We thank Dr. John Ohlfest and Dr. Stacey Decker13 for the generous gift of plasmids and for the training provided to master this method. We thank Marta Dzaman for help with standardizing the Hematoxylin-Eosin staining procedure of paraffin-embedded sections. We also thank Molly Dahlgren for perusing this manuscript and providing helpful suggestions. This work is supported by NIH/NINDS grants to MGC and PRL, Leah&#8217;s Happy Hearts Foundation grant to MCG and PRL. Alex&#8217;s Lemonade Foundation young Investigator Award and the St. Baldrick&#8217;s Foundation Fellowship to CK.

注:所有动物的协议已获得密歇根州委员会的大学动物的使用和护理（UCUCA）。 1.克隆利息的顺序进入新睡美人转座子注意:要插入的基因或使用睡美人转座系统的抑制因素（如shRNA的），克隆感兴趣的序列插入或PKT pT2期质粒的中坚力量。直接克隆这样的调控元件，感兴趣和标记基因仍然受到反向重复（IR / DR）两侧。克隆PDGFβ成PKT骨干下文详述的例子（参见图1b）。 <ol><li>消化5微克质粒载体PKT-IRES-Katushka的4小时，在37℃下用5〜10单位的XbaⅠ位/ XhoI限制酶在50μl反应体积。 </li><li>消化5微克的的pGEM-T-mPDGFβ质粒进行4小时的mPDGFβ的cDNA，在37℃下用5〜10单元的S的NcoI / SacI限制性酶。 </li><li>通过加入2.5倍体积的100％乙醇和1/10体积的3M醋酸钠，pH值5.8沉淀的总DNA并孵育过夜，在-20℃下。 </li><li>离心样品在13800×g离心15分钟。 </li><li>用500μl70％乙醇，离心，在13800×g离心洗DNA沉淀1分钟，重复一次，并重新悬浮于19微升无菌DNA酶的水。 </li><li>按照制造商的在快速钝化试剂盒说明书钝矢量（PKT-IRES-Katushka）的两端，插入（mPDGFβ）。 </li><li>加载钝化载体和插入在1.2％的溴化乙锭染色的琼脂糖凝胶和运行在80伏40分钟。可视化的条带，用干净的剃刀刀片和凝胶切除它们纯化使用根据制造商的协议的凝胶纯化试剂盒的DNA。 </li><li> 1摩尔比（插入:向量），通过在4将它们添加在管结扎插入和载体DNA在步骤1.7纯化。这个管，Pipette 1微升T4连接酶和2微升（含ATP）的T4连接酶缓冲液中，调节音量，以20微升无菌水。孵育18小时，于16℃连接反应。 </li><li>变换化学感受态DH5α细菌细胞与连接产物。 <ol><li>冰上解冻感受态细胞。 </li><li>连接反应的总体积增加至50微升感受态细胞中，轻轻摇动试管并在冰上孵育所述混合物30分钟。热休克细菌45秒，在42℃，并立即返回到冰;在冰上孵育另外2分钟。 </li><li>添加950微升的Luria肉汤中向培养和孵育在37℃下振荡1小时。离心机菌在2400×g离心3分钟，并再悬浮在200μlLB的粒料</li><li>传播转化的细菌到培养皿涂布LB琼脂糖和相应抗生素。过夜孵育板在37℃。 </li><li>最后，收集5-10 bacte里亚尔殖民地文化一夜之间在37℃在5毫升LB培养基的抗生素。 </li><li>纯化使用根据制造商的说明一质粒DNA mini试剂盒的质粒DNA。执行诊断限制性消化来验证插入到载体的适当掺入和提交阳性克隆进行DNA测序，以确保插入物的右方向。 </li></ol></li><li>选择正确的殖民地，并采用了高品质的DNA生产规模可达，无内毒素质粒制备试剂盒。 </li><li>洗脱于无菌水或1毫摩尔Tris - 盐酸该DNA。商店的DNA在-20℃，直到准备注入新生儿。 </li></ol> 2.心室新生儿注射<ol><li>三到四个星期前实验，建立一个养殖老鼠笼一雄一雌老鼠，和一个塑料彩色冰屋。这提供了一个丰富的环境，并有助于在交配过程。 </li><li>当确认怀孕，去除米强麦到一个新的笼子。配种18天后，日常监测笼交付。在产后第1天（P1）进行脑室注射。 </li><li>注射液的制备 注:注射溶液是通过将质粒DNA与转染试剂来创建颗粒用于转染进行。下面是关于注射溶液使用质粒编码睡美人转座和荧光素酶的制备的例子:pT2期/ SB100x吕克和两个座子的质粒:PT / CAGGS-NRASV12和Pt / CMV-SV40-LGT，在一个比1:2:2。所有的质粒具有2微克/微升的浓度。对注射液的配制重要的细节都在讨论。 <ol><li>通过增加准备DNA溶液:4微克（2微升）的pT2 / SB100x吕克，8微克PT / CAGGS-NRASV12（4微升）和8微克（4微升）的pT的/ CMV-SV40-LGT和10微升10％葡萄糖为20微升的最终体积为1微克/毫升的浓度的DNA和5％葡萄糖。 </li><li>通过加入2.8微升在体内喷射的PEI，7.2微升无菌水和10微升10％的葡萄糖，以5％的葡萄糖的终浓度制备PEI的溶液中。 </li><li> PEI的溶液添加到该DNA溶液，混合和涡旋，并让坐在在室温（RT）下持续20分钟。该解决方案现在可以注入。在室温1小时后，保持在冰上的溶液中。 </li><li>适合一个10微升干净注射器装有30号皮下注射针头与12.5°斜角进入微型泵（ 图2＃1）。 </li></ol></li><li>为了验证喷射系统的正常运作中，使用自动注射器（ 图2＃1）撤回10微升的水进入注射器，然后完全排空注射器</li><li>冷却新生儿立体阶段（ 图2＃3）与浆料干冰和醇与2-8℃的温度下进行。 </li><li>通过将幼崽在湿冰诱导麻醉2分钟。麻醉将继续冷冻立体框架的程序的剩余部分上。保持在零度以上的帧的温度，之间2-8℃下将防止热烧伤。由于特殊的预防措施，幼崽可以包着纱布放置在湿冰之前。 </li><li>填用DNA / PEI溶液的注射器。 </li><li>通过将其头部的纱布覆盖耳棒（ 图2b）之间在固定的立体框架的小狗。确保该头骨的背侧是水平的，平行于框架的表面和颅缝都清晰可见。擦拭头，用70％的乙醇。 </li><li>降低注射器的针头和调节使用立体框架的表盘立体坐标直到针触到拉姆达（壁层和枕骨骨的中线相交）（ 图2b）。 </li><li>提起针和调整立体坐标至0.8mm的横向和1.5米。米喙的拉姆达（ 图2b）。 </li><li>降低针，直到它触及稍浅凹皮肤。测量的坐标。放下针头另外150毫米。