Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

Gene targeting by homologous recombination allows the introduction of specific mutations into any cloned gene. This unit provides a protocol in which the gene of interest is inactivated by interrupting its coding sequence with a positive selectable marker. A negative selectable marker is included in the construct outside the region of target gene homology in order to enrich for clones in which the target gene has undergone homologous recombination. The altered target gene is then expressed in embryonic stem cells. A support protocol describes a method for transient expression of Cre recombinase to remove sequences between lox sites, which can also be used as a selection method.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/152629/1/cpns0430.pd

Overview of Gene Targeting by Homologous Recombination

Formerly UNIT 9.15, this unit has been moved to the opening spot of our new chapter on Embryonic Stem Cell technology. The unit has also been updated, and now includes information about the Cre‐lox and FlP/FRT recombinase systems.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/171172/1/cpmb2301.pd

Valuation of closely held and untraded stocks held as collateral by banks in support of loans

It is recognized that one of the primary functions of modern day banking is the extension of credit. Properly administered, the fulfillment of this function is, either directly or indirectly, of importance to the general welfare. Properly loaned money is a source of income to the bank, and hence to the stockholders and employees; it enables the retailer to carry his inventory; the wholesaler to purchase and distribute a variety of products; and the manufacturer to produce a needed finished product. Direct loans to individuals are likewise of vital importance for many reasons. Borrowed funds may enable an individual to meet the demands of an unexpected emergency, to build a home, etc. invest in a business,etc. Generally, the extension of credit is essential to our everyday way of life and has been largely responsible for the high standard of living which we enjoy. It is toward one phase of the broad field of credit administration that the effort of this paper are directed; that is, the area of the secured loan, and, even more specifically, the area relating to loans secured by the pledge of closely held and untraded stocks. The purposes of this study are: 1. To present and briefly discuss the merits and/or faults of tha methods currently used for the valuation of closely held and untraded stocks. 2. To develop and/or represent a suggest:ed procedure for the valuation of closely held and untraded stocks

Stratigraphy and U–Pb Zircon–Titanite Geochronology of the Aley Carbonatite Complex, Northeastern British Columbia: Evidence for Antler-Aged Orogenesis in the Foreland Belt of the Canadian Cordillera

The tectonic significance and age of carbonatite intrusions in the central Foreland Belt of the Canadian Cordillera are poorly constrained. Recent work has demonstrated that one of these carbonatite intrusions, the Aley carbonatite, was emplaced as a syn-kinematic sill, coeval with a major nappe-forming tectonic event. Determining the age of the Aley carbonatite thus provides a means of directly dating syn-tectonic magmatism. Attempts at dating carbonatite units failed due to low U–Pb content in sampled zircon; however, a U–Pb titanite age of 365.9 ± 2.1 Ma was obtained from the Ospika pipe, an ultramafic diatreme spatially and genetically related to the carbonatite. This U–Pb titanite age is further supported by respective 40Ar/39Ar phlogopite ages of 359.4 ± 3.4 Ma and 353.3 ± 3.6 Ma for the pipe and a spatially associated lamprophyre dyke. We interpret the Late Devonian U–Pb titanite age of the Ospikapipe to be the minimum possible age of the carbonatite and syn-magmatic nappe-forming tectonic event. The maximum possible age of the carbonatite is constrained by the Early Devonian age of the Road River Group, the youngest strata intruded by carbonatite dykes and involved in the nappe-forming event. Our dating results for the Aley carbonatite closely correlate with U–Pb zircon and perovskite ages obtained for the Ice River carbonatite complex in the central Foreland Belt of the southern Canadian Cordillera, and support the interpretation of carbonatite intrusions of the western Foreland Belt as genetically linked components of an alkaline-carbonatitic magmatic province. Structural, stratigraphic, and geochronological data from the Aley area indicate that deformation was similar in style to, and coeval with, structures attributable to the Antler orogeny, and are consistent with the Antler orogen having extended the length of the Cordilleran margin from the southern United States to Alaska.La signification tectonique et l’âge des intrusions de carbonatite dans le Domaine de l’avant-pays central de la Cordillère canadienne sont méconnus. Des travaux récents ont démontré qu’une de ces intrusions de carbonatite, la carbonatite d’Aley, a été mise en place en tant que filons-couches syn-cinématiques, contemporains avec un événement tectonique majeur de formation de nappe. La détermination de l’âge de la carbonatite d’Aley fournit ainsi un moyen de datation directe du magmatisme syn-tectonique. Les tentatives de datation des unités de carbonatite ont échoué en raison de la faible teneur en U – Pb du zircon échantillonné; cependant, un âge U–Pb de 365,9 ± 2,1 Ma a été obtenu à partir de titanite provenant de la cheminée d’Ospika, une diatrème ultramafique spatialement et génétiquement apparenté à la carbonatite. Cet âge U–Pb sur titanite est en outre soutenu par des âges 40Ar/39Ar sur phlogopitede 359,4 ± 3,4 Ma et 353,3 ± 3,6 Ma respectivement pour la cheminée et un dyke de lamprophyre spatialement associé. Nous interprétons l’âge U–Pb des titanites du Dévonien supérieur de la cheminée d’Ospika comme étant l’âge minimum possible de la carbonatite et de l’événement tectonique syn-magmatique de formation de nappe. L’âge maximum possible de la carbonatite est limité par l’âge Dévonien inférieur du groupe de Road River, les plus jeunes strates traversées par les intrusions de dykes de carbonatite et impliquées dans l’événement de formation de nappe. Nos résultats de datation pour la carbonatite d’Aley sont étroitement corrélés avec les âges U–Pb sur zircon et pérovskite obtenus pour le complexe de carbonatite de Ice River dans le Domaine de l’avant-pays méridional de la Cordillère canadienne, et appuient l’interprétation des intrusions de carbonatite du Domaine de l’avant-pays occidentalcomme étant des composantes génétiquement liés d’une province magmatique alcalino-carbonatitique. Les données structurales, stratigraphiques et géochronologiques de la région d’Aley indiquent que la déformation était semblable et contemporaine aux structures attribuables à l’orogenèse Antler, et sont compatibles avec l’orogène Antler ayant étendu la longueur de la marge de la Cordillère depuis le sud des États-Unis États jusqu’en Alaska

