The mammalian gene function resource: The International Knockout Mouse Consortium

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed highthroughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research

The mammalian gene function resource: the International Knockout Mouse Consortium.

In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research

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

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

Homeotic Transformation Observed in the 4th Caudal Vertebra of <i>Hotair</i>^{<i>−⁄−</i>} mice.

<p>(A) Visualization of the sacral and caudal region of the mouse skeleton by microCT reveals a homeotic transformation in <i>Hotair</i>^{<i>−⁄−</i>} mice of the 4th caudal vertebra to a structure similar to that of the 3rd caudal vertebra. (B) Dorsal, lateral and ventral comparison of WT and <i>Hotair</i>^{<i>−⁄−</i>} 4th caudal vertebra reveals a structural abnormality in homozygotes indicative of a homeotic transformation.</p

Increased Expression of <i>Lincpint</i> from Postnatal Day 3 to 8 Weeks of Age.

<p><i>LacZ</i> reporter expression (blue) at 3 days, 3 weeks, and 8 weeks in F0 heterozygotes shows increasing <i>Lincpint</i> expression with age. (A) At 3 days, ßgalactosidase staining is only observed in portions of the brain, tendons and ligaments of the hind limb, and some bronchioles in the lung (arrow). (B) At 3 weeks, there is increased staining in the brain, hindlimb, atria of the heart, lung, and liver. (C) By 8 weeks of age, the whole brain, skeletal muscle of the hindlimb and chest, atria and myocardium, lung, and liver tissue all exhibit strong ß-galactosidase staining representative of increased <i>Lincpint</i> expression. Examples shown are representative of n>4 mice per group. (D) RT-PCR analysis of <i>GFP</i> in GA (gastrocnemius) muscle and kidney isolated from 8 week-old and 52 week-old wild type (WT), heterozygous (<i>Lincpint-GFP</i>^{<i>+⁄−</i>}) and homozygous (<i>Lincpint-GFP</i>^{<i>−⁄−</i>}) mice. (E) Comparison of endogenous <i>Lincpint</i> RNA level in GA (gastrocnemius) muscle and kidney of 8 week-old versus 52 week-old wild type (WT) tissues demonstrating increased in <i>Lincpint</i> gene expression. Examples shown are representative of n>4 mice per group.</p