针将刺破皮肤和颅骨，并穿过皮质进入侧脑室（ 图2c）。 </li><li>使用自动注射器，注入0.75微升的DNA / PEI溶液中的0.5微升/分钟的速率</li><li>保持在框架上的小狗一分钟，以允许所述溶液分散到心室。在此期间，麻醉另一个小狗，在冰上，准备下一次注射2分钟。 </li><li>轻轻地，慢慢抬起针，并把正在加热灯下的小狗。监测呼吸和活动。如果有必要，提供四肢轻度刺激，直到第一个呼吸随之而来。 </li><li>返回小狗其母亲后有所回暖，已经达到了红润的肤色，定期呼吸和活跃，一般5-7分钟。如果莫再超过一小狗被同时注入，使所有的幼崽在一起，直到所有注射完成。如果注射时间超过30分钟，在头30分钟，其余的程序结束后返回一半幼仔。 </li><li>监控大坝响应和培育她的幼崽。如果需要的话，一个代理妈妈添加到笼子。 </li><li>确保放置小狗在冰上，将其置于在暖灯之间的间隔小于10分钟。该过程越快越好生存。通常情况下，需要麻醉，并注入小狗的时间为4分钟。 </li></ol>肿瘤的形成和进展3.生物发光监测 3.1）监控质粒注射吸收后<ol><li>注射后24〜72小时，取出从母亲的笼子和地点的所有幼崽在6孔干净的组织培养皿，人们的小狗每口井。 </li><li>制备30毫克/毫升溶液荧光素溶解在生理盐水或phosphatË缓冲区。制备该溶液事先并保持在小等份在-20℃下。 </li><li>用1ml注射器装配有30号皮下注射针注入30微升荧光素皮下的肩胛骨的小狗之间。确保针不会刺穿任何机关，捏皮肤和插入针之前略微抬起。 </li><li>图象立即使用体内生物发光成像系统。对于IVIS频谱，用于收购以下设置:自动曝光，大型分级和光圈f = 1。 </li></ol> 3.2）监测肿瘤的形成和进展注:动物将开始形成宏观的肿瘤检测的生物发光和组织学内2.5-6周，这取决于注入的致癌质粒。 <ol><li>用1ml注射器装有26号针头注射100微升的30毫克/毫升的荧光素溶液腹膜内。 </li><li>放置在麻醉室中的动物。注入到五只动物在同一时间。 </li><li>等待5分钟，然后启动该氧气/异氟烷流量（1.5-2.5％异氟烷）麻醉动物。后3-4分钟，从麻醉室取出动物，开始在生物发光室中的氧气/异氟烷流动。将动物在装有玻璃鼻锥体，以允许麻醉和光的通畅通道的相应的槽。 </li><li>通常情况下，获得了一系列的6幅图像，在2分钟的间隔有自动曝光时间，平均分档和f = 1的开放光圈。 </li><li>设定的关注区域的所有成像，通常为椭圆的头区域中的动物，并使用校准的单位光子/秒/厘米2 / SR测量的发光强度。 </li><li>记录每只动物的最大价值。接着，记录可以在肿瘤每只动物之间和/或动物的进展期间进行比较，例如磨片n个不同的治疗方法使用。 </li></ol>新的GBM 4.组织学和免疫组化分析注:当肿瘤已经达到所希望的试验时间点，可以将动物处死，将脑灌注，固定和分析。为生存分析垂死阶段表示在实验的终点，当动物被人道地处死的肿瘤负荷的第一个迹象，由临床分期的定义，当动物变成症状表现受损的流动性，驼背姿势和破旧毛皮。所用的标准的组织学和免疫组化方法的简要说明如下。 4.1）灌注和固定<ol><li>麻醉小鼠的腹膜内注射氯胺酮100毫克/公斤和10毫克/公斤甲苯噻嗪。 </li><li>按捏鼠标的脚检查踏板反射。当这种反射被取消，这表明深度麻醉，immobilize鼠标上的夹层板与销，打开腹腔，解剖隔膜并打开胸腔，以可视化的心脏。 </li><li>插入一个钝20号插管进入心脏的左心室。