这项工作是由科学技术部资助项目（MOST 103-2633-B-002-002; 104-2320-B-002-048）。我们感谢博士。贝蒂吴和谢建库·李在台大，利·泽博尼在斯坦福大学和彼得·奥夫曼博士在夏威夷大学读书的手稿，尤恩·奇恩台大技术援助，以及文奇·韦博士在农业生物技术研究中心中央研究院的技术咨询。

在8只雌性小鼠 - 12周龄被用于这项研究。 C57 / BL6野生型（WT）和IL-15缺陷（IL15 - / - ）小鼠分别从国家实验动物中心（NLAC），台湾和Taconic的农场购买。 IL-15缺陷型（IL15 - / - ）和IL-15-显性（IL15 +/-和IL15 + / +）显示出1:3的比例在第二代从IL15 +/-杂合子的横。 IL-15的基因型- / -小鼠通过PCR分析证实。小鼠保持在实验动物中心（LAC），国立台湾大学医学院（NTU）学院无特定病原（SPF）设施。所有的动物程序，包括麻醉材料和方法按照由医学国立台湾大学和学院公共卫生机构动物护理和使用委员会（IACUC）（动物协议20130225批准的证明）批准的动物方案进行。 &lt;P类= "jove_title"&gt; 1。针灸针的制备注:针灸针是一手柄和一针端（ 图1A）构成的医疗器械。针刺针的直径为0.2毫米（36 G）至0.35毫米（28 G）范围内。我们选择针的最好的点，以避免刺皮肤期间可能出现的过度的细胞损伤。有4个不同的0.2毫米的针的长度包括13毫米（0.5英寸），25毫米（1.0英寸），40毫米（1.5英寸），并在长度为50毫米（2.0英寸）。的10针13毫米长的bundle上C57BL / 6小鼠皮肤振荡针期间提供最佳舒适的手柄。如果该方法适用于较大的动物的皮肤或由研究人员巨掌执行的参数可以修改。 <ol><li>从无菌的，无热原的包装中取出针灸针。将针在无菌铺巾。 </li><li>使用粘合剂标签带10针结合我一起NA束（ 图1B）。 注:10针几何方式布置，以形成圆柱状。为了方便起见，使用一块石蜡膜的涂敷粘合带之前稳定布置。确保所有的针点在同一平面上。使用束而不是单个针通过增加针和皮肤之间的接触面积有利于最大DNA输注。 </li></ol> 2.大量的制备和无内毒素的质粒DNA <ol><li>变换用质粒DNA化学感受细菌细胞。 </li><li>选择并在3mL LB的含有抗生素的培养基接种单一菌落，并在摇床中在37℃下孵育过夜。 </li><li>用抗生素100在250ml的LB培养基中并在37℃振荡培养过夜C.:通过在1稀释细菌培养物扩大培养</li><li>通过使用高qualit制备DNA的大量Y，按照制造商的说明书，无内毒素的质粒制备试剂盒。 注:Acufection协议需要高纯度和质粒DNA的量大。高质量的使用，无内毒素和柱纯化的质粒DNA的建议。 </li><li>洗脱在无菌水中DNA。商店DNA以小等分试样在-20℃下直至准备acufection。 注意:避免DNA的反复冻结与解冻，以确保一致的质粒DNA的表达。 </li><li>稀释质粒DNA至1毫克/毫升在无菌PBS中进行acufection。 </li></ol> 3.程序Acufection 注:DNA的最佳量和目标皮肤表面的面积可以针对不同的基因变化，需要进一步优化。穿刺力和次数松开皮肤的角质层也可以根据皮肤的厚度而变化。这些条件必须评估acufected格涅·特兰斯克里的表达水平后小心地确定PTS通过qRT-PCR。 <ol><li>称量小鼠和给予腹腔注射三溴乙醇（每克体重400微克）。 注:2,2,2-三溴是被普遍用于小鼠注射的麻醉剂。溶液是通过溶解在含有2-甲基-2-丁醇的2.5％的蒸馏水（200mL）中的非医药级2,2,2-三溴乙醇（2.5克）制备。 </li><li>使用在眼睛兽医眼科软膏以防止麻醉下干燥。 注: - 2分钟麻醉作用将在1来诱导。检查脚趾捏反射，以保证手术麻醉前的足够的深度。 </li><li>剃须脊侧皮肤，适用脱毛霜，以溶解角质蛋白在毛干。 注:使用的脱毛霜去除头发将促进DNA的注射入皮肤。 </li><li>用清水浸泡棉球擦去脱毛膏。 注:脱毛膏是易溶于水。