Name: vascular gene transfer from metallic stent surfaces using adenoviral vectors tethered through hydrolysable cross-linkers
Uploaded: 2014-08-12T13:00:00.000+00:00
Duration: 12 min 30 s
Description: Watch this Scientific Journal Video about Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers at JoVE.com

The authors do not have competing financial interests to disclose.

1，准备Cy3标记的腺病毒为实验版<ol><li> （; pH值9.3 CBB）在650微升的碳酸盐/碳酸氢盐缓冲液暂停2×10 12颗粒广告空 （约2×10 11感染单位）。 </li><li>溶解的1瓶（0.2毫克）的胺反应性的荧光染料（Cy3标记（NHS）2）在1ml CBB的含量为0.2毫克/毫升的最终浓度。 </li><li>加入100微升的染料溶液，以病毒悬浮液，涡旋5秒并保温1小时，在28℃振荡（100-200转）。 </li><li>平衡用PBS 20ml的琼脂糖凝胶6B柱。加入750微升的Cy3标记的广告在悬浮液中逐滴的凝胶床的中心。 </li><li>经过病毒悬液渗透树脂，加入5毫升的PBS。弃去洗脱液。 </li><li>加入0.5毫升的PBS，并收集在一个标有玻璃容器的洗脱液。重复此步骤，收集10倍的共有100.5毫升分数。 </li><li>法日E按分光光度法（260和280 nm）的病毒DNA含量收集的部分。 </li><li>汇集含有总（F1通过F10总和）的光密度（OD值）的10％以上在260nm处的级分。注意:典型地，馏分F3，F4，F5和F6合并并混合。 </li><li>再测定该混合合并的级分通过分光光度法（260和280nm）和测定用荧光（550 EX / 570 青霉 nm）的病毒DNA含量和Cy3标记的衣壳蛋白。注意:Cy3标记（NHS）2校准曲线覆盖10 -9 -10 -13 mol / L的范围内，制备和fluorimetrically测定在同一平板上的汇集级分。 </li><li>计算标记的病毒产量和标签密度。假定1.19×10 12病毒颗粒/ ml的对应于1的OD单位为1厘米路径长度29。使用公式:收益率= [（外径260nm处 /1.19)*10 12 * V /广告投入量] * 100，其中，外径为260nm的光德将合并的制剂在260nm的nsity和V是汇集制剂（毫升）来计算广告回收率（％）的体积。使用公式:标记密度= C * 6.02 * 10 23 /（OD 260nm处 /1.19)*10 12，其中C是共用的制剂的Cy3标记的浓度（摩尔/毫升）和OD 260nm处是共用的制剂的光密度260纳米，计算平均的Cy3标记的密度。注意:标有病毒的典型复苏的输入剂量&gt; 70％。标记密度为每一个病毒颗粒600-800荧光分子。 </li><li>等分Cy3标记的广告空成更小的部分（一般为5×10 11颗粒）适于个体释放实验。注:当前协议规定仅在释放实验使用Cy3标记信息的。所有转导的研究进行了与非标记的载体中。 </li></ol> 2，激活金属样品<ol><li>清洗不锈钢Š蒂尔网状盘或不锈钢支架连续在异丙醇（5分钟×2）氯仿（5分钟×2）在55℃下振荡（100转）。除去溶剂。 </li><li>加热样品30分钟，在200℃下。 </li><li>溶解的聚烯丙基胺的双膦酸盐与安装潜硫醇基（PABT 27,28,30）的水（1-2％重量/体积）在72℃下振荡培养（100rpm下）。调节pH至4.5-5与KHCO 3。 </li><li>孵育金属样品在2-4小时，在72℃的PABT溶液振摇（100-200转）。注:该过程可在此时被暂停。样品是稳定3天，在4℃下。 </li><li>三次漂洗的样品用双蒸水（DDW）。 </li><li>露出所述样本以三（2 - 羧乙基）膦（TCEP，15毫克/毫升的0.1M醋酸缓冲液）15分钟，在28℃下振荡（100-200转）。 </li><li>在脱气DDW冲洗5倍。 </li><li>揭露样品2％聚乙烯安装吡啶基团（PEI <sub&gt; PDT 27,28,30）的脱气DDW在氩气气氛下搅拌2小时，在28℃下振荡（100-200转）。注:该过程可在此时被暂停。样品是稳定2周，在4℃。 </li><li>冲洗样本三次DDW。 </li><li>暴露样品到10毫克/毫升二硫苏糖醇/ DDW到吡啶基二硫代基团转化为硫醇。 </li><li>在脱气DDW再次冲洗5倍。 </li></ol> 3，腺病毒激活和金属表面固定<ol><li> （; pH值9.3 CBB）在487.5-495微升碳酸盐/碳酸氢盐缓冲液暂停5×10 11颗粒无论是绿色荧光蛋白的广告或广告吕克 （约5×10 10感染单位）。注意:对于发布的研究中，使用5×10 11 Cy3标记的广告空颗粒（音量调节到487.5-495微升用CBB）。 </li><li>慧聪溶解有不同的水解率28 [（快速（T 2 = 5天），中等（T 1 /2 = 12 d）和慢（T 2 = 50 D）HC， 即右心导管检查，免疫组化或SHC; 图1]或非水解交联剂（NHC），磺基-LC-SPDP在CBB为20毫米。 </li><li>立即加入5-12.5微升交联剂溶液的病毒悬浮液为500微升（交联剂200-500μM终浓度），旋涡的总体积，并培育1小时，在28℃下振荡（100〜 200转）。 </li><li>平衡与脱气5毫米EDTA / PBS（EPBS）20ml的琼脂糖凝胶6B柱。逐滴加入500微升的交联剂改性的广告在悬浮到树脂床的中心。 </li><li>加入5 mL脱气EPBS的。弃去洗脱液。 </li><li>加入0.5毫升脱气EPBS，并收集在一个标有玻璃容器的洗脱液。重复此步骤10次收集总共10 0.5毫升的级分。 </li><li>确定采用分光光度法收集到的馏分的外径在260和280 nm和转换OD到病毒滴度（1.19×10 12 / ml的CORREsponds到1外径）29。 </li><li>汇集含有洗脱病毒的&gt; 10％的级分（注意:典型地，馏分F3-F6）。重复分光光度法测定效价为汇集停牌。 </li><li>转移的病毒悬浮液的小瓶中与活化的金属样品（如每2.11）。孵育在28℃氩气氛下进行1小时以摇动（100〜200转）。注意:在此步骤中得到的广告 - 拴系的金属试样中的协议部分5-7所描述的后续版本，以及转导实验中被进一步使用。 </li></ol> 4，定量表面相关的广告载体的PCR <ol><li>准备网制定与表面固定广告绿色荧光蛋白 ，通过NHC，特困，免疫组化和右心导管检查在第2和第3条所述（N = 3，每型）束缚。 </li><li>使用的QIAamp DNA微套件隔离病毒DNA。单独放置网格到含有200μl的混合物的180微升ATL的B组成的1.5ml塑料管uffer和20μl蛋白酶K（均来自该试剂盒）。添加广告的eGFP颗粒的已知量（如用于校准曲线的标准）为含有相同的混合物的分离的管。孵育56℃过夜没有动摇。 </li><li>加入200μl的AL缓冲液（由试剂盒），通过涡旋混合，加入200微升的100％乙醇，并在室温下孵育5分钟。 </li><li>从每管中的混合物转移到一个单独的MinElite柱（从盒）和自旋在8000 rpm离心1分钟。冲洗含网状管180微升新鲜ALT缓冲器，添加到各个列和自旋在8000 rpm离心1分钟。弃去洗脱液。 </li><li>加入500μl的AW1缓冲液（由试剂盒）装入柱和旋在8000 rpm离心1分钟。弃去洗脱液。 </li><li>加入500μl的AW2缓冲液（由试剂盒）装入柱和旋在8000 rpm离心1分钟。 </li><li>弃去洗脱液。 </li><li>旋在14000转另外的3分钟unti升列是完全干燥。弃去洗脱液。 </li><li>加入50微升的MilliQ级水入列。转速为14000 rpm离心5分钟。收集50微升洗脱液从每列进（从工具包）单独收集管中。 </li><li>准备PCR反应混合液的解决方案通过结合以下（12.5微升电源的SYBR Green PCR扩增预混，0.63微升10微米绿色荧光蛋白检测引物[5&#39;-ACG的TAA ACG海合会ACA的AGT TC-3&#39;]，10 0.63微升乘μMeGFP的反义引物[5&#39;-AAG TCG TGC TGC TTC ATG TG-3&#39;]，6.3微升的MilliQ级水）。通过计划的反应，包括"无DNA"控制的数量乘以这些卷。 </li><li>负载5微升的广告eGFP的 DNA（来自步骤4.8）和20μl的PCR主混合物的成微安光学96孔反应板的一式三份的孔中。封板微安光学胶薄膜。旋转该板在1,000 rpm离心1分钟，以消除气泡。 </li><li>将板到7500实时定量PCR发动机的插座。在7500系统SDS软件（V1.4或更高版本）选择主菜单中的"创建新的实验。"单击"下一步"。