支持这项工作是由美国国立卫生研究院（7R01HL098528-03）和FAMRI的临床创新奖。

1。蛋白质转染试剂的制备<ol><li>含干膜管加入250μl的甲醇或氯仿中溶解的Pro-Ject的试剂。 </li><li>涡以最快的速度为10-20秒。 </li><li>项目临试剂吸取20微升到单独的微量离心管。 </li><li>减压蒸除溶剂，通过将含有至少6小时，在室温下在层流罩下的Pro-Ject的试剂的微量离心管中。它必须是完全干燥的。另外，干燥的Pro-Ject的试剂使用真空干燥器。 </li><li>管，于-20℃下保存他们是很好的一年，在此温度下。 </li></ol> 2。临项目试剂添加蛋白质<ol><li>吸取50微升PBS，含2.0μg的感兴趣的蛋白质，含干燥的Pro-Ject的转染试剂20μl的成筒。 </li><li>加入蛋白后，在较低速度f振荡混合溶液或3-5秒，然后在室温下孵育30分钟。 </li></ol> 3。气管内注射蛋白质<ol><li>麻醉小鼠腹腔注射氯胺酮/甲苯​​噻嗪（100毫克/公斤和10毫克/公斤）。 </li><li>镇静后，小鼠采用特殊设计的平台（ 图1）上切牙暂停。 </li><li>使用镊子和一个文件夹作为一个喉镜，口咽打开，使得该导管可以被引入（ 图2）。气管的鼠标放置一个光源对声带的可视化。 </li><li>蛋白质/项目试剂混合物（50微升）注射通过气管使用宾州世纪Microsprayer。 </li></ol>

<ol>
	<li>Glasser, S. W., Korfhagen, T. R., Wert, S. E., Whitsett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+models+for+study+of+pulmonary+development+and+disease.">Transgenic models for study of pulmonary development and disease.</a> Am. J. Physiol. 267, 489-497 (1994).</li><li>D'Armiento, J., Dalal, S. S., Okada, Y., Berg, R. A., Chada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagenase+expression+in+the+lungs+of+transgenic+mice+causes+pulmonary+emphysema.">Collagenase expression in the lungs of transgenic mice causes pulmonary emphysema.</a> Cell. 71, 955-961 (1992).</li><li>Foronjy, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+dismutase+expression+attenuates+cigarette+smoke-+or+elastase-generated+emphysema+in+mice.">Superoxide dismutase expression attenuates cigarette smoke- or elastase-generated emphysema in mice.</a> Am. J. Respir Crit. Care Med. 173, 623-631 (2006).</li><li>Foronjy, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+divergent+roles+of+secreted+frizzled+related+protein-1+(SFRP1)+in+lung+morphogenesis+and+emphysema.">The divergent roles of secreted frizzled related protein-1 (SFRP1) in lung morphogenesis and emphysema.</a> Am. J. Pathol. 177, 598-607 (2010).</li><li>Costa, R. H., Kalinichenko, V. 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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. 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+(Pp2a)+Regulates+Innate+Immune+and+Proteolytic+Responses+to+Cigarette+Smoke+Exposure+in+the+Lung.">Protein Phosphatase 2a (Pp2a) Regulates Innate Immune and Proteolytic Responses to Cigarette Smoke Exposure in the Lung.</a> Toxicol. Sci. , (2012).</li><li>Brown, R. H., Walters, D. M., Greenberg, R. S., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+of+endotracheal+intubation+and+pulmonary+functional+assessment+for+repeated+studies+in+mice.">A method of endotracheal intubation and pulmonary functional assessment for repeated studies in mice.</a> J. Appl. Physiol. 87, 2362-2365 (1999).</li></ol>

作者有没有利益冲突的披露。

与其他方法相比，这种技术的优点是，它产生在肺组织内本身的蛋白水平和活性增加。此外，它渗透到肺，而不是只是停留于气道的最前端区域。我们测量蛋白活性增加我们的注射液，甚至气道灌洗后用生理盐水15。组织活动的分析表明，该蛋白进入细胞，并不会只留在空气空间的鼠标。通过免疫组化显示弥漫我们注入的蛋白质染色肺泡内和呼吸道的鼠标（ 图3），这进一步?...