连接用的塑料管穿过蠕动泵的容器具有含氧和肝素台氏溶液（0.8％氯化钠，0.0264％的CaCl 2·2H 2 O中，套管0.005％的NaH 2 PO 4，0.1％葡萄糖，0.1％的NaHCO 3，0.02 ％氯化钾）。通过调节蠕动泵的速度，以确保一个缓慢而不断流动，每秒或更小大约一滴如果动物是较小的启动台氏液的流动。 </li><li>做一个小切口进入心脏的右心房，允许血液和台氏漏。 </li><li>通过观察内部器官（最明显的，在肝和肾），因为它们变得缺乏血液的热烫监视灌注效率。 </li><li>当溶液从心脏排出清晰（3-5分钟），切换从台氏溶液至4％多聚甲醛固定（4％PFA中，pH值7.34的磷酸盐缓冲盐水）。 </li><li>由颈部和尾部的增加的刚度监视组织的良好的固定。 </li><li>斩首鼠标，仔细解剖大脑从头骨。 <ol><li>拉覆盖颅骨吻侧，对鼻子的皮毛，并抓住它牢牢地稳住头部，背部朝上。 </li><li>用锋利的解剖刀切下沿轨道的边缘，以横切轨道的肌肉和底层颧弓。 </li><li>回头腹朝上，用咬骨钳去除附着颈部肌肉。暗恋的第一椎骨和脑干中删除。 </li><li>形象化耳蜗，抢在咬骨钳上覆颞骨，并创建一个开放的时间和枕骨之间。从小脑的表面剥离枕骨。 </li><li&gt;粉碎骨覆鼻用咬骨钳。小心除去剩余的额叶和从脑中被注意到不要损伤大脑表面的表面顶骨。在轨道方面特别注意。 </li><li>转动头部腹面朝上。大脑就会滑下从头骨的剩余部分，​​现在连只通过颅神经。用小剪刀剪的嗅觉，视神经和三叉神经。这将释放大脑。 </li></ol></li><li>放置脑在4％PFA中过夜，在4℃下进行后固定。 </li><li>用于组织学分析和苏木精 - 伊红染色，大脑转移到磷酸盐缓冲液24小时，然后进行石蜡包埋。 </li><li>对于免疫组织学分析，转移到大脑的磷酸盐缓冲盐水在30％蔗糖溶液中24-48小时（直到大脑沉到试管底部） </li><li>通过肿瘤区域的中间部分大脑的一半，将切面朝下放入一冷冻模具中，嵌入到冷冻包埋介质（OCT化合物中），以及闪存冻结在干冰乙醇，干冰异戊烷的浆料或入液氮。保持冷冻块在-80°C，直到切片和染色。 </li></ol> 4.2）石蜡苏木伊红染色嵌入式脑的内在的GBM的组织病理学分析<ol><li>在4微米厚部分石蜡包埋的大脑，跌幅在42℃水浴和安装到载玻片上。 </li><li>脱paraffinize滑动通过将它们放置10分钟在60℃的烘箱中，然后在5分钟的间隔，通过一系列的三个滑动容器装满二甲苯传送它们。 </li><li>通过一系列乙醇浴水合幻灯片:100％乙醇2分钟，重复一次，95％乙醇2分钟，重复一次，70％乙醇中2分钟，然后转移到幻灯片水。 </li><li>通过浸渍他们在填充有哈里斯苏木2分钟的滑动容器弄脏的幻灯片。 </li><li>冲洗自来水，直到水是清的。 </li><li>浸滑两次到烧蓝溶液（0.1％碳酸氢钠）。 </li><li>让载玻片放置在自来水中2分钟，然后转移到80％乙醇中2分钟。 </li><li>通过浸渍他们在一个容器填充有曙红5分钟染色的幻灯片。 </li><li>脱水的幻灯片在一系列乙醇浴（80％乙醇3-4骤降，95％乙醇2分钟，重复一次，100％乙醇2分钟，重复一次）。 </li><li>清除连续用3浴室二甲苯，每次3分钟。 </li><li>盖玻片用二甲苯基的安装介质，并让干燥在平坦表面上，在室温下，直到准备用于成像。室温保存。 </li></ol> 4.3）免疫组化冷冻保存完好的嵌入式大脑从头诱导GBM中的分子和细胞表征<ol><li>检索来自-80℃储存冻结的块，放置到低温恒温器，并允许温度平衡30分钟。 </li><li>第12节-14微米厚的部分和安装2-3节上带正电的载玻片上。切段串联，通过收集相邻部分上的不同幻灯片。这允许与对肿瘤内的相邻组织切片的不同抗体的染色。 </li><li>干片在干燥器中至少1小时切片，以允许所述组织的良好的粘附之后。 </li><li>创建在滑动的每个端部的疏水性屏障（与疏水标记物），以允许液体以覆盖部分和留在在洗涤的幻灯片。覆盖用磷酸盐缓冲盐水中含0.1％的Triton-X的溶液区段（PBS 0.1％Tx）和轻轻摇动在水平摇动器5分钟以透化组织。重复两次。 </li><li> 30分钟轻微振荡方框的10％正常山羊血清（或血清从其中第二抗体升高的动物）中的溶液的非特异性结合</li><li>制备在2％正常山羊血清初级抗体溶液在PBS中0.1％的Tx和吸管将溶液过的章节。放置载玻片在黑暗中一个覆盖湿润腔室，并保持在室温下过夜。 </li><li>第二天，洗涤切片3次，每次5分钟，用PBS 0.1％Tx和孵育在稀释于2％正常山羊血清的PBS 0.1％的Tx一小时的荧光第二抗体。 </li><li>为5分钟，用PBS 0.1％TX，染液用核染色（ 如 DAPI）进行2分钟，根据封固剂将保留荧光的水冲洗切片3次，洗一次，明确在水和盖玻片。放置幻灯片在一个平面上，并让它们固化至少24小时，在黑暗中，直到准备用于成像。保持在4℃之后。 </li></ol> 5.生成与特定的基因的改变原发肿瘤细胞系。 <ol><li>从睡美人荷瘤小鼠产生细胞系选择鼠标证实大的肿瘤（生物发光10 7 -10 8光子/秒/厘米2 / SR）。 </li><li>安乐死小鼠异氟烷麻醉过量，杀头的动物和小心地从头骨解剖大脑（如第4节所述）。把大脑用一种清洁的培养皿。 </li><li>用剃刀刀片，使冠状切割前，后的肿瘤。肿瘤的位置通常是可识别的，由于脑的透明度。 </li><li>解剖的肿瘤，通常是5-7毫米，直径与细镊子并放入1.5ml的管中填充有300μl的神经干细胞培养基（NSC媒体:DMEM / F12与B-27补充剂，N2添加剂，和Normocin，补充有人重组表皮生长因子和bFGF在20纳克/ ml的浓度各自）。 </li><li>轻轻均质肿瘤用塑料杵，其配合到管的壁上。 </li><li>加入1毫升的酶 - 自由组织离解溶液和孵育在37℃下5分钟。 </li><li>通过一个70微米的细胞滤网通过细胞悬浮液，用10毫升的清洗过滤器NSC媒体，离心机在300 XG为4分钟，倾析上清液和再悬浮颗粒进入7毫升NSC媒体。 </li><li>板到T25培养瓶中并培养，在37℃在组织培养培养箱中95％的空气和5％的CO 2与和气氛。 3天之后，一些细胞已经死亡，一些附着到培养皿的底部，有些是形成神经球，自由浮动的在媒体上。 </li><li>去除这些悬浮细胞和重制版到T75组织培养瓶。 </li><li>培养三天后，收集神经球，游离如步骤5.6中所述，应变通过细胞过滤和计数细胞。冷冻细胞在胎牛血清10％DMSO中，并传代细胞的等分试样在一百万个细胞于14ml NSC的密度补充EGF和FGF的T75烧瓶中。生长速率将取决于用来产生肿瘤的致癌基因。适应细胞培养条件，以防止过度生长，并在相对高的密度维持细胞。 </li></ol>

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The authors have nothing to disclose.