彻底清除霜西港岛线升防止油腻的表面，并确保更好的刺结果。 </li><li>使用70％的乙醇浸泡消毒棉签消毒皮肤表面。 </li><li>标记目标面积（1cm×1cm的）与预先测定的模版的脱毛皮肤。注:标记目标站点，将有助于应用针束向上和向下的限定区域内。这是重要的，以确保相同量的DNA递送至的比表面积，以尽量减少实验对实验变异。 </li><li>放置质粒DNA（10微克）的到显着皮肤上的10μL降。 注:DNA为acufection的量随不同的基因和表达载体的类型而变化。使用DNA的量应该进行感兴趣的实验前应仔细滴定。 10μL是小液滴和它位于井的剃和脱掉的皮肤无滚下而穿刺。包括用10μL空载体DNA处理的小鼠的对照组小鼠比较cufected与研究的基因。 </li><li>握住针刺针束（ 图1B），并用100次，或直到从DNA在PBS中的水分在皮肤上消失的向上和向下运动刺破标记表面。注:可能出现皮肤表面的有些发红。不要制定那些流血大幅削减。当从DNA在PBS中的水分（10μL）在皮肤上消失可操作地确定向上和向下的在皮肤表面上穿刺运动的数量。我们在30秒决定100倍，因为它给了从acufection交付基因最一致的结果。如果适用，该针可重复使用多达6个目标皮肤刺（1 1cm×1cm的每个）。冲洗在70％乙醇和PBS acufection之间的针。更改为新的针时它们索然无味或不同的质粒DNA，以避免交叉污染。 </li><li>放置在加热垫上acufected小鼠以维持体温，直到清醒。 </li><li>回到老鼠笼子里时，他们有恢复意识充足。注意:保持acufected小鼠单独笼（每笼少于5只动物）的未acufected小鼠的笼子。 </li><li>把水瓶到位，笼返回IVC（独立通风笼）机架。 </li></ol> 4.术后护理<ol><li>有关步骤后的第一个48个小时，密切监控任何不适，包括行为变化（坐立不安，搅动或进食障碍）或外观异常（粗头发涂层或驼背姿势）小鼠。 </li></ol> IL-15ΔE7-acufected皮肤5.咪喹莫特（IMQ）处理注:转录表达和蛋白质生产acufected基因是第3天小鼠的兴趣质粒可转染后3天不同类型的兴奋剂来治疗acufected检测。我们举例说明这里通过用IMQ霜IL-15ΔE7-acufected小鼠皮肤。 IMQ是imidazoquinolin胺批准˚F或治疗外生殖器和围产期疣13。局部治疗IMQ示出以诱导人银屑病样皮肤病症与片状皮肤，表皮增殖和真皮中性粒细胞浸润12，14，15的表现小鼠。 注意:所述的质粒载体包含全长小鼠IL-15的cDNA，延伸因子1α的调节（PEF），具有在C末端（PEF-IL-的IL-2的信号肽和FLAG标签侧翼下15，5859个碱基对），将其作为由班福德等人所描述构造。 16。该质粒被用作IL-15模板删除第一16个氨基酸在残基33 - 通过SOE 48在IL-15基因，IL-15ΔE7（PEF-IL-15ΔE7，5811个碱基对）的外显子7（合成通过重叠延伸）PCR方法17。细菌菌落已成功用IL-15ΔE7转化体通过限制性酶消化验证，随后通过DNA测序12,18。在协议中步骤2所述制备无内毒素的质粒DNA的大量。 <ol><li>由三溴乙醇（每克体重400微克）的腹膜内注射麻醉acufected小鼠和眼睛应用兽医眼科软膏，以防止麻醉下干燥。检查脚趾捏反射的执行程序开始前，要确保麻醉足够的深度。注意:由于2厘米×2cm的皮肤将用于IMQ进行处理，2个剂量的pIL-15ΔE7质粒DNA（10微克每剂量10μLPBS）的被acufected 2个靶位点（1 1cm×1cm的每个）。 </li><li>标记侧翼皮肤（2厘米×2cm）上覆盖所述acufected区域。 </li><li>用棉签涂抹上标记的表面积局部涂抹奶油IMQ。注意:步骤5.1，只在第一个剂量（60毫克/剂量）进行。接下来连续的剂量适用于非麻醉小鼠。 </li><li>用高清晰度摄像机每天记录IMQ治疗背部皮肤的变化。注意:一个人持有，而另一个正在录制鼠标。静态图片拍摄，并在以后的时间编辑。 </li><li>通过注入，然后在治疗IMQ后第4天或7天颈椎脱位三溴过量的（每克体重800微克）安乐死的小鼠。