在"新实验向导"画面从下拉菜单中选择的SYBR Green，点击"添加"。点击"下一步"来获得板的布局。突出显示所有井进行分析，选择的SYBR Green。突出连续无DNA对照孔中，信息eGFP的 DNA标准品和未知量，并使用各自的名称从下拉菜单将它们标记。 </li><li>切换到仪器标签。选择"添加解离相"，改变以及量由默认的50微升至25微升。单击开始。 </li><li>检查质控摘要离群等违规使用后分析PCR结果。 </li></ol> 5，释放动力学水解的交联剂拴载体颗粒从塑钢网<ol><li>摇动（100-200转）洗经由RHC，IHC SHC和NHC衍生的Cy3标记的广告在网状样品（按照3.9）中的1％BSA / PBS中（1小时×3）。 </li><li>使用无菌细镊子，将网格到96孔板中预充有200微升的洗脱缓冲液的各孔中（0.1％BSA / 0.1％吐温20 / PBS）中。 </li><li>取各网格的中央部的荧光图像。记录显微镜和用于图像采集的CCD摄像机的设置。 </li><li>测定法以及扫描模式，最大限度的降低了板fluorimetrically（ 前 550/570 EM）。使用井非衍生网格为背景的控制。 </li><li>孵育板在37℃振荡（50rpm下）。 </li><li>在预定的时间（1-30天范围内）吸出洗提缓冲液，而不会干扰的网眼，并添加200μl的新鲜的洗脱缓冲液。更换缓冲液后，重复步骤4.3-4.5。 </li></ol> 6，转导培养切尔的LS通过网格固定的广告载体<ol><li>洗净的广告EGFP -或广告吕克 -derivatized网格三次用无菌PBS 5分钟振荡（100转）。 </li><li>使用细无菌镊子取出筛网盘逐个地和单独将它们放置到一个96孔板用在生长对数期，感兴趣的细胞类型的孔中。 </li><li>孵育细胞24小时，在37℃下，在5％CO 2。 </li><li>使用相应的非末端的端点检测（荧光显微镜，荧光计为绿色荧光蛋白或生物发光成像的荧光素酶），以确定的范围和基因表达的孔中的空间分布。 </li><li>可选地，代替培养基中的PBS以增加荧光测定法（535分之485纳米），如果低的eGFP表达预期的灵敏度。注意:如果使用荧光配有良好的扫描功能，读取该板在一个良好的扫描模式来评估EGFP-表达细胞的孔中的空间分布。交易所PBS代表完成后，荧光介质。 </li><li>图片用的FITC滤光片组的转导细胞中井广告绿色荧光蛋白 -eluting网（包括底层和外围网）。以代表图像的40-200X放大。记录在荧光显微镜和用于采集图像的CCD照相机的确切设置。 </li><li>加入5微升荧光素股票的PBS中（10毫克/毫升）直接向井用Ad 卢克 -eluting啮合到500微克/毫升和终浓度在37℃和5％CO 2的10分钟前，生物发光成像。吸介质和成像后，用荧光素的培养基更换。 </li><li>重复端点检测在预定的时间（长达2周）研究报告基因表达的以下基介导的基因转移的动力学。 </li></ol>在延迟时间7点确认保存传导能力<ol><li>准备广告绿色荧光蛋白去使用RHC，IHC SHC和NHC矢量圈养rivati​​zed目（如每2.1-2.11和3.1-3.9）及个别地将其放置在96孔板中的60-80％汇合的牛主动脉内皮细胞的孔的网眼（BAEC ）。 </li><li>在1和2天后网格放置使用荧光显微镜和荧光（如每6.4-6.5）分析培养的BAEC转导与网状固定广告的eGFP。 </li><li>开始转了48小时后洗净含网状孔用PBS两次。 </li><li>加入200微升的0.25％胰蛋白酶/ EDTA至每个孔，并孵育15分钟，在37℃振荡（100转）。洗涤3次，用PBS。 </li><li>使用荧光显微镜和荧光，以确定完全除去与网格关联的所有的eGFP阳性细胞。 </li><li>新鲜种子传代BAEC入井带部分释放网格在60-80％的初始接种密度（2-2.7×10 4个细胞/孔）。 </li><li>孵育板在37℃和5％CO <suB&gt; 2的时间之前，通过荧光显微镜和荧光"新"传导事件评估预定的时间（1-10天）。 </li></ol> 8，广告洗脱支架在血管成形术的大鼠颈动脉支架型部署<ol><li>在这个协议中描述的所有动物的程序符合有关实验动物使用联邦法规，批准了费城儿童医院的IACUC。要坚持以无菌手术条件的所有仪器都釜消毒。为了保证连续不育珠灭菌器使用的仪器之间使用的最多连续五禽戏。 </li><li>准备广告吕克根据2.1-2.11和3.1-3.9 -eluting支架。存储所述病毒衍生的支架在无菌PBS中，在4℃下在使用前不超过24小时。 </li><li>麻醉的雄性Sprague-Dawley大鼠（400-450克）与IP注射氯胺酮（100毫克/千克）的，和甲苯噻嗪（5毫克/千克）。德由爪夹紧响应和肌紧张termine麻醉深度。适用于眼科兽医药膏，以防止角膜和巩膜干燥。注意:吸入麻醉，用4％和2％异氟醚（1升/分钟）用于诱导和维持麻醉，分别是可能的，但阻碍自由进入动物的颈部区域，从而不推荐用于一个没有经验的用户。 </li><li>由脚趾捏重新评估麻醉深度。刮胡子，准备无菌颈部和上胸部区域。施用抗生素（头孢唑啉; 20毫克/千克; IM），镇痛药（美洛昔康; 0.5毫克/公斤; SC）和盐水（10毫升/千克; SC）。导尿尾静脉与地下24导管和管理肝素（200国际单位/千克;四）。注:可以用来代替头孢唑啉10毫克/千克（IM）的恩诺沙星（拜有利）的剂量。避免使用抗生素缺乏显著活性，对革兰氏阳性菌。 10-15毫克/公斤卡洛芬（Rimadyl）可被用来代替美洛昔康作为超前镇痛。麻醉性镇痛药（E.G。，吗啡，丁丙诺啡）应该是因为他们的呼吸，压抑性的回避。 </li><li>执行经皮肤和筋膜颈部正中切口。使用钝性分离技术来分离左侧颈外动脉。扎断颈外动脉在最前端亲和力位点。应用滑动临时结扎，以颈内动脉起始。 </li><li>使一个2毫米动脉切开切口在左侧颈外动脉。 </li><li>通过切口在颈外动脉插入2法国Fogarty的导管插入颈总动脉。膨胀导管用生理盐水的前端，并从主动脉弓到颈动脉分叉以剥蚀内皮通过3倍。 </li><li>滑动的管件的（1.04毫米外径，0.99毫米内径）在Fogarty的导管并进入颈总动脉。撤回福格蒂导管。 </li><li>安装和压接广告吕克 -derivatized支架上的1.5气球毫米直径的血管成形术导管。通过聚四氟乙烯管插入所述支架和推进其插入颈总动脉的中间部分。避免摩擦管或容器壁的支架。 </li><li>展开支架，在12个大气压下30秒和撤回的血管成形术导管。 </li><li>扎断颈外动脉近端动脉切开术位点和释放临时结扎于颈内动脉。 </li><li>修复手术伤口层与运行4.0薇乔缝合线和缝合皮肤。 </li><li>恢复对气候变暖垫动物，直到门诊，并返回到它的隔离笼。而疼痛或不适的迹象通常通过手术后的动物显示出超出该过程后的第12小时，72小时，考虑延长美洛昔康治疗（0.5毫克/公斤，SC每日）。 </li></ol>动脉基因表达九生物发光成像<ol><li>在预定时间点（第1天 - 3周范围）基因洗脱支架部署在颈总动脉后，用异氟醚吸入麻醉（在氧气中2-4％的异氟烷​​）麻醉大鼠。 </li><li>取出手术钉书针，无菌准备场地并重新​​打开手术伤口。使用到左颈总动脉钝性分离，再增益获取和它来自迷走神经与相邻的结缔组织中分离出来。 </li><li>制备50mg荧光素在PBS和25％的Pluronic F-127的PBS中的混合物/毫升（1:4体积/体积），并将其存储在冰上。注意:该制剂表现为在4℃下的粘性溶液，并立即接通时与组织接触的凝胶在37℃下。 </li><li>应用200微升冷却的萤光素/尼克混合物直接向颈总动脉的暴露部分和验证的凝胶坚固性。 </li><li>将动物在IVIS谱装置的成像室仰卧位并保持异氟烷麻醉用面罩。 </li><li>在acquisiti在控制面板窗口（生活图像，版本4.2或更高版本）选择位置"B"（6.6厘米相机被摄物距离）和题为"视野"和"装箱"，分别下拉菜单分档系数"中等"。键入在主题高度盒子"2.5厘米"。选择"分钟"为单位时间内，在"曝光时间"复选框，并选择"2"的数值。 </li><li>三分钟的应用程序的萤光素/尼克凝胶后，通过点击屏幕上的"获取"按钮启动图像采集。注:图像采集时间可以有所不同，从1到6分钟取决于预期的信号强度。 </li><li>获取图像后，用生理盐水冲洗凝胶关闭并轻轻用无菌纱布和棉花涂药动脉周围的空间。 </li><li>关闭与薇乔缝线和缝合皮肤上的伤口。 </li><li>恢复动物，回到笼子里。在后来吨重复成像IME点，研究动脉表达的时间进程由所述支架固定的Ad载体带来的。 </li><li>另外，请执行下列成像荧光素的全身用药。麻醉和预习动物按9.1-9.2。导尿尾静脉与地下24导管和安全的导管，手术胶带。 </li><li>制备荧光素的PBS溶液（50毫克/毫升）并在10秒的时间间隔注入1ml溶液通过导管。注射后1分钟开始图像采集按9.7-9.8。注:一个较快的注射速率（&lt;10秒）可引发癫痫发作和呼吸骤停。如果成像过程中出现抽搐或呼吸停止的动物必须被安乐死。 </li><li>按照步骤9.10。 </li></ol>

