KI mice engrafted with human CD34+ hematopoietic cells had improved human myeloid cell reconstitution in the lung. In particular, hIL-3/GM-CSF KI mice supported the development of human alveolar macrophages that partially rescued the pulmonary alveolar proteinosis syndrome. Moreover, human alveolar macrophages mounted correlates of a human innate immune response against influenza virus. The hIL-3/GM-CSF KI mice represent a unique mouse model that permits the study of human mucosal immune responses to lung pathogens

Human thrombopoietin knockin mice efficiently support human hematopoiesis in vivo

Hematopoietic stem cells (HSCs) both self-renew and give rise to all blood cells for the lifetime of an individual. Xenogeneic mouse models are broadly used to study human hematopoietic stem and progenitor cell biology in vivo. However, maintenance, differentiation, and function of human hematopoietic cells are suboptimal in these hosts. Thrombopoietin (TPO) has been demonstrated as a crucial cytokine supporting maintenance and self-renewal of HSCs. We generated RAG2−/−γc−/− mice in which we replaced the gene encoding mouse TPO by its human homolog. Homozygous humanization of TPO led to increased levels of human engraftment in the bone marrow of the hosts, and multilineage differentiation of hematopoietic cells was improved, with an increased ratio of myelomonocytic verus lymphoid lineages. Moreover, maintenance of human stem and progenitor cells was improved, as demonstrated by serial transplantation. Therefore, RAG2−/−γc−/− TPO-humanized mice represent a useful model to study human hematopoiesis in vivo

The Exon 8-Containing Prosaposin Gene Splice Variant Is Dispensable for Mouse Development, Lysosomal Function, and Secretion

Prosaposin is a multifunctional protein with diverse functions. Intracellularly, prosaposin is a precursor of four sphingolipid activator proteins, saposins A to D, which are required for hydrolysis of sphingolipids by several lysosomal exohydrolases. Secreted prosaposin has been implicated as a neurotrophic, myelinotrophic, and myotrophic factor as well as a spermatogenic factor. It has also been implicated in fertilization. The human and the mouse prosaposin gene has a 9-bp exon (exon 8) that is alternatively spliced, resulting in an isoform with three extra amino acids, Gln-Asp-Gln, within the saposin B domain. Alternative splicing in the prosaposin gene is conserved from fish to humans, tissue specific, and regulated in the brain during development and nerve regeneration-degeneration processes. To elucidate the physiological role of alternative splicing, we have generated a mouse lacking exon 8 by homologous recombination. The exon 8 prosaposin mutant mice are healthy and fertile with no obvious phenotype. No changes were detected in prosaposin secretion or in accumulation and metabolism of gangliosides, sulfatides, neutral glycosphingolipids, neutral phospholipids, other neutral lipids, and ceramide. These data strongly indicate that the prosaposin variant containing the exon 8-encoded three amino acids is dispensable for normal mouse development and fertility as well as for prosaposin secretion and its lysosomal function, at least in the presence of the prosaposin variant missing the exon 8-encoded three amino acids

Embryonic stem cell tumor model reveals role of vascular endothelial receptor tyrosine phosphatase in regulating Tie2 pathway in tumor angiogenesis

Inhibiting angiogenesis has become an effective approach for treating cancer and other diseases. However, our understanding of signaling pathways in tumor angiogenesis has been limited by the embryonic lethality of many gene knockouts. To overcome this limitation, we used the plasticity of embryonic stem (ES) cells to develop a unique approach to study tumor angiogenesis. Murine ES cells can be readily manipulated genetically; in addition, ES cells implanted subcutaneously in mice develop into tumors that contain a variety of cell types (teratomas). We show that ES cells differentiate into bona fide endothelial cells within the teratoma, and that these ES-derived endothelial cells form part of the functional tumor vasculature. Using this powerful and flexible system, the Angiopoietin/Tie2 system is shown to have a key role in the regulation of tumor vessel size. Endothelial differentiation in the ES teratoma model allows gene-targeting methods to be used in the study of tumor angiogenesis

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

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