皮肤切除并处理（步骤4.1 - 4.5）。 </li></ol> 6.匀浆小鼠皮肤<ol><li>使用一对外科剪刀，使从尾的基部的水平切割。双边前往后肢的基地，并继续沿侧翼前肢的底座垂直切割。 </li><li>小心剥离从后部到前部下面的组织的皮肤上。使用剪刀剪下背部皮肤。放置在无菌培养皿皮肤。 </li><li>用手术刀切除目标皮肤并在液氮立即冻结它。注意:acufected皮肤无相同的尺寸（1厘米×1cm）的或与治疗IMQ（2厘米×2cm）上是固定的，每只小鼠以最小化实验对实验变体。 </li><li>将冷冻的皮肤组织在与手柄和杵预冷的2室砂浆的中心。使用的砂浆铅锤粉碎组织。 </li><li>粉碎的组织快速放置于含有细胞裂解试剂500μLRNA提取或到含有PBS的50μL和1x蛋白酶抑制剂混合物，以获得蛋白裂解液的管子的新管中。 </li></ol> 7. H＆E和免疫组化皮肤科染色<ol><li>将皮肤组织在录像带并在4％多聚甲醛浸泡在一夜之间。 </li><li>嵌入和部分组织，这对我们来说是由病理实验室在LAC，台大进行。注意:部分被切割，其厚度为5μm。地方组织secti附件上带正电的幻灯片。 </li><li>加热载玻片在65℃下15分钟。 </li><li>请将幻灯片染色架。沉浸齿条成相应的含二甲苯替代5分钟两次，接着在100％，95％，80％，75％，60％和50％乙醇（每次5分钟），用于补液顺序浸渍槽中。浸泡在自来水中的幻灯片染色前3分钟。 </li><li>执行苏木和伊红染色。注意:在LAC，NTU在病理实验室根据请求进行H＆E染色。 </li><li>对于免疫组织化学染色，框在室温下用封闭溶液区段5分钟以减少非特异性背景染色。注:该块解决方案与商业免疫技术开发。没有动物血清被包含在该产品。 </li><li>浸在PBS中的滑动来冲块溶液。 </li><li>稀释在PBS块溶液在1:10和添加2％胎牛血清（FBS）（稀释溶液）。 </li><lI&gt;移液器的适当稀释的初级抗体到所述部分和孵育在室温下60分钟。注:抗体在稀释液稀释。分别为200 Ki67的（克隆SolA15）和Ly6G（克隆1A8），:抗体浓度为这种染色已经在1优化:800和1。这些浓度被发现不显示背景染色明显的污渍。 <ol><li>染色在同一个运行一个串行部分用相同的溶液省略初级抗体，以控制用于次级抗体的非特异性结合。 </li></ol></li><li>洗涤，每次5分钟滑动在TBS三次加0.1％吐温20（TBST溶液）。洗涤之间变化解决方案。 </li><li>移液器标记的聚合物的每个部分中的一个降和在室温下孵育40分钟。注意:标记的聚合物在商业上通过结合用过氧化物酶和第二抗体的氨基酸聚合物的量减少到Fab"片段制备。该试剂已准备好使用小鼠组织切片的免疫组织化学染色。 </li><li>洗涤滑动在TBST中三次，每次5分钟。洗涤之间变化解决方案。 </li><li>吸管3,3&#39;- diaminobenzindine四盐酸盐（DAB）溶液到组织切片，并让在室温下放置直到棕色沉淀下一个亮视野显微镜是可见的。注意:时间DAB在过氧化物酶的存在沉淀大约30分钟。 </li><li>浸泡幻灯片水停止过氧化物酶活性。浸幻灯片在核染色甲基绿的解决方案。洗涤载玻片在PBS中3次，每次5分钟。 </li><li>放置在覆盖有永久安装平台（每部分8μL）染色的截面的盖玻片。 </li></ol> 8.照片彩绘组织切片分析<ol><li>可视化亮视野显微镜下H＆E染色和IHC的组织切片。使用附连到显微镜电荷耦合器件（CCD）照相机捕获的图像。对于q分析的定量的，成像软件可用于<ol><li>扫描使用scanscope与20倍的目标整个染色切片。 </li><li>测量所述表皮的1毫米段内的表皮厚度和段内计数的Ki67免疫反应性细胞的数量。 使用显微镜自动化及图像分析软件进行定量分析:注意。表皮厚度由基底层和表皮的最外层之间的距离限定。 </li><li>计数Ly6G免疫反应细胞在皮肤区域（230 X 270微米2矩形）的正下方的局部IMQ处理皮肤的数量。 </li></ol></li></ol>