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Protocol development and studies presented in this paper were supported in part by American Heart Association Scientist Development grants (I.F. 0735110N and M.C. 10SDG4020003), U.S. National Heart, Lung, and Blood Institute grant HL 72108 (R.J.L.), and U.S. National Heart, Lung, and Blood Institute T32 007915 (S.P.F.). Angioplasty catheters were kindly donated by NuMED (Hopkinton, NY). The authors wish to acknowledge secretarial assistance of Ms. Susan Kerns.

<html>所提出的协议描述了一种用于通过腺病毒载体的可逆附着取得到coatless不锈钢表面衬底介导的基因递送的操作方法。而开发出用于血管再狭窄的支架为基础的基因疗法的特定目的，该技术具有在生物材料，生物医学植入物和基因治疗等领域的更广泛的应用。 虽然提出的研究仅使用不锈钢作为一个典型的金属基板，PABT结合其他金属的合金，如钴/铬和镍钛合金具有相当的亲?...

基因疗法作为治疗方法的有效性是通过基因治疗的载体1,2的差的定位能力受到阻碍。适当的定位结果中的转基因表达在目标位置的亚治疗水平的缺乏，导致载体对非靶器官3，包括那些负责安装针对两个矢量和编码的治疗产物4的免疫应答的广泛传播， 5。一电位是指，以抵消转导的滥交和促进目标是引入基因的载体在所需位置处在于，通过血液和淋巴排除其自由传播的形式。典型地，这样的努力依赖于局部注射的递送系统，包括混有纤维蛋白，胶原蛋白或透明质酸水凝胶基质6-10，其能够在注射部位短暂维持基因的载体通过物理截留第要么病毒或非病毒载体的时间在聚合网络。 用于局部基因治疗另一普遍接受的范例利用基因载体的固定化植入假体装置11,12的表面上。永久性医疗植入物（血管，支气管，泌尿系统和胃肠道支架，起搏器，人工关节，手术和妇科网等 ），每年都用在数以千万计的患者13。虽然通常是有效的，这些设备很容易出现并发症，是由当前的医疗行为14-17控制不佳的。植入假体装置提出了一个独特的机会，作为代理平台局部基因治疗。从药物动力学观点来看，医疗植入物与基因载体的结果相对低的输入剂量植入物/组织界面上实现既高局部浓度基因载体和减缓兵卫的动力学的表面衍生ř消除从这个位置。至于长期居留的后果和增强摄取目标细胞群，固定化载体的最小基因载体的传播。因而不慎接种的非靶组织中被减少。 在植入生物材料的基因的载体（也称为基底介导的基因递送或固相的基因递送）的表面束缚已在细胞培养和动物实验使用实施两个特定（抗原-抗体18-20生物素蛋白-生物素21,22）和非特异性23-26（充电，范德华力）的相互作用。共价连接的载体，以植入装置的表面之前已被视为非功能由于与表面过于强键排除向量的内化通过靶细胞。最近已证明，这种限制可以通过使用作为四面体的使用自发水解的交联剂来克服所述腺病毒载体27,28的支架和壳体蛋白的经修饰的金属表面之间存。此外，该载体释放速率和在体外和体内转基因表达的时间进程可以调制与使用可水解的交联剂呈现不同水解28的动力学。 本文件提供了腺病毒载体活化金属表面的可逆共价连接的详细协议，并引入了一个有用的实验装置来研究在体外随后传导事件在培养的平滑肌和内皮细胞和体内的支架成形术的大鼠颈动脉模型。

