Este trabajo fue financiado por los Institutos Nacionales de Salud (7R01HL098528-03) y el Premio a la Innovación Clínica de FAMRI.

1. Preparación de Proteína reactivo de transfección <ol><li> Disolver el reactivo Pro-Ject mediante la adición de 250 l de metanol o cloroformo al tubo que contiene la película seca. </li><li> Vortex durante 10-20 segundos a velocidad máxima. </li><li> Pipetear 20 l de reactivo de Pro-Ject en tubos de microcentrífuga separados. </li><li> Evaporar el disolvente mediante la colocación de los tubos de microcentrífuga que contiene el reactivo Pro-Ject bajo una campana de flujo laminar durante un mínimo de 6 horas a temperatura ambiente. Debe estar completamente seco. Alternativamente, secar el reactivo Pro-Ject utilizando un secador de vacío. </li><li> Guarde los tubos a -20 ° C. Son válidos por un año a esta temperatura. </li></ol> 2. Añadir proteína a reactivo Pro-Ject <ol><li> Pipetear 50 l de PBS, que contenía 2,0 g de la proteína de interés, en un tubo que contiene 20 l de reactivo de transfección Pro-Ject seca. </li><li> Después de agregar la proteína, mezcle la solución mediante agitación a baja velocidad fo 3-5 seg seguido por incubación durante 30 min a temperatura ambiente. </li></ol> 3. Administración intratraqueal de Proteína <ol><li> Anestesiar a los ratones por inyección intraperitoneal de ketamina / xilazina (100 mg / kg y 10 mg / kg). </li><li> Después se logra la sedación, los ratones son suspendidos de sus incisivos superiores utilizando una plataforma especialmente diseñado (Figura 1). </li><li> El uso de pinzas y un clip de papel como un laringoscopio, se abre la orofaringe modo que el catéter puede ser introducido (Figura 2). Las cuerdas vocales se visualizan mediante la colocación de una fuente de luz frente a la tráquea del ratón. </li><li> La mezcla de reactivos proteína / Proyecto (50 l) se inyecta a través de la tráquea utilizando un siglo Microsprayer Penn. </li></ol>

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+humoral+immune+responses+to+viral+antigens+create+barriers+to+lung-directed+gene+therapy+with+recombinant+adenoviruses.">Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.</a> J. Virol. 69, 2004-2015 (1995).</li><li>Liu, Q., Muruve, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+inflammatory+response+to+adenovirus+vectors.">Molecular basis of the inflammatory response to adenovirus vectors.</a> Gene Ther. 10, 935-940 (2003).</li><li>Pfeifer, C., Aneja, M. K., Hasenpusch, G., Rudolph, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+serotype+9-mediated+pulmonary+transgene+expression:+effect+of+mouse+strain,+animal+gender+and+lung+inflammation.">Adeno-associated virus serotype 9-mediated pulmonary transgene expression: effect of mouse strain, animal gender and lung inflammation.</a> Gene Ther. 18, 1034-1042 (2011).</li><li>Wallace, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+phosphatase+2A+regulates+innate+immune+and+proteolytic+responses+to+cigarette+smoke+exposure+in+the+lung.">Protein phosphatase 2A regulates innate immune and proteolytic responses to cigarette smoke exposure in the lung.</a> Toxicol. Sci. 126, 589-599 (2012).</li><li>Wallace, A. 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Los autores no tienen conflictos de intereses a revelar.

La ventaja de esta técnica sobre otros métodos es que produce aumentos en los niveles de proteína y la actividad en el tejido pulmonar en sí. Por otra parte, penetra a las regiones más distales del pulmón en lugar de alojarse sólo dentro de las vías respiratorias. Hemos medido aumento de la actividad de proteínas de nuestra inyectado incluso después lavaging las vías respiratorias con solución salina 15. Los análisis de actividad del tejido indican que la proteína está entrando en las células ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Pro-Ject</td>
 <td>Pierce Bio</td>
 <td>89850</td>
 <td></td>
 </tr>
 <tr>
 <td>Microsprayer</td>
 <td>Penn Century</td>
 <td>FMJ-250</td>
 <td></td>
 </tr>
 <tr>
 <td>Beta-galactosidase</td>
 <td>Pierce Bio</td>
 <td>89850</td>
 <td></td>
 </tr>
 <tr>
 <td>Beta-galactosidase antibody</td>
 <td>Santa Cruz Bio</td>
 <td>SC-19119</td>
 <td></td>
 </tr>
 <tr>
 <td>Mouse serum albumin</td>
 <td>Sigma Aldrich</td>
 <td>A3139</td>
 <td></td>
 </tr>
 <tr>
 <td>Ketamine/xylazine</td>
 <td>Sigma Aldrich</td>
 <td>K113</td>
 <td></td>
 </tr>
 </tbody>
</table>