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N., DeFilippis, A. P., Azimi, N., Kurys, G., Waldmann, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+5'+untranslated+region,+signal+peptide,+and+the+coding+sequence+of+the+carboxyl+terminus+of+IL-15+participate+in+its+multifaceted+translational+control.">The 5' untranslated region, signal peptide, and the coding sequence of the carboxyl terminus of IL-15 participate in its multifaceted translational control.</a> J Immunol. 160, 4418-4426 (1998).</li><li>Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., Pease, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+hybrid+genes+without+the+use+of+restriction+enzymes:+gene+splicing+by+overlap+extension.">Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension.</a> Gene. 77, 61-68 (1989).</li><li>Tan, X., Lefrancois, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+IL-15+isoforms+generated+by+alternative+splicing+are+expressed+in+the+intestinal+epithelium.">Novel IL-15 isoforms generated by alternative splicing are expressed in the intestinal epithelium.</a> Genes Immun. 7, 407-416 (2006).</li><li>Basner-Tschakarjan, E., Mirmohammadsadegh, A., Baer, A., Hengge, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+trafficking+of+DNA+in+keratinocytes:+evidence+for+DNA-binding+proteins.">Uptake and trafficking of DNA in keratinocytes: evidence for DNA-binding proteins.</a> Gene Ther. 11, 765-774 (2004).</li><li>Fan, H., Lin, Q., Morrissey, G. R., Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+via+hair+follicles+by+topical+application+of+naked+DNA+to+normal+skin.">Immunization via hair follicles by topical application of naked DNA to normal skin.</a> Nat Biotechnol. 17, 870-872 (1999).</li><li>Kennedy-Crispin, M., 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=Human+keratinocytes'+response+to+injury+upregulates+CCL20+and+other+genes+linking+innate+and+adaptive+immunity.">Human keratinocytes' response to injury upregulates CCL20 and other genes linking innate and adaptive immunity.</a> J Invest Dermatol. 132, 105-113 (2012).</li></ol>

