Este trabalho foi financiado pelo National Institutes of Health (7R01HL098528-03) e Innovator Award Clínica FAMRI.

1. Preparação de Proteína Reagente de Transfecção <ol><li> Dissolve-se o reagente Pro-Ject por adição de 250 ul de metanol e clorofórmio, para o tubo contendo a película seca. </li><li> Vortex para 10-20 segundos a velocidade máxima. </li><li> Pipetar 20 ul de reagente Pro-Ject em tubos de microcentrífuga separados. </li><li> Evapora-se o solvente por meio de colocação dos tubos de microcentrífuga contendo o reagente Pro-Ject sob uma câmara de fluxo laminar durante um mínimo de 6 horas à temperatura ambiente. Ele deve estar completamente seca. Alternativamente, secar o reagente Pro-Ject usando um secador de vácuo. </li><li> Armazenar os tubos a -20 ° C. Eles são bons para um ano a esta temperatura. </li></ol> 2. Adicionando a proteína reagente Pro-Ject <ol><li> Pipetar 50 ul de PBS, contendo 2,0 ug de proteína de interesse, dentro de um tubo contendo 20 ul de reagente de transfecção secas Pro-Ject. </li><li> Após a adição da proteína, misturar a solução num vórtice a baixa velocidade fou 3-5 seg seguido de incubação durante 30 min à temperatura ambiente. </li></ol> 3. Administração Intratraqueal de Proteína <ol><li> Anestesiar ratos por injecção intraperitoneal de cetamina / xilazina (100 mg / kg e 10 mg / kg). </li><li> Após sedação é alcançado, os ratos estão suspensos os incisivos superiores, utilizando uma plataforma especialmente concebidos (Figura 1). </li><li> Utilizando uma pinça e um clipe de papel, como um laringoscópio, orofaringe é aberto de modo que o cateter pode ser introduzido (Figura 2). As cordas vocais são visualizados através da colocação de uma fonte de luz contra a traqueia do rato. </li><li> A mistura reagente de proteína / projeto (50 ul) é injetado através da traquéia usando uma Penn Century microsprayer. </li></ol>

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Os autores não têm conflitos de interesse de divulgar.

A vantagem desta técnica em relação a outros métodos é que produz aumentos dos níveis de proteína e de actividade dentro do próprio tecido do pulmão. Além disso, ele penetra para as regiões distais do pulmão em vez de ficar apenas dentro das vias respiratórias. Nós medimos o aumento da atividade da proteína do nosso injetado mesmo depois lavaging as vias aéreas com soro fisiológico 15. As análises de actividade de tecido indicam que a proteína é entrar nas células e não apenas permanecem...

<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

O aumento da expressão da proteína permite aos pesquisadores entender melhor o papel funcional desta proteína na regulação de processos biológicos fundamentais 1. No pulmão, isto foi conseguido tipicamente através de abordagens genéticas que utilizam ratinhos transgénicos 2,3 ou vectores virais ou não virais, que elevam os níveis de proteínas por meio de aumento da expressão do gene 4. Os ratinhos transgénicos são dispendiosas e demoradas para a geração e a inserção aleatória de um gene de expressão do transgene ou crónica pode alterar o desenvolvimento normal dos pulmões e, portanto, limitar a utilidade do modelo de cinco. Enquanto transgénicos condicionais evitam os problemas associados com a expressão do gene crónica 6, os transactivador (rtTA) tetraciclina ratinhos controlados reversa, que são utilizados para gerar a expressão condicional, desenvolver espontânea alargamento espaço de ar 7. Tal como acontece com transgénicos, a utilização de vectores virais e não virais é caro 8 e pode provocar a dose drespostas inflamatórias ependent que confundem os resultados 9 e dificultar a expressão 10. Além disso, a eficácia de doses repetidas são limitados por respostas imunes reforçadas ao vetor de 11,12. Pesquisadores estão desenvolvendo adeno-associado vetores virais (AAV) que provocam menos inflamação e têm mais expressão dentro do pulmão 13. Usando β-galactosidase, apresentamos um processo para o aumento da expressão de proteínas rápida e eficazmente dentro do pulmão, usando uma técnica de transfecção directa de proteínas. Este protocolo de mistura uma quantidade fixa de proteína purificada com 20 ul de um reagente de transfecção à base de lípidos (Pro-Ject, Pierce Bio) para permitir a penetração no próprio tecido do pulmão. A mistura de proteína lipossomal é então injectado para dentro dos pulmões dos ratos através da traqueia usando uma microsprayer (Penn Century, Filadélfia, PA). O microsprayer gera uma multa nuvem de aerossol líquido ao longo dos pulmões. Usando a técnicademonstrámos deposição uniforme da proteína injectada ao longo das vias aéreas e alvéolos dos ratinhos 14. A técnica de transfecção de lípidos permite o uso de uma pequena quantidade de proteína para alcançar o efeito. Isto limita a resposta inflamatória, que de outra forma seria provocada por administração da proteína. De fato, usando esta técnica nós publicamos que fomos capazes de aumentar significativamente a atividade PP2A no pulmão sem afetar pulmão celularidade do lavado 15. Lavagem pulmonar celularidade feita 24 horas após o desafio foi comparável com os controlos (27 ± 4 Controlo vs 31 ± 5 transfectada albumina, N = 6 por grupo). Além disso, aumenta os níveis de proteína sem induzir mudanças de desenvolvimento do pulmão ou mudanças de arquitectura que pode ocorrer em modelos transgénicos. No entanto, a necessidade de administrações repetidas podem tornar esta técnica menos favorável para estudos examinando os efeitos dos aumentos a longo prazo na expressão da proteína. Isto seria particularmente true de proteínas com uma meia-vida curta.