<table><tbody><tr><td>316 stainless steel mesh disks</td><td>Electon Microscopy Sciences</td><td>E200-SS</td><td></td></tr><tr><td>Generic 304-grade stainless steel stents</td><td>Laserage</td><td>custom order</td><td></td></tr><tr><td>Ad&lt;sub&gt;eGFP&lt;/sub&gt;</td><td>University of Pennsylvania Vector Core</td><td>AD-5-PV0504</td><td></td></tr><tr><td>Ad&lt;sub&gt;Luc&lt;/sub&gt;</td><td>University of Pennsylvania Vector Core</td><td>AD-5-PV1028</td><td></td></tr><tr><td>Ad&lt;sub&gt;EMPTY&lt;/sub&gt;</td><td>University of Pennsylvania Vector Core</td><td>A858</td><td></td></tr><tr><td>Cy3(NHS)&lt;sub&gt;2&lt;/sub&gt;</td><td>GE Healthcare</td><td>PA23000</td><td></td></tr><tr><td>Sepharose 6B</td><td>Sigma-Aldrich</td><td>6B100-500ML</td><td></td></tr><tr><td>UV 96-well plates</td><td>Costar</td><td>3635</td><td></td></tr><tr><td>Fluorometry 96-well plates</td><td>Costar</td><td>3915</td><td></td></tr><tr><td>Cell culture 96-well plates</td><td>Falcon</td><td>353072</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)</td><td>Pierce Thermo Scientific</td><td>20490</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Pierce Thermo Scientific</td><td>20290</td><td></td></tr><tr><td>sulfo-LC-SPDP</td><td>Pierce Thermo Scientific</td><td>21650</td><td></td></tr><tr><td>Spectrophotometer</td><td>Molecular Devices&amp;nbsp;</td><td>SpectraMax 190</td><td></td></tr><tr><td>Spectrofluorometer</td><td>Molecular Devices</td><td>SpectraMax Gemini EM</td><td></td></tr><tr><td>Orbital shaker incubator</td><td>VWR</td><td>1575R</td><td></td></tr><tr><td>Horizontal airflow oven</td><td>Shel Lab</td><td>1350 FM</td><td></td></tr><tr><td>Centra-CL2 centrifuge&amp;nbsp;</td><td>International Equipment Company</td><td>426</td><td></td></tr><tr><td>Digital vortex mixerer</td><td>Fisher Thermo Scientific</td><td>02-215-370</td><td></td></tr><tr><td>Eclipse TE300 fluorescence microscope</td><td>Nikon&amp;nbsp;</td><td>TE300</td><td></td></tr><tr><td>DC 500 CCD camera</td><td>Leica</td><td>DC-500</td><td></td></tr><tr><td>7500 Real-Time PCR system</td><td>Applied Biosystems</td><td>not available</td><td></td></tr><tr><td>IVIS Spectrum bioluminescence station</td><td>Perkins-Elmer</td><td>not available</td><td></td></tr><tr><td>EDTA dipotassium salt</td><td>Sigma-Aldrich</td><td>ED2P</td><td></td></tr><tr><td>Bovine serum albumin fraction V (BSA)</td><td>Fisher Thermo Scientific</td><td>BP1600-100</td><td></td></tr><tr><td>Tween-20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Dumont forceps</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>A10 cell line&amp;nbsp;</td><td>ATCC</td><td>CRL-1476</td><td></td></tr><tr><td>Bovine aortic endothelial cells</td><td>Lonza</td><td>BW-6002</td><td></td></tr><tr><td>Luciferin, potassium salt</td><td>Gold Biotechnology</td><td>LUCK-1Ge</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443-250G</td><td></td></tr><tr><td>PBS without calcium and magnesium</td><td>Gibco</td><td>14190-136</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gemini Bio-Products</td><td>100-106</td><td></td></tr><tr><td>Penicillin/Streptomycin solution</td><td>Gibco</td><td>11540-122</td><td></td></tr><tr><td>DMEM, high glucose</td><td>Corning cellgro</td><td>10-013-CV</td><td></td></tr><tr><td>0.25% Trypsin/EDTA</td><td>Gibco</td><td>25200-056</td><td></td></tr><tr><td>QIAamp DNA micro kit</td><td>Qiagen</td><td>56304</td><td></td></tr><tr><td>Power Sybr Green PCR Master Mix</td><td>Applied Biosystems</td><td>4367659</td><td></td></tr><tr><td>MicroAmp Optical 96-well Reaction Plate</td><td>Applied Biosystems</td><td>N8010560</td><td></td></tr><tr><td>MicroAmp Optical Adhesive Film</td><td>Applied Biosystems</td><td>4360954</td><td></td></tr><tr><td>Cephazolin&amp;nbsp;</td><td>Apotex</td><td>not available</td><td></td></tr><tr><td>Loxicom (Meloxicam)</td><td>Norbrook</td><td>not available</td><td></td></tr><tr><td>Heparin sodium</td><td>APP Pharmaceuticals</td><td>not available</td><td></td></tr><tr><td>Ketavet (Ketamine)</td><td>VEDCO</td><td>not available</td><td></td></tr><tr><td>Anased (Xylazine)&amp;nbsp;</td><td>Lloid</td><td>not available</td><td></td></tr><tr><td>Forane (Isoflurane)&amp;nbsp;</td><td>Baxter</td><td>not available</td><td></td></tr><tr><td>Curved Moria iris forceps</td><td>Fine Science tools</td><td>11370-31</td><td></td></tr><tr><td>Curved extra-fine Graefe forceps</td><td>Fine Science Tools</td><td>11152-10</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11252-20</td><td></td></tr><tr><td>Vannas spring scissors</td><td>Fine Science Tools</td><td>15018-10</td><td></td></tr><tr><td>Fine scissors - ToughCut</td><td>Fine Science Tools</td><td>14058-09</td><td></td></tr><tr><td>Surgical scissors</td><td>Fine Science Tools</td><td>14101-14</td><td></td></tr><tr><td>Vicryl suture (5-0)</td><td>Ethicon</td><td>J385</td><td></td></tr><tr><td>Suture thread (4/0 silk)&amp;nbsp;</td><td>Fine Science Tools</td><td>18020-40</td><td></td></tr><tr><td>Michel suture clips</td><td>Fine Science Tools</td><td>12040-02</td><td></td></tr><tr><td>Wound dilator (Lancaster eye specula)</td><td>KLS Martin</td><td>34-149-07</td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Michel suture clip applicator</td><td>Fine Science Tools</td><td>112028-12</td><td></td></tr><tr><td>Insyte Autoguard 24 G IV catheter</td><td>Beckton-Dickinson</td><td>381412</td><td></td></tr><tr><td>2F Fogarty catheter</td><td>Edwards Lifesciences</td><td>120602F</td><td></td></tr><tr><td>Teflon tubing</td><td>Vention</td><td>041100BST</td><td></td></tr><tr><td>PTA catheter</td><td>NuMed</td><td>custom order</td><td></td></tr><tr><td>Gauze pads</td><td>Kendall Healthcare</td><td>9024</td><td></td></tr><tr><td>Cotton applicators</td><td>Solon Manufacturing</td><td>WOD1003</td><td></td></tr><tr><td>Saline</td><td>Baxter</td><td>281321</td><td></td></tr><tr><td>10 ml syringe (Luer-Lok)</td><td>Beckton-Dickinson</td><td>309604</td><td></td></tr><tr><td>1 ml syringe (Luer-Lok)</td><td>Beckton-Dickinson</td><td>309628</td><td></td></tr><tr><td>Clippers with #40 blade</td><td>Oster&amp;nbsp;</td><td>78005-314</td><td></td></tr><tr><td>Transpore surgical tape</td><td>3M</td><td>MM 15271</td><td></td></tr><tr><td>Puralube vet ointment</td><td>Pharmaderm</td><td>not available</td><td></td></tr></tbody></table>