作者没有财务权益披露。

以确保acufected质粒DNA的表达中最关键的步骤是均匀振荡并松开皮肤的角质层。同时轻轻推针不切开皮肤，用力要够硬踏面。为了便于在上1厘米×1cm的表面积10μLDNA溶液吸收，针应当振荡在30秒为约100倍向上和向下。可以确定需要刺破皮肤上的力和通过注意的是需要得到acufected基因的最佳表达水平的次数。 修饰质粒DNA的量施加到达到表达的令人满意的程度，可能需要acufection后，...

皮肤是机体防御的第一线。角质形成细胞（KCS）在人类和小鼠的皮肤中的主要细胞类型。响应环境刺激（ 例如阳光，氧气，化学品和病原侵入），枯否细胞被活化并产生宽促炎细胞因子和趋化因子如IL-8，IL-6，IL-1α，IL-1β，TNF-α的阵列α和GM-CSF 1。他们一起引发免疫细胞在皮肤的招聘。加重皮肤炎症常常与许多人类疾病，包括急性异位接触性皮炎，慢性T细胞介导的炎症（ 例如过敏性接触性皮炎和银屑病）2相关联。通过抑制KC活化调节皮肤炎症反应是治疗皮肤炎症一个可行的办法。本协议描述了一种新方法，瞬时表达在表皮细胞因子基因，以研究免疫RESponse继发于皮肤这样的处理。 表皮组成的皮肤最表面的一层。它作为一个物理屏障保持外部物质如核酸和病原体进入皮肤的更深层这样。一些无针技术已经建立了表皮DNA转移3，4。在溶液中的DNA或与阳离子脂质体或腺病毒载体相关联的已被直接施加到与技术修饰表皮如除去角质层的或与脱毛试剂的治疗。角化上皮的去除促进DNA交叉的表皮屏障，并与角质形成细胞和朗格汉斯细胞相互作用以诱导免疫应答5。虽然大的表面积可用于DNA转移而这种做法不涉及针，也有缺点，包括要求对于大批量DNA（10 - 100微克），在皮肤上的苛刻处理通过汽提，并且在没有诱导DNA递送增压器6在免疫动物的免疫应答的结果不一致。裸DNA到皮肤的微粒介导的细胞内递送已被证明是高效的和可再现的用于诱导免疫应答7,8。一种手持式基因枪已被用于轰击皮肤目标部位用质粒DNA包被的金颗粒直径微米大小2通过加压氦气气流9的装置。质粒DNA被想必在皮肤采取了由KCsand树突细胞（DC）和蛋白质被局部表示或树突10被输送到引流淋巴结。尽管基因枪系统简单，需要有限的技术经验，为准备和提供的"DNA BUL成本让"（ 即金粉末，氦气）和基因枪本身限制了它的一般应用。另外，当实验在不同的位置进行了氦气输送系统的要求限制了便于基因枪交通工具。Microseeding已经证明，以实现基因转移比单次注射和粒子轰击11的更高的效率。Microseeding使用纹身枪无需使用微粒珠递送DNA至皮肤11。振荡上使用该方法对皮肤的微针是有可能直接刮细胞膜，从而在同一时间传送到DNA多个小区。然而，随着大量注射用0.254毫米直径针相关的疼痛是不利的。 在这里，我们提供一种替代的方法来在体内 DNA递送。该"acufection" </str翁&gt;我们在这里描述的方案提供了一种经济和有效的方式来在小鼠皮肤递送质粒DNA。简言之，将10针灸针（直径0.2mm×13毫米长）在通过胶粘带束被束缚。将10μL的PBS用含有感兴趣的转基因的质粒DNA的10微克体积置于剃毛，脱毛霜处理的侧面上的皮肤。通过使用针刺针束产生振荡的皮肤表面（1厘米×1cm）的，在角质层的角蛋白被松开，并且施加到所述表面上的质粒DNA被吸收进入表皮。皮肤损害进行监测，发现是最小的。在acufected皮肤转基因的表达通过定量实时PCR（定量RT-PCR）和ELISA证实。使用IMQ处理的小鼠模型12 acufection递送IL-15ΔE7对皮肤炎症的免疫调节效果进行了论证。虽然acufection证明是一个简单的，少DEMAnding方法用于体内 DNA递送，DNA为不同的基因和时代刺破皮肤的最佳量必须仔细优化以获得可再现的结果。

<table><tbody><tr><td>Accu Handy Needle</td><td>Accu</td><td>36Gx0.5</td><td>Medical device</td></tr><tr><td>Nair&lt;sup&gt;TM&lt;/sup&gt; Lotion</td><td>Church &amp;amp; Dwight</td><td>N/A</td><td>Use to remove hair</td></tr><tr><td>Shandon Xyline substitute</td><td>Thermo Fisher Scientific</td><td>6764506</td><td>Deparaffinization</td></tr><tr><td>Avertin (2,2,2-Tribromethanol)</td><td>Sigma-Aldrich</td><td>T4,840-2</td><td>Anesthesia</td></tr><tr><td>2-methyl-2-butanol</td><td>Sigma-Aldrich</td><td>152463</td><td>Solvent for the dissolution of avertin</td></tr><tr><td>Difco&lt;sup&gt;TM&lt;/sup&gt; LB broth, Miller</td><td>BD</td><td>244620</td><td>Propagation and maintenance of &lt;em&gt;E. coli&lt;/em&gt; for molecular biology</td></tr><tr><td>Mouse IL-15/IL-15R Complex ELISA Ready-SET-Go kit</td><td>eBioscience</td><td>88-7215</td><td>Measure IL-15 protein</td></tr><tr><td>Anti-mouse Ly6G</td><td>BioLegend</td><td>127601</td><td>Antibody used for immunohistochemical stain</td></tr><tr><td>anti-mouse Ki-67</td><td>eBioscience</td><td>14-5698-80</td><td>Antibody used for immunohistochemical stain</td></tr><tr><td>Simple Stain Mouse MAXPO (Rat)</td><td>Nichirei Biosciences</td><td>414341F</td><td>Reagent to block background signal in mouse-on-mouse stain</td></tr><tr><td>DAB Peroxidase (HRP) Substrate Kit</td><td>Vector Laboratories</td><td>SK-4100</td><td>Peroxidse substrate for immunohistochemical stain</td></tr><tr><td>Ultra V block</td><td>Thermo Fisher Scientific</td><td>TA-060-UB</td><td>Reduce nonspecific background staining</td></tr><tr><td>Methyl green</td><td>Sigma-Aldrich</td><td>M8884</td><td>Nuclear staining</td></tr><tr><td>Vecta Mount&lt;sup&gt;TM&lt;/sup&gt;</td><td>Vector Laboratories</td><td>M-5000</td><td>Permanently preserving histochemical stains</td></tr><tr><td>Protease inhibitor cocktail tablets</td><td>Roche</td><td>04-693-116-001</td><td>Inhibit protease activity in cell lysate</td></tr><tr><td>Aldara cream</td><td>3M Parmaceuticals</td><td>N/A</td><td>5% imiquimod cream</td></tr><tr><td>Bessman Tissue Pulverizer</td><td>Spectrum Labs</td><td>189475</td><td>Use to pulverize skin tissue</td></tr><tr><td>ABI7900HT cycler</td><td>Thermo Fisher Scientific</td><td>N/A</td><td>quantitative real-time PCR assay</td></tr><tr><td>Camcorder</td><td>Panasonic</td><td>HDC-SD60</td><td>Image documentation</td></tr><tr><td>Axio Scope.A1</td><td>ZEISS</td><td>N/A</td><td>Bright-field microscopy</td></tr></tbody></table>