<html> Para demonstrar a eficácia da nossa técnica foi utilizada uma microsprayer (Penn Century) para injectar a traqueia de ratinhos com 2 mg de albumina de rato (Sigma) dissolvidos em 50 mL de PBS que continha 20 ul de reagente de transfecção Pro-Ject. Os ratinhos tratados com albumina foram comparados com os ratos que foram tratados de modo idêntico, com 2 ug de proteína de beta-galactosidase (Pierce Bio). Depois de vinte e quatro horas, os ratos foram sacrificados e os pulmões foram processados ​​para aná...

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

Protein Transfection of Mouse Lung

Os ratinhos transgénicos ou vectores virais têm sido utilizados para aumentar a expressão de proteína dentro do pulmão. No entanto, estas técnicas são demoradas e tecnicamente desafiante e têm efeitos fora do alvo, que pode confundir os resultados. O protocolo de transfecção de proteínas utiliza um reagente de transfecção à base de lipidos e uma microsprayer ultrafina para entregar uniformemente proteína activa a células do pulmão.

A transfecção de proteína de rato Lung

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 visualizações.  St. Luke's Roosevelt Medical Center. Os ratinhos transgénicos ou vectores virais têm sido utilizados para aumentar a expressão de proteína dentro do pulmão. No entanto, estas técnicas são demoradas e tecnicamente desafiante e têm efeitos fora do alvo, que pode confundir os resultados. O protocolo de transfecção de proteínas utiliza um reagente de transfecção à base de lipidos e uma microsprayer ultrafina para entregar uniformemente proteína activa a células do pulmão.

Vídeo: Protein Transfection of Mouse Lung

Gene-gun Transfection of Hippocampal Neurons

Gene-gun Transfection de neurônios do hipocampo

Biologia

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.

Coloração de proteínas em géis com Coomassie G-250 sem ácido acético e Solventes Orgânicos

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.

Usando o sistema de fracionamento GELFREE 8100 para Fracionamento baseada no peso molecular com Recovery 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.

Instilação traqueal direta de Solutos em pulmão de rato

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 do mouse pulmão embrionário, um sistema para avaliar os mecanismos moleculares de Ramificação

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.

In vivo de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

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.

Caracterização do isolado, ventilado, Lung Mouse e Instrumentados perfundidos com fluxo 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.

Isolamento &amp; Caracterização da Hoechst Baixa CD45 Negativo Mouse Células-Tronco Mesenquimais de pulmão

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.

Geração de camundongos derivadas de células-tronco pluripotentes induzidas

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.

Função Pulmonar Fenotipagem Rato<em&gt; In Vivo</em&gt; Com a capacidade pulmonar Diffusing

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 ...