vascular gene transfer from metallic stent surfaces using adenoviral vectors tethered through hydrolysable cross-linkers

支架内再狭窄呈现的广泛使用，以重新建立血液穿过冠状动脉和外周动脉的极为狭窄段流基于支架的血管成形术的主要并发症。可调谐释放基因具有抗再狭窄活性可能存在另一种战略血管内支架，以目前使用的药物洗脱支架。为了达到临床平移，基因洗脱支架必须具有支架固定化基因载体释放和血管系统的位点特异性转导的可预测的动力学，同时避免通常与用于所述载体的物理截留在聚合物涂层相关联的严重的炎症反应。本文描述了一种腺病毒基因载体coatless束缚到基于所述腺病毒颗粒的可逆结合​​支架的详细方法，以聚烯丙胺二膦酸盐（PABT）经由水解的交联剂（HC）改性不锈钢表面。一个家庭的双官能团（胺和硫醇反应性）的HC与中链酯水解测距5-50天的平均吨半被用于与所述支架连接的载体中。该载体的固定化过程典型地是在9小时，包括几个步骤:1）温育的金属样品在PABT（4小时）的水溶液; 2）脱保护以三（2 - 羧乙基）膦（20分钟）安装在PABT硫醇基; 3）膨胀的金属表面的巯基反应的能力通过使与聚乙烯亚胺衍生的吡啶基二硫（PDT）基团（2小时）的样品; 4）变换PDT基与硫醇与二硫苏糖醇（10分钟）; 5）修改腺病毒与HC（1小时）; 6）纯化修饰的腺病毒颗粒通过尺寸排阻柱色谱法（15分钟）和在硫醇化钢表面（1小时），巯基反应性的腺病毒颗粒7）固定。该技术具有超越支架宽的应用潜力，通过促进生物假体装置的表面工程通过基板介导的基因递送到细胞中的接口的植入外来物质，以提高其生物相容性。

矢量释放实验 腺病毒载体到植入物的表面，包括介入器械如血管内支架的束缚，近似向量到疾病部位，部分避免了缺乏矢量"物理定位。然而，为了能够实现通过靶组织的转导的治疗效果，所述载体必须释放从表面（ 图2）。使用水解的交联剂是假设为允许通过该交联剂的水解介1）〜polybisphosphonate改性的有效附着载体，硫醇 - 安装金属植入物和2）的?...

Watch this Scientific Journal Video about Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers at JoVE.com

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

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 ...

vascular-gene-transfer-from-metallic-stent-surfaces-using-adenoviral

Research

JoVE Journal

Medicine

9.8K 次观看.  The Children's Hospital of Philadelphia, University of Pennsylvania. 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. 

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

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 ...

科研

医学

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

腺病毒基因治疗对糖尿病性角膜病变:对伤口愈合和干细胞标记表达的人体器官培养角膜和角膜缘上皮细胞

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

发育生物学

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

编程干细胞的治疗性血管新生使用可生物降解高分子纳米微球

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

生物工程

Fabrication of Small Caliber Stent-grafts Using Electrospinning and Balloon Expandable Bare Metal Stents

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and perforations in vasculature. Such stent-grafts are manufactured by covering a stent with a polymer membrane. An ideal stent-graft should have a biocompatible stent covered by a porous, thromboresistant, and biocompatible polymer membrane which mimics the extracellular matrix thereby promoting injury site healing. The goal of this protocol is to manufacture a small caliber stent-graft by encapsulating a balloon expandable stent within two layers of electrospun polyurethane nanofibers. Electrospinning of polyurethane has been shown to assist in healing by mimicking native extracellular matrix, thereby promoting endothelialization. Electrospinning polyurethane nanofibers on a slowly rotating mandrel enabled us to precisely control the thickness of the nanofibrous membrane, which is essential to achieve a small caliber balloon expandable stent-graft. Mechanical validation by crimping and expansion of the stent-graft has shown that the nanofibrous polyurethane membrane is sufficiently flexible to crimp and expand while staying patent without showing any signs of tearing or delamination. Furthermore, stent-grafts fabricated using the methods described here are capable of being implanted using a coronary intervention procedure using standard size guide catheters.