development of an economical dna delivery system by "acufection" and its application to skin research

在皮肤的免疫反应的失调与许多人的皮肤疾病有关。免疫相关基因直接转移到皮肤组织是一个令人着迷的方法来研究人类疾病的小鼠模型皮肤炎症的免疫调节。这里，我们提出具有成本效益的协议，它在小鼠皮肤递送裸DNA，并导致转基因表达。该方法被精压"acufection"，表示ACU穿刺介导的DNA转染 。为了执行acufection，小鼠皮肤首先用DNA注入在磷酸盐缓冲盐水（PBS），然后与针灸针束轻轻一竖以促进DNA的吸收和转染到细胞中。质粒DNA被推测采取了由角质形成细胞和皮肤中的树突细胞（DC），并表示成蛋白质。用针本身的机械刺没有造成皮肤损伤或诱导角质细胞活化。表达方式转染基因的是在皮肤在以下acufection 2天转录和翻译水平检测和维持达7天。这种acufection方法发展的主要目标是调查IL-15的先前未定义的亚型。使用这种方法，一个可变剪接IL-15同种型有部分缺失的外显子7（IL-15ΔE7）是在皮肤中表达，随后与Toll样受体7（TLR7）激动剂，咪喹莫特（IMQ）处理，以诱导炎症。 Acufection递送皮肤IL-15ΔE7抑制角质形成细胞增殖，表皮厚度和中性粒细胞募集中IMQ诱导的皮肤炎症。与在确定皮肤炎症的调节机制越来越大的兴趣，所述这里描述的协议提供了一种成本效益的和通用的替代基因枪系统或microseeding用于体内 DNA递送。这可能潜在地允许一个新颖的功能的发现基因在皮肤或调查的用于皮肤疾病的新疗法。

虽然一些发红与针灸针刺后的第一个小时之内是明显的，皮肤清除在第2天和穿刺（ 图2A）后，观察到高达至第7天无不良性皮肤反应。穿刺针与未经处理的皮肤（ 图2B）相比，H＆E分析诱导表皮正角化和最小的真皮浸润的非常低的水平。表皮厚度（ 图2C）和Ki67 +细胞（ 图2D）?...

Watch this Scientific Journal Video about Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research at JoVE.com

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

这个协议是用于在小鼠皮肤中表达裸质粒DNA具有成本效益的选择。该协议的总体目标是提供免疫相关基因植入皮肤组织划定的皮肤炎症特定基因的功能作用。

通过"Acufection"和它的皮肤研究中的应用的经济型DNA递送系统的开发

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

development-an-economical-dna-delivery-system-acufection-its

Research

JoVE Journal

Immunology and Infection

7.4K 次观看.  National Taiwan University College of Medicine. 这个协议是用于在小鼠皮肤中表达裸质粒DNA具有成本效益的选择。该协议的总体目标是提供免疫相关基因植入皮肤组织划定的皮肤炎症特定基因的功能作用。 

视频: Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，<em&gt;地中海实蝇</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

科研

遗传学

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

发育生物学

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the engineered pDNA is administered to the vaccines, it is transcribed and translated into immunogen proteins that can elicit responses from the immune system. Many ways of delivering DNA vaccines have been investigated; however each delivery route has its own advantages and pitfalls. Skin tattooing is a novel technique that is safe, cost-effective, and convenient. In addition, the punctures inflicted by the needle could also serve as a potent adjuvant. Here, we a) demonstrate the intradermal delivery of plasmid DNA encoding enhanced green fluorescent protein (pCX-EGFP) in a mouse model using a tattooing device and b) confirm the effective expression of EGFP in the skin cells using confocal microscopy.

