この作品は、国立衛生研究所（7R01HL098528-03）とFAMRIの臨床イノベーター賞によってサポートされていました。

1。タンパク質トランスフェクション試薬の調製<ol><li>ドライフィルムを含む試験管にメタノールやクロロホルムを250μlを加えることによりプロジェクト試薬を溶解する。 </li><li>トップスピードで10〜20秒間ボルテックスする。 </li><li>別個のマイクロ遠心チューブにプロジェクト試薬のピペットを20μl。 </li><li>室温で6時間以上は、層流フードの下プロジェクト試薬を含有するマイクロチューブを配置することによって、溶媒を蒸発する。それは完全に乾燥していなければならない。また、真空デシケーターを用いたプロジェクト試薬を乾燥させます。 </li><li> -20℃でチューブを保管彼らは、この温度で​​1年間に適しています。 </li></ol> 2。プロジェクト試薬にプロテインを追加する<ol><li> PBSのピペットを50μl、乾燥プロジェクトのトランスフェクション試薬を20μlを含むチューブに、目的のタンパク質の2.0アンピシリンを含む。 </li><li>タンパク質を追加した後、低速fでボルテックスして、ソリューションをミックスまたは3-5秒室温で30分間インキュベートした。 </li></ol> 3。プロテインの気管内投与<ol><li>ケタミン/キシラジン（100 mg / kg体重および10 mg / kg体重）の腹腔内注射によってマウスを麻酔。 </li><li>鎮静が達成された後、マウスを、特別に設計されたプラットフォーム（ 図1）を使用して上顎から吊り下げられている。 </li><li>喉頭鏡として鉗子やクリップを使用して、中咽頭（ 図2）カテーテルを導入することができるように開放される。声帯は、マウスの気管に対して光源を配置することによって可視化される。 </li><li>タンパク質/プロジェクト試薬混合物（50μl）をペンセンチュリーMicrosprayerを使って気管を介して注入されています。 </li></ol>

<ol>
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著者らは、開示する利害の衝突を持っていません。

他の方法に比べて、この技術の利点は、肺組織自体の中のタンパク質レベルおよび活性の増加を生じることである。また、それだけで気道内に滞在するのではなく、肺の最も遠​​位の地域に浸透。私たちも、生理食塩水15で気道を洗浄助後に注入液の増加したタンパク質の活性を測定した。組織アクティビティ分析は、タンパク質が細胞に入るとき、ちょうどマウスの空気空間内に...

<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

増やすタンパク質の発現は、より良いキー生物学的プロセス1を調節するそのタンパク質の機能的役割を理解するために研究者を可能にします。肺では、これは、トランスジェニックマウス2,3又は増加した遺伝子発現4を介してタンパク質レベルを上昇させるウイルスまたは非ウイルスベクターを利用する遺伝的アプローチを介して典型的に達成された。トランスジェニックマウスを生成するためにコストと時間がかかり、導入遺伝子又は慢性の遺伝子発現のランダム挿入は、正常な肺の発達を変更し、従って、モデル5の有用性を制限することができる。条件式を生成するために使用される慢性の遺伝子発現6、逆テトラサイクリン制御型トランス（rtTA）マウスに関連する問題AVERT条件トランスジェニックながら、自発的なエアスペースの拡大7を開発する。トランスジェニックと同様に、ウイルス性および非ウイルスベクターの使用は、8高価であり、用量dのを誘発することができ結果9を混乱し、式10を妨げるependent炎症反応。また、反復投与の有効性は、ベクトル11,12に強化された免疫応答によって制限されます。研究者は少なく炎症を誘発し、肺13内に長い式を持つアデノ随伴ウイルス（AAV）ベクターの開発を進めています。 β-ガラクトシダーゼを用いて、迅速かつ効果的に直接タンパク質トランスフェクション技術を用いて肺内のタンパク質の発現を増加させるための方法を提案する。このプロトコルは、肺組織自体への浸透を可能にする脂質ベースのトランスフェクション試薬20μlの（プロジェクト、ピアースバイオ）を用いて精製タンパク質の一定量を混合する。リポソームタンパク質混合物を次いmicrosprayer（ペン世紀、フィラデルフィア、PA）を用いて気管を介してマウスの肺に注入される。 microsprayerは、肺全体に液体エアロゾルの微プルームを生成します。技術を用いて私たちは、気道やマウス14の肺胞を通して注入されたタンパク質の均一な付着を実証している。脂質トランスフェクション手法は、効果を達成するために少量のタンパク質の使用を可能にする。これは、そうしないと高タンパク投与によって引き起こされる炎症反応を制限します。実際、この手法使って、我々はかなり肺胞細胞性15に影響を与えることなく、肺のPP2A活性を高めることができたことを発表した。チャレンジ後24時間を取られ、肺胞細胞充実性は、コントロール（;グループ当たりN = 6 27±4制御対31±5アルブミントランスフェクションされた）に匹敵するものであった。また、トランスジェニックモデルで発生する可能性があり、肺の発達変更やアーキテクチャの変更を誘発することなく、タンパク質レベルを増加させます。しかし、反復投与の必要性は、タンパク質発現の長期的増加する効果を調べる研究のためにこの技術はあまり好ましく行うことができる。これはTRU特にだろう短い半減期を有するタンパク質の電子。