在这篇文章中，我们详细介绍了产生GBM的新车型使用的致癌质粒DNA SB transposase-介导的融入周围的新生小鼠脑室下区细胞的多功能和可重复的方法。我们提出了一个协议，以产生转座子的质粒与感兴趣的新基因，说明如何监测肿瘤的进展在活的动物，以及如何表征这些肿瘤的组织病理学和免疫组织化学特征。 由于我们的实验室 （ 图3）和其他13</su...

胶质母细胞瘤（GBM）是最常见的（60％）的原发性脑成人肿瘤，具有15-21个月的中位生存当与外科手术，放射治疗和化疗1治疗。新疗法的GBM势在必行。实验性疗法需要测试在动物模型中能充分地再现了人类疾病的显着特征。策略诱导GBM在啮齿类动物中包括化学诱变烷化剂，生殖细胞或体基因变异，或移植使用神经胶质瘤细胞系2。最常用的模型采用的神经胶质瘤细胞系的植入，或者原位，进入脑或皮下使用同源细胞在动物中具有相同基因型;或异种细胞，最常用的人的GBM细胞系中，植入免疫受损小鼠中3。移植提供了许多优势，为颅内肿瘤的研究:方便重复性，standardized增长率，死亡和肿瘤的定位时间。然而，这些模型有局限性，由于用于植入和准确地再现组织能力有限的人工，微创手术的方法提供人类GBM（IV级WHO）的特点:伪pallisading坏死，核atypias，弥漫性浸润，微血管增生和肾小球样的血管的形成异常4-7。感应GBM的通过改变体细胞的基因组中与致癌基因DNA，无论是用病毒载体8-12，或与转座子介导的整合13，再现更接近人类疾病的病因和概括人类GBM的组织病理学特征。 从历史上看，CNS的肿瘤已经基于原点的感知单元，其被观察到的分类，将是一个预测因子为生存14。 GBM中分为原发性和继发性。新出现的证据TODAY指向主要胶质母细胞瘤15的高度异质性。二级GBM（15％），低级别星形细胞瘤（WHO I级），并anaplasic星形细胞瘤恶变的结果（WHO II级），与较早出现的疾病，预后较好和基因表达的"前神经"模式相关，而初级GBM（85％）表现出迟发性，预后差，胶质（古典），神经或间质的表达模式。无论是基因表达的这些模式相关的肿瘤起源细胞的实际仍在积极调查。积累的数据表明，与GBM的相关基因突变的组合预测生存。例如，heterozygocity损失染色体1P / 19Q，IDH1基因突变，PDGFRα扩增，对（LOH）与二级GBM，前神经表达模式和更好的预后相关，而EGFR表达，缺口和Sonic hedgehog通路的激活和NF1和PTEN的损失和p53的突变相关的神经，古典，或间质的主要GBM和预后更差16,17。大规模测序项目的出现和可用于测试许多患者标本的积累带来了丰富的新的信息相对于基因突变和途径牵连GBM发生发展和个体化用药，在治疗可以专门针对的可能性患者的遗传异常。最终，以评估这些突变和途径以系统的方式的预测值，并在每一种情况下，测试可能的治疗，需要的GBM的动物模型中与预先确定的遗传改变。基因组DNA的转座子介导的整合提供了一个可行的方法。 睡美人转座子制，Tc1的/ 航海类转座子的成员，被"唤醒"（构建）的多步骤多项式从鲑转座酶基因，这成为休眠超过10万年前18,19 SS位点特异性诱变的。在本质上，通过特定序列（反向重复/直接重复:IR / DR）的侧翼DNA的转座子可由睡美人转座活性的方式整合入基因组中"切割和粘贴"的方式。转座识别的IR部位的端部，切除转座子随机整合入碱基T和A，其在转（ 图1a）被复制，在转座子的每个末端碱基之间的另一DNA位点。睡美人转座由三个结构域，转座子结合结构域，核定位序列和催化结构域的。四个转座分子需要转座子的两端汇集并允许换位，但是，如果是本转座的过多的分子，它们可以二聚化和tetramerize抑制换位反应20。一个高效的换位反应需要转座到转座子的最佳比例。编码转座的DNA可被传递在相同的质粒用转座子（顺式），或在不同的质粒（反式）。为了确保转座和座子之间的最佳比率，有足够的活性的启动子可被选择用于转座的表达（为"顺式"的模式），或在注射液中的质粒可以被优化的比例（对于"反式"模式）。睡美人座子系统可成功地用于功能基因组学，插入诱变，转基因和体细胞基因治疗21。作为一种合成的结构重新设计，从休眠鲑变体功能分子，睡美人转座不绑定到人类或其他哺乳动物的其他20个座子。自从被发现以后，分子工程有enhanc编了SB座子系统通过改变在IR序列和另外的TATA二核苷酸侧翼的座子，导致中PT2座子的转座功效。这些转座子已经优化了SB的结合到IR部位和增加的切除的效果。