Skin tattooing is a potent and safe way to delivery DNA vaccine intradermally. Here, a DNA plasmid encoding EGFP is delivered by tattooing to the skin of a laboratory mouse, and the expression of EGFP in the skin cells is then inspected by confocal microscopy.

皮肤纹身作为一种新的方法DNA疫苗交货

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

生物工程

High-Throughput DNA Plasmid Multiplexing and Transfection Using Acoustic Nanodispensing Technology

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often performed at low throughput, it is moreover time-consuming and error-prone, even more so when multiplexing several plasmids. We developed an easy, fast, and accurate method to perform cell transfection in a 384-well plate layout using acoustic droplet ejection (ADE) technology. The nanodispenser device used in this study&#160;is based on this technology and allows precise nanovolume delivery at high speed from a source well plate to a destination one. It can dispense and multiplex DNA and transfection reagent according to a predesigned spreadsheet. Here we present an optimal protocol to perform ADE-based high-throughput plasmid transfection which makes it possible to reach an efficiency of up to 90% and a nearly 100% cotransfection in cotransfection experiments. We extend initial work by proposing a user-friendly spreadsheet-based macro, able to manage up to four plasmids/wells from a library containing up to 1,536 different plasmids, and a tablet-based pipetting guide application. The macro designs the necessary template(s) of the source plate(s) and generates the ready-to-use files for the nanodispenser and tablet-based application. The four-steps transfection protocol involves i) a diluent dispense with a classical liquid handler, ii) plasmid distribution and multiplexing, iii) a transfection reagent dispense by the nanodispenser, and iv) cell plating on the prefilled wells. The described software-based control of ADE plasmid multiplexing and transfection allows even nonspecialists in the field to perform a reliable cell transfection in a fast and safe way. This method enables rapid identification of optimal settings for a given cell type and can be transposed to higher-scale and manual approaches. The protocol eases applications, such as human ORFeome protein (set of open reading frames [ORFs] in a genome) expression or CRISPR-Cas9-based gene function validation, in nonpooled screening strategies.

This protocol describes high-throughput plasmid transfection of mammalian cells in a 384-well plate using acoustic droplet ejection technology. The time-consuming, error-prone DNA dispensing and multiplexing, but also the transfection reagent dispensing, are software-driven and performed by a nanodispenser device. The cells are then seeded in these prefilled wells.

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

Cell transfection, indispensable for many biological studies, requires controlling many parameters for an accurate and successful achievement. Most often ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

设计和人工细胞外基质的构建（AECM）蛋白质<em&gt;大肠杆菌</em&gt;对皮肤组织工程

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

建立高通量表皮球体培养系统，以模拟角膜细胞干细胞可塑性

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Assembly and Operation of an Acoustofluidic Device for Enhanced Delivery of Molecular Compounds to Cells

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. Ultrasound-mediated sonoporation is an emerging technique for rapid intracellular delivery of biomolecules. Sonoporation occurs when cavitation of gas-filled microbubbles forms transient pores in nearby cell membranes, which enables rapid uptake of biomolecules from the surrounding fluid. Current techniques for in vitro sonoporation of cells in suspension are limited by slow throughput, variability in the ultrasound exposure conditions for each cell, and high cost. To address these limitations, a low-cost acoustofluidic device has been developed which integrates an ultrasound transducer in a PDMS-based fluidic device to induce consistent sonoporation of cells as they flow through the channels in combination with ultrasound contrast agents. The device is fabricated using standard photolithography techniques to produce the PDMS-based fluidic chip. An ultrasound piezo disk transducer is attached to the device and driven by a microcontroller. The assembly can be integrated inside a 3D-printed case for added protection. Cells and microbubbles are pushed through the device using a syringe pump or a peristaltic pump connected to PVC tubing. Enhanced delivery of biomolecules to human T cells and lung cancer cells is demonstrated with this acoustofluidic system. Compared to bulk treatment approaches, this acoustofluidic system increases throughput and reduces variability, which can improve cell processing methods for biomedical research applications and manufacturing of cell-based therapeutics.

This protocol describes the assembly and operation of a low-cost acoustofluidic device for rapid molecular delivery to cells via sonoporation induced by ultrasound contrast agents.

