这项工作得到了斯洛文尼亚的研究机构（项目，基建中心IP J2 - 9770 - 0510和程序P2 - 0249）。这个视频代表的"电穿孔技术为基础的技术和处理"的科学研讨会，并在斯洛文尼亚的卢布尔雅那大学电气工程学院举办的研究生课程，补充材料。作者还感谢Duša Hodžič请提供质粒DNA。

1。细胞培养，质粒和缓冲区，实验的准备<ol><li>在这个实验中，使用中国仓鼠卵巢细胞（CHO - K1）。细胞是生长在一种营养混合物的HAM - F12辅以2毫米L -谷氨酰胺，10％胎牛血清，400μL/ L庆大霉素（所有自Sigma - Aldrich化学公司，Deisenhofen，德国），和1（PAA）的毫升/升crystacilin（Pliva公司，萨格勒布，克罗地亚）。细胞都保持在37 ° C，饱和湿度5％的CO 2，在孵化器的气氛为24小时。 </li><li>放大质粒pEGFP - N1（Clontech公司Laboratories公司，山景，加利福尼亚，美国）编码大肠杆菌DH5α菌株绿色荧光蛋白（GFP）和隔离HiSpeed​​质粒马克西试剂盒（QIAGEN，Hilden的，德国）。质粒DNA的浓度（质粒在TE缓冲液溶解）应在260 nm分光光度计确定，并经凝胶电泳证实。 </li><li>准备isoosmolar磷酸钠缓冲液（10毫米的Na 2 HPO 4，10毫米的NaH 2 PO 4，MgCl 2的 1毫米，250毫米蔗糖，pH值7.4）。 </li><li>在实验当天准备由胰蛋白酶细胞悬液，用0.25％胰酶/ EDTA溶液（Deisenhofen，德国化学公司，Sigma - Aldrich公司）。离心机细胞5分钟在1000转（180 XG）4 ° C（Sigma公司，德国）和重悬细胞沉淀isoosmolar磷酸钠缓冲液到一个细胞密度为5 × 10 6细胞/ ml。 </li></ol> 2。硬件设备<ol><li>细胞暴露在枪头电场（图1）与集成电极连接到一个高电压的原型发电机。针尖与电极几何形状允许应用相对均匀的电场和电脉冲发生器允许在不同的方向传递。卢布尔雅那大学电气工程学院，生物控制论，在实验室开发的尖端和发电机。 </li></ol><img src="/files/ftp_upload/3309/3309fig1.jpg" alt="图1"/> 图1。垂直和水平（一）横截面和照片（B）与集成电极尖端吸管。在灰色的截面是用于塑料外壳和电极的黑色。集成了电极的枪头由四个圆棒电极。电极由不锈钢制成，其直径为1.4毫米，相邻电极相距1毫米，而相反的电极是2毫米。电极粘成并行的塑料尖，其适用的长度是30 毫米 2 。 3。基因electrotransfer协议<ol><li>质粒pEGFP - N1的细胞悬液，在浓度为10微克/毫升。 </li><li>为2-3分钟，在室温下孵育的混合物，在提出申请前电脉冲。 </li><li> ASPIRE 100μL细胞悬液与集成电极枪头。 </li><li>为了达到最佳的基因electrotransfer疗效，并保持细胞活力，应使用最佳参数的电脉冲。在这个实验中，8矩形脉冲（每1毫秒，振幅225 V的重复频率为1 Hz的时间）的火车是适用于每个样本，使用高电压原型发生器。使用了两种不同的电场协议（图2）:在第一个协议，所有的脉冲传递在同一个方向，而在第二个协议脉冲通过改变或者电场方向和方向交付。第二个协议只能使用适当的脉冲发生器，它允许应用程序在不同方向的电脉冲。 </li><li>后立即脉冲的应用程序转移到6孔板枪头的细胞，并添加胎牛血清（FCS - 美国Sigma公司）（样本量的25％）。 </li><li> 5分钟的细胞在37 ° C至使细胞膜上再加盖印章。 </li><li>每个样品中加入2毫升的HAM - F12在6孔，并在37 ° C孵育24小时的细胞在饱和湿度5％的CO 2在孵化器的气氛。 </li></ol><img src="/files/ftp_upload/3309/3309fig2.jpg" alt="图2"/> 图2电场的协议:（一）所有脉冲是在同一个方向传递，（二）脉冲传递改变或者电场方向和方向。 4。图像采集和测定基因electrotransfer疗效<ol><li>基因electrotransfer疗效的细胞表达GFP的24小时后的脉冲应用程序的百分比确定。 </li><li>使用与单色仪系统（彩瓷四，Visitron，德国）和排放量在507 nm处检测产生的488 nm的光激发荧光显微镜（在我们的例子中，AXIOVERT，ZR德国蔡司200）观察细胞。使用成像系统（MetaMorph成像系统，Visitr影像记录也可用于上，德国），但其他类似的数据采集软件。 </li><li>收购至少5张图像放大20倍的目标（相衬和绿色荧光）。 </li><li>计数相衬图像的细胞和细胞表达GFP绿色荧光图像。确定基因electrotransfer疗效的百分比除以细胞表达GFP的所有细胞，在每个相应的图像（图3）。 </li></ol> 5。代表性的成果: <img src="/files/ftp_upload/3309/3309fig3.jpg" alt="图3"/> 图3时所有的脉冲是在同一个方向，并交付脉冲或者改变电场方向和方向，提出交付时表达GFP细胞的百分比。细胞暴露于列车的8个脉冲振幅225伏，持续时间为1 ms，重复频率为1 Hz。结果获得通过荧光显微镜。图中的每个值代表三个独立实验的±标准差的平均值。通过改变电场的方向和方向的细胞表达GFP增加的百分比。