protein transfection of mouse lung

El aumento de la expresión de proteínas permite a los investigadores a entender mejor el papel funcional de esta proteína en la regulación de procesos biológicos clave 1. En el pulmón, esto se ha logrado típicamente a través de enfoques genéticos que utilizan ratones transgénicos 2,3 o vectores virales o no virales que elevan los niveles de proteína a través de aumento de la expresión del gen 4. Los ratones transgénicos son costosos y requiere mucho tiempo para la generación y la inserción aleatoria de un gen de expresión del transgen o crónica pueden alterar el desarrollo normal de los pulmones y por lo tanto limitar la utilidad del modelo 5. Mientras que los transgénicos condicionales evitar problemas asociados con la expresión crónica gen 6, los ratones transactivador controlado por tetraciclina inverso (rtTA), que se utilizan para generar la expresión condicional, se desarrollan espontánea aire ampliación espacio 7. Al igual que con los transgénicos, el uso de vectores virales y no virales es caro 8 y puede provocar dosis-drespuestas inflamatorias ependent que confunden los resultados 9 y dificultan la expresión 10. Por otra parte, la eficacia de las dosis repetidas están limitados por las respuestas inmunes mejoradas a el vector de 11,12. Los investigadores están desarrollando adeno-asociado vectores virales (AAV) que provocan una menor inflamación y tener una expresión más larga dentro del pulmón 13. Uso de β-galactosidasa, se presenta un método para incrementar la expresión rápida y eficazmente la proteína dentro del pulmón utilizando una técnica directa de transfección de proteína. Este protocolo se mezcla una cantidad fija de proteína purificada con 20 l de un reactivo de transfección a base de lípidos (Pro-Ject, Pierce Bio) para permitir la penetración en el tejido pulmonar en sí. La mezcla de proteína liposómica se inyecta entonces en los pulmones de los ratones a través de la tráquea utilizando un microsprayer (Penn Century, Filadelfia, PA). El microsprayer genera una fina nube de aerosol líquido a través de los pulmones. Utilizando la técnicahemos demostrado la deposición uniforme de la proteína inyectada a través de las vías respiratorias y los alvéolos de los ratones 14. La técnica de transfección de lípidos permite el uso de una pequeña cantidad de proteína para lograr el efecto. Esto limita la respuesta inflamatoria que de otro modo sería provocado por alta administración de la proteína. En efecto, el uso de esta técnica hemos publicado que hemos sido capaces de aumentar significativamente la actividad de PP2A en el pulmón sin afectar pulmón celularidad lavado 15. Lavado pulmonar celularidad tomada 24 horas después de la exposición fue comparable a los controles (27 ± 4 Control vs 31 ± 5 albúmina transfectadas, N = 6 por grupo). Además, aumenta los niveles de proteína sin inducir cambios en el desarrollo del pulmón o cambios en la arquitectura que pueden ocurrir en modelos transgénicos. Sin embargo, la necesidad de administraciones repetidas puede hacer esta técnica menos favorable para los estudios que examinan los efectos de los aumentos a largo plazo en la expresión de la proteína. Esto sería particularmente true de las proteínas con una vida media corta.

<html> Para demostrar la eficacia de nuestra técnica se utilizó una microsprayer (Penn Century) para inyectar la tráquea de los ratones con 2 mg de albúmina de ratón (Sigma) disuelto en 50 l de PBS que contenía 20 l de reactivo de transfección Pro-Ject. Los ratones tratados con albúmina se compararon con ratones que fueron tratados de manera idéntica con 2 g de proteína beta-galactosidasa (Pierce Bio). Después de veinticuatro horas los ratones fueron sacrificados y los pulmones se procesaron para el análisis ...