增强分子化合物输送到细胞的声透流体装置的组装和操作

Efficient intracellular delivery of biomolecules is required for a broad range of biomedical research and cell-based therapeutic applications. ...

Absorbent Microbiopsy Sampling and RNA Extraction for Minimally Invasive, Simultaneous Blood and Skin Analysis

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, we describe the protocol for using&#160;an absorbent microneedle-based device, absorbent microbiopsy, for minimally invasive sampling of skin and blood mixture. Our goal is to help facilitate rapid progress in clinical research, the establishment of biomarkers for skin disease and reducing the risk for clinical research participants. In contrast to conventional skin biopsy techniques, the absorbent microbiopsy can be performed within seconds and does not require intensive training due to its simple design. In this report, we describe the use of absorbent microbiopsy, including loading and application, on a&#160;volunteer. Then, we show how to isolate RNA from the absorbed sample. Finally, we demonstrate the use of quantitative reverse transcription PCR (RT-qPCR) to quantify mRNA expression levels of both blood (CD3E and CD19) and skin (KRT14 and TYR). The methods that we describe utilize off the shelf kits and reagents. This protocol offers a minimally invasive approach for simultaneous sampling of skin and blood within the same absorbent microbiopsy matrix. We have found human ethics committees, clinicians and volunteers to be supportive of this approach to dermatological research.

In this article, we demonstrate how the absorbent microbiopsy technique is performed and how the sample can be used for RNA extraction for simple and simultaneous sampling of skin and blood in a minimally invasive manner.

吸收性微活检取样和 rna 提取用于微创同时进行血液和皮肤分析

Conventional skin biopsy limits the clinical research that involves cosmetically sensitive areas or pediatric applications due to its invasiveness. Here, ...

医学

细胞培养物中的粗载体生产和高效 DNA 递送

Transgene Expression in Cultured Cells Using Unpurified Recombinant Adeno-Associated Viral Vectors

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue types, making them a popular choice for gene delivery in animal models and in clinical settings. In addition to therapeutic applications, rAAVs are a useful laboratory tool for delivering transgenes tailored to the researcher&#39;s experimental needs and scientific goals in cultured cells. Some examples include exogenous reporter genes, overexpression cassettes, RNA interference, and CRISPR-based tools, including those for genome-wide screens. rAAV transductions are less harmful to cells than electroporation or chemical transfection and do not require any special equipment or expensive reagents to produce. Crude lysates or conditioned media containing rAAVs can be added directly to cultured cells without further purification to transduce many cell types&#8212;an underappreciated feature of rAAVs. Here, we provide protocols for basic transgene cassette cloning and demonstrate how to produce and apply crude rAAV preparations to cultured cells. As proof of principle, we demonstrate the transduction of three cell types that have not yet been reported in rAAV applications: placental cells, myoblasts, and small intestinal organoids. We discuss appropriate uses for crude rAAV preparations, the limitations of rAAVs for gene delivery, and considerations for capsid choice. This protocol outlines a simple, low-cost, and effective method for researchers to achieve productive DNA delivery in cell culture using rAAV without the need for laborious titration and purification steps.

作者聚焦:揭示未纯化重组 AAV 在细胞培养研究中的潜力 

Recombinant adeno-associated virus (rAAV) is widely used for clinical and preclinical gene delivery. An underappreciated use for rAAVs is the robust transduction of cultured cells without the need for purification. For researchers new to rAAV, we provide a protocol for transgene cassette cloning, crude vector production, and cell culture transduction.

使用未纯化的重组腺相关病毒载体在培养细胞中进行转基因表达

Recombinant adeno-associated viral vectors (rAAV) can achieve potent and durable transgene expression without integration in a broad range of tissue ...

通过"Acufection"和它的皮肤研究中的应用的经济型DNA递送系统的开发

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此视频中的章节

核酸的传递通过显微注射胚胎在世界范围内的农业害虫，

转基因器官皮肤培养使用灭活人真皮代

皮肤纹身作为一种新的方法DNA疫苗交货

使用声学纳米分配技术实现高通量DNA质粒多路复用和转染

设计和人工细胞外基质的构建（AECM）蛋白质

建立高通量表皮球体培养系统，以模拟角膜细胞干细胞可塑性

增强分子化合物输送到细胞的声透流体装置的组装和操作

吸收性微活检取样和 rna 提取用于微创同时进行血液和皮肤分析

使用未纯化的重组腺相关病毒载体在培养细胞中进行转基因表达

使用未纯化的重组腺相关病毒载体在培养细胞中进行转基因表达