<ol>
	<li>Rebersek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporator+with+automatic+change+of+electric+field+direction+improves+gene+electrotransfer+in-vitro.">Electroporator with automatic change of electric field direction improves gene electrotransfer in-vitro.</a> Biomed Eng Online. 6, 25-25 (2007).</li><li>Trontelj, K., Rebersek, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tip+electrode+chamber+for+small+volume+electroporation,+electrofusion,+and+gene+transfection.">Tip electrode chamber for small volume electroporation, electrofusion, and gene transfection.</a> 18, (2006).</li><li>Neumann, E., Schaefer-Ridder, M., Wang, Y., Hofschneider, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+into+mouse+lyoma+cells+by+electroporation+in+high+electric+fields.">Gene transfer into mouse lyoma cells by electroporation in high electric fields.</a> EMBO J. 1, 841-841 (1982).</li><li>Daud, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+trial+of+interleukin-12+plasmid+electroporation+in+patients+with+metastatic+melanoma.">Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma.</a> J Clin Oncol. 26, 5896-5903 (2008).</li><li>Pavlin, M., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+conductivity+of+a+suspension+of+permeabilized+cells:+a+theoretical+analysis.">Effective conductivity of a suspension of permeabilized cells: a theoretical analysis.</a> Biophys J. 85, 719-729 (2003).</li><li>Xie, T., Sun, L., Zhao, H., Fuchs, J., Tsong, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+mechanisms+of+electric+field-induced+DNA+transfection.+IV.+Effects+of+DNA+topology+on+cell+uptake+and+transfection+efficiency.">Study of mechanisms of electric field-induced DNA transfection. 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Effects of DNA topology on cell uptake and transfection efficiency.</a> Biophys J. 63, 1026-1031 (1992).</li><li>Rols, M., Delteil, C., Serin, G., Teissie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+effects+on+electrotransfection+of+mammalian+cells.">Temperature effects on electrotransfection of mammalian cells.</a> Nucleic Acids Res. 22, 540-540 (1994).</li><li>Golzio, M., Teissie, J., Rols, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+synchronization+effect+on+mammalian+cell+permeabilization+and+gene+delivery+by+electric+field.">Cell synchronization effect on mammalian cell permeabilization and gene delivery by electric field.</a> Biochim Biophys Acta. 1563, 23-28 (2002).</li><li>Haberl, S., Miklavcic, D., Pavlin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Mg+ions+on+efficiency+of+gene+electrotransfer+and+on+cell+electropermeabilization.">Effect of Mg ions on efficiency of gene electrotransfer and on cell electropermeabilization.</a> Bioelectrochemistry. 79, 265-271 (2010).</li><li>Rols, M., Teissie, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electropermeabilization+of+mammalian+cells+to+macromolecules:+control+by+pulse+duration.">Electropermeabilization of mammalian cells to macromolecules: control by pulse duration.</a> Biophys J. 75, 1415-1423 (1998).</li><li>Kanduser, M., Miklavcic, D., Pavlin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+involved+in+gene+electrotransfer+using+high-and+low-voltage+pulses-An+in+vitro+study.">Mechanisms involved in gene electrotransfer using high-and low-voltage pulses-An in vitro study.</a> Bioelectrochemistry. 74, 265-271 (2009).</li><li>Pavlin, M., Flisar, K., Kanduser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+electrophoresis+in+gene+electrotransfer.">The role of electrophoresis in gene electrotransfer.</a> J Membr Biol. 236, 75-79 (2010).</li><li>Sersa, G., Cemazar, M., Semrov, D., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+electrode+orientation+improves+the+efficacy+of+electrochemotherapy+of+solid+tumors+in+mice.">Changing electrode orientation improves the efficacy of electrochemotherapy of solid tumors in mice.</a> Bioelectrochem Bioenerg. 39, 61-66 (1996).</li><li>Faurie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electro-mediated+gene+transfer+and+expression+are+controlled+by+the+life-time+of+DNA/membrane+complex+formation.">Electro-mediated gene transfer and expression are controlled by the life-time of DNA/membrane complex formation.</a> J Gene Med. 12, 117-125 (2010).</li></ol>