Automated Spacecraft Communications Service Demonstration Using NASA's SCaN Testbed

The traditional paradigm for space mission operations relies on inefficient, highly scripted pre-planned activities between space communications & navigation service providers and user ground mission operations centers. Typically there is limited or non-existent automation capabilities on the user spacecraft for requesting space communications services, and on the provider network for request dispositioning and service provisioning. In the past, using these processes for space networks was sufficient with the relatively small number of user spacecraft requesting services. However, with an ever increasing number of satellites being launched to orbit, more complex event-driven science objectives, exploration missions involving collaborative platforms, and more distant missions, approaches that improve automation, flexibility and efficiency are needed. This paper describes NASA's recently completed on-orbit demonstration results of a new space communications service concept called User Initiated Service, and a discussion for infusing this innovation into operations

PPAR-γ in the Cardiovascular System

Peroxisome proliferator-activated receptor-γ (PPAR-γ), an essential transcriptional mediator of adipogenesis, lipid metabolism, insulin sensitivity, and glucose homeostasis, is increasingly recognized as a key player in inflammatory cells and in cardiovascular diseases (CVD) such as hypertension, cardiac hypertrophy, congestive heart failure, and atherosclerosis. PPAR-γ agonists, the thiazolidinediones (TZDs), increase insulin sensitivity, lower blood glucose, decrease circulating free fatty acids and triglycerides, lower blood pressure, reduce inflammatory markers, and reduce atherosclerosis in insulin-resistant patients and animal models. Human genetic studies on PPAR-γ have revealed that functional changes in this nuclear receptor are associated with CVD. Recent controversial clinical studies raise the question of deleterious action of PPAR-γ agonists on the cardiovascular system. These complex interactions of metabolic responsive factors and cardiovascular disease promise to be important areas of focus for the future

The Space Communications and Navigation Testbed aboard International Space Station: Seven Years of Space-based Reconfigurable Software Defined Communications, Navigation, and Networking

The adoption of software defined radios offers space missions a new way to develop and operate space transceivers for communications and navigation.The SCaN Testbed on-board the ISS led groundbreaking efforts to champion use of software defined radios for space communications. The SCaN Testbed has allowed NASA, industry, academia, and international partners to pursue their respective interests in joint collaboration with NASA, and move this technology and it's applications to the space domain. Launched in 2012, The SCaN Testbed has logged over 4000 hours of operation exploring the development, reconfiguration, and operation of software defined radios and their software applications. Over the past seven years, experimenters and organizations from across the United States and around the world, have advanced the applications of software defined radios and networks using the SCaN Tested. Some of SCaN Testbed's successful experiments include the demonstration of the first Ka-band full duplex space transceiver, which became an R&D 100 award winning technology, and was inducted into the Space Technology Hall of Fame, following the launch and space deployment of a successful commercial product line based on the Testbed radios.Experiments have focused on new software development and operations concepts for understanding how to manage and apply this relatively new technology to space to improve communications and navigation for space science and exploration missions. The advanced capabilities of the software radios allow for multiple applications or functions (e.g., communication and navigation) to operate from the same radio platform. Multiple software waveform applications enable software component reuse and improve efficiency for multiple applications operating over different mission phases. The new capabilities of software defined radios such as on-orbit reconfiguration, also present new challenges such as increased operational complexity. Experiments of the SCaN testbed include more intelligent or cognitive applications to improve communications efficiency and manage the complexity of the radios, the communication channels, and the network itself. The software defined radios on the SCaN Testbed are each compliant to NASA's Space Telecommunications Radio System (STRS) Architecture. The STRS Architecture provides commonality among radio developments from different providers and different mission applications, and is designed to reduce the cost, risk, and complexity of unique and custom radio developments. This radio architecture standard defines common waveform software interfaces, methods of instantiation, operation, and documentation. As the SCaN Testbed concludes its operations on ISS, this presentation explores the advancements and accomplishments made to advance software defined radio technology and its applications for exploration. The accomplishments cover a number of experiment areas in Ka-band and S-band communications with TDRS, high rate communications, adaptive waveform operation, navigation using both GPA and Galileo constellations, complex networking and disruptive tolerant link protocols, user initiative service, and initial experiments with intelligent and cognitive applications which demonstrate the significant potential of software defined and cognitive radios