Watch this Scientific Journal Video about Protein Transfection of Mouse Lung at JoVE.com

Protein Transfection of Mouse Lung

Los ratones transgénicos o vectores virales se han utilizado para aumentar la expresión de la proteína en el pulmón. Sin embargo, estas técnicas son mucho tiempo, y técnicamente difícil tener efectos fuera de objetivo que pueden confundir los resultados. Nuestro protocolo de transfección de proteína utiliza un reactivo de transfección basado en lípidos y un microsprayer ultrafino para entregar de manera uniforme proteína activa a las células pulmonares.

La transfección de proteínas de ratón de pulmón

Increasing protein expression enables researchers to  better understand the functional role of that protein in regulating key  biological processes1. In ...

Research

JoVE Journal

Biology

9.2K vistas.  St. Luke's Roosevelt Medical Center. Los ratones transgénicos o vectores virales se han utilizado para aumentar la expresión de la proteína en el pulmón. Sin embargo, estas técnicas son mucho tiempo, y técnicamente difícil tener efectos fuera de objetivo que pueden confundir los resultados. Nuestro protocolo de transfección de proteína utiliza un reactivo de transfección basado en lípidos y un microsprayer ultrafino para entregar de manera uniforme proteína activa a las células pulmonares.

Video: Protein Transfection of Mouse Lung

Gene-gun Transfection of Hippocampal Neurons

Gene-gun Transfección de las neuronas del hipocampo

Investigación

Biología

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Tinción de las proteínas en geles con Coomassie G-250, sin ácido disolvente orgánico y el acético

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Direct Tracheal Instillation of Solutes into Mouse Lung

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice. Using this approach, one can target delivery of test solutes or solids (such as lung therapeutics, surfactants, viruses, and small oligonucleotides) into the distal lung. Tracheal instillations may be the preferred methodology, over inhalation protocols that may primarily target the upper respiratory tract and possibly expose the investigator to potentially hazardous substances. Additionally, in using the tracheal instillation protocol, animals can fully recover from the non-invasive procedure. This allows for making subsequent physiological measurements on test animals, or reinstallation using the same animal. The amount of instillate introduced into the lung must be carefully determined and osmotically balanced to ensure animal recovery. Typically, 30-75 &mu;L instillate volume can be introduced into mouse lung.

Intratracheal instillations deliver solutes directly into the lungs. This procedure targets the delivery of the instillate into the distal regions of the lung, and is therefore often incorporated in studies aimed at studying alveoli. We provide a detailed survival protocol for performing intratracheal instillations in mice.

La instilación directa traqueal de solutos en pulmón de ratón

Intratracheal instillations deliver solutes directly into the lungs.  This procedure targets the delivery of the instillate into the distal regions of the ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Cultura de embriones de ratón de pulmón, un sistema para evaluar los mecanismos moleculares de la ramificación

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

En vivo de imágenes y tratamientos terapéuticos en un modelo de ratón ortotópico de cáncer de ovario

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

Caracterización de los aislados y ventilados, y pulmón de ratón instrumentado perfundidos con flujo pulsátil

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Aislamiento y Caracterización de Hoechst Baja CD45 Negativo Mouse células madre mesenquimales de pulmón

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generación de ratones derivados de células madre pluripotentes inducidas

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods are needed that can quantify the pathologic changes. Furthermore, to provide translational relevance to the mouse models, such measurements should be tests that can easily be done in both humans and mice. Unfortunately, in the present literature few phenotypic measurements of lung function have direct application to humans. One exception is the diffusing capacity for carbon monoxide, which is a measurement that is routinely done in humans. In the present report, we describe a means to quickly and simply measure this diffusing capacity in mice. The procedure involves brief lung inflation with tracer gases in an anesthetized mouse, followed by a 1 min gas analysis time. We have tested the ability of this method to detect several lung pathologies, including emphysema, fibrosis, acute lung injury, and influenza and fungal lung infections, as well as monitoring lung maturation in young pups. Results show significant decreases in all the lung pathologies, as well as an increase in the diffusing capacity with lung maturation. This measurement of lung diffusing capacity thus provides a pulmonary function test that has broad application with its ability to detect phenotypic structural changes with most of the existing pathologic lung models.

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

Función Fenotipificación ratón pulmonar<em&gt; En Vivo</em&gt; Con la capacidad de difusión pulmonar

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...