没有利益冲突的声明。

基因electrotransfer是一个多功能的生物科技技术，使进入细胞的DNA转移，通过应用短，高电压电脉冲，并由于其安全性，有效性和易于应用的病毒载体，代表了更安全的替代。虽然在今天的基因electrotransfer被广泛用于转染所有类型的细胞和第一阶段的临床试验，使用这种方法，已报道4，底层的机制仍然没有完全理解。据了解，应用足够的实力来的细胞的电脉冲，导致跨膜电位的增加，从而?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> HAM - F12 </td><td>临时机场管理局</td><td> E15 - 016 </td><td>培养基</td></tr><tr><td> L -谷氨酰胺</td><td> Sigma - Aldrich公司</td><td> G7513 </td><td></td></tr><tr><td>胎牛血清</td><td>临时机场管理局</td><td> A15 - 151 </td><td></td></tr><tr><td>庆大霉素</td><td> Sigma - Aldrich公司</td><td> G1397 </td><td>抗生素</td></tr><tr><td> crystacilin </td><td>普利瓦</td><td> 625110 </td><td>抗生素</td></tr><tr><td>以pEGFP - N1 </td><td> Clontech公司实验室</td><td> 6085-1 </td><td>质粒DNA </td></tr><tr><td> HiSpeed​​质粒马克西套件</td><td> Qiagen公司</td><td> 12662 </td><td></td></tr><tr><td> NA 2 HPO 4 </td><td>默克公司</td><td> F640786 933 </td><td></td></tr><tr><td>的NaH 2 PO 4 </td><td> TKI的Hrastnik </td><td> 0795 </td><td></td></tr><tr><td> MgCl 2的 </td><td> Sigma - Aldrich公司</td><td> M - 8266 </td><td></td></tr><tr><td>蔗糖</td><td> Sigma - Aldrich公司</td><td> 16104 </td><td></td></tr><tr><td>胰蛋白酶/ EDTA溶液</td><td> Sigma - Aldrich公司</td><td> T4174 </td><td></td></tr><tr><td>枪头</td><td>定制</td><td></td><td></td></tr><tr><td>电脉冲发生器</td><td>定制</td><td></td><td></td></tr><tr><td> 6孔板</td><td> TPP </td><td> 92406 </td><td></td></tr><tr><td> 15 mL离心管</td><td> TPP </td><td> 91015 </td><td></td></tr><tr><td> 75厘米的培养瓶中</td><td> TPP </td><td> 90076 </td><td></td></tr></tbody></table>

changing the direction and orientation of electric field during electric pulses application improves plasmid gene transfer in vitro

基因electrotransfer是一种物理方法，用于提供短期和强烈的电脉冲，从而导致细胞膜不稳定的应用进入细胞的基因，使其渗透到小分子，并允许转让大分子如DNA。它代表了一个病毒载体，由于其安全性，有效性和易于应用。对于基因electrotransfer使用不同的电脉冲协议，以达到最大的基因转染，其中之一是在脉冲传递改变电场的方向和方位。改变电场方向和方向，增加主管DNA进入细胞的膜面积。在这段视频中，我们展示了基因electrotransfer疗效差异时所有的脉冲是在同一个方向，并交付，脉冲时，通过改变或者电场方向和方向交付。对于这个集成了电极和高电压的原型生成器，它允许在电脉冲的应用不同方向的电场变化的目的端，分别使用。基因electrotransfer疗效是确定的脉冲数量除以所有细胞表达绿色荧光蛋白与细胞的数量的应用后的24小时。结果表明，基因转染增加时，在电脉冲传递的电场方向改变。

Watch this Scientific Journal Video about Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro at JoVE.com

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

基因转染，电是提高约两​​次脉冲的应用过程中改变电场方向时，细胞活力不受影响。基因转染的增加是由主管DNA进入细胞的膜面积增加。

的方向转变，并在电脉冲电场方向的应用，提高质粒的基因转移在体外</em

Gene electrotransfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses, which cause ...

changing-direction-orientation-electric-field-during-electric-pulses

Research

JoVE Journal

Medicine

Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

10.4K 次观看.  University of Ljubljana. 基因转染，电是提高约两​​次脉冲的应用过程中改变电场方向时，细胞活力不受影响。基因转染的增加是由主管DNA进入细胞的膜面积增加。

视频: Changing the Direction and Orientation of Electric Field During Electric Pulses Application Improves Plasmid Gene Transfer in vitro

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

科研

医学

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

缺血性心力衰竭在临床前模型的转基因

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

在小鼠永久性大脑中动脉结扎术中的应用

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

在癌症的生产和检测活性氧（ROS）的

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

在动物模型揭示神经，血液，创伤后应激障碍和行为相关因素的生物标志物

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

从金属支架表面使用的腺病毒载体栓通过水解的交联剂血管基因转移

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

一种新型的<em&gt;在体内</em&gt;基因转移技术和<em&gt;在体外</em&gt;细胞分析为肌肉骨骼疾病骨质流失的研究

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

以显微镜为基础的方法评估上皮细胞迁移过程中的体外创面愈合

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

体内兔颈总动脉内皮的基因转移

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

的方向转变，并在电脉冲电场方向的应用，提高质粒的基因转移在体外

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