Direct monitoring pressure overload predicts cardiac hypertrophy in mice

Pressure overload (POL) is a classical model for studying cardiac hypertrophy, but there has been no direct measure of hemodynamics in a conscious ambulatory mouse model of POL. We used abdominal aortic constriction to produce POL and radiotelemetry to measure the blood pressure and heart rate for three weeks. The cardiac size correlated with the systolic pressure in the last week is better than other hemodynamic parameters. Cardiac fibrosis was more correlated to the cardiac size than to the systolic pressure. The expression of the cardiac genes that are typically associated with cardiac hypertrophy was correlated with both cardiac size and systolic pressure. In conclusion, the systolic pressure is the major determinant of cardiac hypertrophy in the murine POL model. In contrast, cardiac fibrosis shows the influence of other factors besides systolic pressure. The combination of the POL model with continuous direct measurements of hemodynamics represents a significant technological advance and will lead to an extended usefulness of POL methodologically.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58150/2/pm7_11_001.pd

Robust acinar cell transgene expression of CreErT via BAC recombineering

Pancreatic acinar cells are critical in gastrointestinal physiology and pancreatitis and may be involved in pancreatic cancer. Previously, a short rat pancreatic elastase promoter has been widely utilized to control acinar cell transgene expression. However, this partial sequence does not confer robust and stable expression. In this study, we tested the hypothesis that a transgene employing bacterial-artificial-chromosome (BAC) technology to express a tamoxifen-regulated Cre recombinase from a full-length mouse elastase gene (BAC-Ela-CreErT) would be more robust and stable. When founders were crossed with Rosa26 reporter mice nearly 100% of acini expressed Β-galactosidase after tamoxifen treatment. The expression was specific for pancreatic acinar cells and these characteristics have remained stable for 2 years. However, because of high levels of expression in differentiated acinar cells, this construct is tamoxifen independent in ∼50% of adult acinar cells. This model of pancreatic acinar specific Cre expression is a powerful tool for future transgenic and knockout studies. genesis 46:390–395, 2008. © 2008 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/60895/1/20411_ftp.pd

Osteoblast‐Targeted suppression of PPARγ increases osteogenesis through activation of mTOR signaling

Nuclear receptor peroxisome proliferator‐activated receptor‐γ (PPARγ) is an essential transcription factor for adipocyte differentiation. In mesenchymal stem cells, PPARγ has been assumed to play a negative role in osteoblastic differentiation, by working in an adipogenesis dependent manner, due to the reciprocal relationship between osteoblast and adipocyte differentiation. However, the direct role of PPARγ in osteoblast function is not fully understood, due in part to inadequate model systems. Here, we describe an adenoviral‐mediated PPARγ knockout system in which suppression of PPARγ in mesenchymal stem cells enhanced osteoblast differentiation and inhibited adipogenesis in vitro. Consistent with this in vitro observation, lipoatrophic A‐ZIP/F1 mice, which do not form adipocytes, displayed a phenotype in which both cortical and trabecular bone was significantly increased compared with wild‐type mice. We next developed an inducible osteoblast‐targeted PPARγ knockout ( Osx Cre/flox‐ PPARγ ) mouse to determine the direct role of PPARγ in bone formation. Data from both in vitro cultures of mesenchymal stem cells and in vivo µCT analysis of bones suggest that suppression of PPARγ activity in osteoblasts significantly increased osteoblast differentiation and trabecular number. Endogenous PPARγ in mesenchymal stem cells and osteoblasts strongly inhibited Akt/mammalian target of rapamycin (mTOR)/p70S6k activity and led to decreased osteoblastic differentiation. Therefore, we conclude that PPARγ modulates osteoblast differentiation and bone formation through both direct and indirect mechanisms. The direct mode, as shown here, involves PPARγ regulation of the mTOR pathway, while the indirect pathway is dependent on the regulation of adipogenesis. S tem C ells 2013;31:2183–2192Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/100308/1/stem1455.pd