In the protocol, we present a method to manufacture a small caliber stent-graft by sandwiching a balloon expandable stent between two electrospun nanofibrous polyurethane layers.

小口径支架移植物使用静电及球囊扩张裸金属支架的制作

Stent-grafts are widely used for the treatment of various conditions such as aortic lesions, aneurysms, emboli due to coronary intervention procedures and ...

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 ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

颈动脉介入移植中颈静脉持久性转基因表达的家兔模型

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the treatment of many hematopoietic diseases in humans, as their use offers stable transgene expression due to the vector&#39;s ability to integrate into the host genome. This method demonstrates the application of ex vivo gene therapy of hepatocytes to a large animal model of hereditary tyrosinemia type I. This process consists of 1) isolation of primary hepatocytes from the autologous donor/recipient animal, 2) ex vivo gene delivery via hepatocyte transduction with a lentiviral vector, and 3) autologous transplant of corrected hepatocytes via portal vein injection. Success of the method generally relies upon efficient and sterile removal of the liver resection, careful handling of the excised specimen for isolation of viable hepatocytes sufficient for re-engrafting, high-percentage transduction of the isolated cells, and aseptic surgical procedures throughout to prevent infection. Technical failure at any of these steps will result in low yield of viable transduced hepatocytes for autologous transplant or infection of the donor/recipient animal. The pig model of human type 1 hereditary tyrosinemia (HT-1) chosen for this approach is uniquely amenable to such a method, as even a small percentage of engraftment of corrected cells will lead to repopulation of the liver with healthy cells based on a powerful selective advantage over native-diseased hepatocytes. Although this growth selection will not be true for all indications, this approach is a foundation for expansion into other indications and allows for manipulation of this environment to address additional diseases, both within the liver and beyond, while controlling for exposure to viral vector and opportunity for off-target toxicity and tumorigenicity.

Lentiviral Vector-mediated Gene Therapy of Hepatocytes Ex Vivo for Autologous Transplantation in Swine

This protocol is intended to describe porcine hepatocyte isolation and ex vivo gene delivery to cure models of metabolic diseases via autologous cell transplantation. Although this particular model enjoys unique advantages that favor successful therapy, the application is a relevant foundation to address additional diseases and indications.

猪自体移植肝细胞外细胞外细胞基因治疗

Gene therapy is an ideal choice to cure many inborn errors of metabolism of the liver. Ex-vivo, lentiviral vectors have been used successfully in the ...

Implantation of Tissue-Engineered Vascular Graft in Mouse Carotid Artery via Cuff Technique

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently,&#160;the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.

Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.

通过 Cuff 技术将组织工程血管移植物植入小鼠颈动脉

The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal ...

Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.

The protocol described here enables researchers to specifically modify adenovirus capsids at selected sites by simple chemistry. Shielded adenovirus vectors particles and retargeted gene transfer vectors can be generated, and vector host interactions can be studied.

联合遗传和化学衣壳改性腺病毒基因转移载体用于屏蔽和靶向

Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host ...

生物学

Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction

In addition to forming a high-resistance barrier, the urothelium lining the renal pelvis, ureters, bladder, and proximal urethra is hypothesized to sense and transmit information about its environment to the underlying tissues, promoting voiding function and behavior. Disruption of the urothelial barrier, or its sensory/transducer function, can lead to disease. Studying these complex events is hampered by lack of simple strategies to alter gene and protein expression in the urothelium. Methods are described here that allow investigators to generate large amounts of high-titer adenovirus, which can then be used to transduce rodent urothelium with high efficiency, and in a relatively straightforward manner. Both cDNAs and small interfering RNAs can be expressed using adenoviral transduction, and the impact of transgene expression on urothelial function can be assessed 12 h to several days later. These methods have broad applicability to studies of normal and abnormal urothelial biology using mouse or rat animal models.

Methods are described for the generation of large amounts of recombinant adenoviruses, which can then be used to transduce the native rodent urothelium allowing for expression of transgenes or downregulation of endogenous gene products.

使用腺病毒介导的转导在天然膀胱尿氦中表达转基因

In addition to forming a high-resistance barrier, the urothelium lining the renal pelvis, ureters, bladder, and proximal urethra is hypothesized to sense ...