<html>私たちの技術の有効性を実証するために、我々はプロジェクトのトランスフェクション試薬20μlのが含まれていたPBS50μlの中に溶解させたマウスの2μgのアルブミン（Sigma）でマウスの気管に注入するmicrosprayer（ペンセンチュリー）を使用していました。アルブミン処置したマウスは、β-ガラクトシダーゼタンパク質2μgの（ピアースバイオ）と同一の方法で処理したマウスと比較した。二?...

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

Protein Transfection of Mouse Lung

トランスジェニックマウスまたはウイルスベクターは、肺内のタンパク質の発現を増加させるために使用されてきた。しかしながら、これらの技術は、技術的に困難な、時間がかかり、その結果を混乱させることができオフターゲット作用を有する。当社タンパク質トランスフェクションプロトコルは、脂質ベースのトランスフェクション試薬と一様に肺細胞に活性なタンパク質を提供する超microsprayerを使用しています。

マウス肺のタンパク質トランスフェクション

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

Research

JoVE Journal

Biology

9.3K ビュー.  St. Luke's Roosevelt Medical Center. トランスジェニックマウスまたはウイルスベクターは、肺内のタンパク質の発現を増加させるために使用されてきた。しかしながら、これらの技術は、技術的に困難な、時間がかかり、その結果を混乱させることができオフターゲット作用を有する。当社タンパク質トランスフェクションプロトコルは、脂質ベースのトランスフェクション試薬と一様に?...

ビデオ: Protein Transfection of Mouse Lung

Gene-gun Transfection of Hippocampal Neurons

Hi, I'm Kma. I work in Case Lab at Brown University Clean Slicer stage with 70%ethanol. Place plate insert into six.Well plate with medium and incubate at 37 degree whole brain with tweezers between midbrain and four brainin. Isolate hippocampus on one side, isolate hippocampus from the second side. Place hippocampi onto A PBS drop.Aspirate out the PBS make sagittal sections using the tissue slicer. Repeat transfer slicers onto a perish by squatting with PBS wash sections by adding and aspirating PBS and medium gently. Separate sections with tweezers.Wash sections with PBS and media. Again, transfer sections onto the plate and insert. Aspirate out the media in sections using tweezers.Attach gene gun hose to the helium cylinder. Attach barrel to gene gun. Fill the magazine with bullets.Insert magazine into the Jing gun. Adjust the pressure on the helium tank shoot. EGFP coated beads onto the sections.And now.

海馬ニューロンの遺伝子郡のトランスフェクション

生物学

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.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

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.

液相の回復と分子量ベース画用GELFREE 8100分別システムの使用

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.

マウス肺への溶質の直接気管点眼

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.

マウス胎児肺の文化、分岐の分子機構を評価するシステム

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.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

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

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

鶏胚における単一細胞のトランスフェクション

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

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.

絶縁型、ベンチレーテッド、および計装のマウス肺の特性評価は、拍動流を灌流

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 & 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.

ヘキストの分離および特性評価低 CD45負マウス肺間葉系幹細胞

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.

人工多能性幹細胞に由来するマウスの作製

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