<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

增加蛋白质表达，使研究人员能够更好地了解这种蛋白质的功能作用，在调控的关键生物过程1。在肺中，这已被典型地实现通过遗传方法，利用转基因小鼠的2,3或蛋白水平提升的病毒或非病毒载体，通过基因表达增加4。是昂贵的和费时的，以产生转基因小鼠，和随机插入 ​​的转基因或慢性的基因的表达，可以改变正常肺发育，从而限制在模型5的效用。虽然避免与慢性基因表达6，反向四环素控制的反式激活（rtTA）小鼠，这是用来生成条件表达式相关联的问题的条件的转基因品种，开发自发空气空间扩大7。与转基因植物，病毒和非病毒载体的使用是昂贵的，并可引发剂量-D炎症反应，ependent混淆的结果，阻碍表达10。此外，反复给药的功效的增强免疫反应的载体11,12的限制。研究人员正在开发的腺相关病毒（AAV）载体，引起炎症较轻，并有较长的表达式中肺13。 使用β-半乳糖苷酶，我们提出了一个方法，迅速和有效地提高蛋白表达在肺部直接使用蛋白转染技术。此协议混合固定量的纯化的蛋白质，用脂质体转染试剂20μl的（亲Ject的皮尔斯生物技术），以允许渗透到肺组织本身。的蛋白脂质体混合物，然后注入经由气管使用microsprayer（宾州世纪，费城，宾夕法尼亚州）的小鼠的肺中。的microsprayer产生罚款羽整个肺部的液体气溶胶。使用该技术我们已经证明了均匀注入的蛋白质沉积，整个呼吸道和肺泡的小鼠14。的脂质转染技术允许使用一个小的量的蛋白质来实现的效果。这限制了炎症反应，否则将挑起高蛋白管理。事实上，使用这种技术，我们发表，我们能够显着提高PP2A活性而不影响在肺肺灌洗细胞型15。肺灌洗细胞构成挑战后24小时可比对照组（27±4控制为31±5白蛋白转;每组N = 6）。此外，它增加蛋白水平的而诱导肺发育变化或架构变更，可发生在转基因模型。然而，需要重复给药可能使这种技术逊于长期增加蛋白表达的影响研究。这将是特别是TRUE蛋白质半衰期短。

<html>为了证明我们的技术的有效性，我们使用了microsprayer（宾州世纪）注入小鼠的小鼠用2微克白蛋白（Sigma公司）溶解在50μlPBS中含有临Ject的转染试剂20μl的气管。白蛋白处理的小鼠相比，与β-半乳糖苷酶蛋白的2微克（皮尔斯生物技术），以相同的方式处理的小鼠。二十四小时后，将小鼠处死并肺部组织学分析处理。福尔马林固定，白蛋白和β-半乳糖苷酶处理的小鼠的肺组织切片进行免疫组化β-?...

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

Protein Transfection of Mouse Lung

转基因小鼠或病毒载体已用于肺内，以增加蛋白质的表达。然而，这些技术是费时，技术上具有挑战性，并有脱靶效应，可以混淆结果。蛋白转染协议使用基于脂质的转染的试剂和超细microsprayer的均匀地提供肺细胞的活性的蛋白质。

蛋白转染小鼠肺

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 次观看.  St. Luke's Roosevelt Medical Center. 转基因小鼠或病毒载体已用于肺内，以增加蛋白质的表达。然而，这些技术是费时，技术上具有挑战性，并有脱靶效应，可以混淆结果。蛋白转染协议使用基于脂质的转染的试剂和超细microsprayer的均匀地提供肺细胞的活性的蛋白质。

视频: 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.

海马神经元的基因枪转

科研

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.

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

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.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

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.

溶质直接气管滴入小鼠肺

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.

小鼠胚胎的龙文化，一个系统来评价分枝的分子机制

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.

活体成像和治疗卵巢癌的原位小鼠模型

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.

单细胞转染鸡胚

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.

表征隔离，通风，和仪表小鼠肺灌注与脉动流

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.

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

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.

诱导多能干细胞来源于小鼠的生成

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