在SB转座也经历了显著的改善;在本文中所呈现的实验中使用的转座是SB100X，通过DNA改组的策略，随后通过在哺乳动物细胞中22大规模遗传筛选产生的活动过度的转座。 在本报告中，我们提出了一种快速，灵活和可再现的方法来诱导固有GBM小鼠非病毒，转座子介导的整合的质粒DNA进入新生小鼠23的亚脑室区的细胞。我们提出了一个简单的方法来创建转座子与新基因适合用于使用睡美人转座基因组整合和演示如何监视质粒的摄取和使用生物发光疾病进展。我们也刻画转载本模型GBM的组织学和免疫组化特点。此外，我们提出了一个快速的方法，以产生从这些肿瘤原发性GBM细胞系。睡美人模型，其中肿瘤细胞从原来的动物诱导的，允许候选GBM基因的诱导和肿瘤进展中的作用的功能性评估。此系统也适用于良好的新颖的GBM治疗，包括在免疫感受态小鼠的免疫疗法的检测，而不需要侵入性炎症的外科手术，其可以改变局部微环境。

<table><tbody><tr><td>Stoelting&#039;s Lab Standard with Rat and Mouse Adaptors</td><td>Stoelting</td><td>51670</td><td>Other companies produce similar frames, any of them with small mouse adaptors are suitable&amp;nbsp;</td></tr><tr><td>Quintessential Stereotaxic Injector (QSI)</td><td>Stoelting</td><td>53311</td><td>Injections can be made without it, ,but&amp;nbsp;&amp;nbsp;&amp;nbsp; the automatic injector allows for increased reproducibility and convenience</td></tr><tr><td>10 &amp;mu;l 700 series hand fitted MICROLITER syringe</td><td>Hamilton</td><td>Model 701</td><td>This syringe model will deliver from 0.5 to 500 ul of solution reliably. Other syrnges (fro example 5 ul, Model 75) may also be used</td></tr><tr><td>30 gauge hypodermic needle&lt;br/&gt; (1.25&quot;, 15&lt;sup&gt;o&lt;/sup&gt; bevel)&amp;nbsp;</td><td>Hamilton</td><td>S/O# 197462</td><td>This needle is ideal to pierce the skin and the skull of a neonatal mouse without the need of other invasive procedures. If it gets dull (doesn&#039;t easily enter the skin), it needs to be replaced.</td></tr><tr><td>in vivo-jetPEI</td><td>Polyplus Transfection</td><td>201-10G</td><td>Aliquot in small volumes and keep at -20 oC&amp;nbsp;</td></tr><tr><td>D-Luciferin, Potassium Salt</td><td>Goldbio.com</td><td>LuckK-1g</td><td>Other sources of firefly lucifierase are just as adequate</td></tr><tr><td>Hematoxylin Solution, Harris Modified</td><td>Sigma Aldrich</td><td>HHS128-4L</td><td></td></tr><tr><td>Tissue-Tek O.C.T. Compound</td><td>Electron Microscopy Sciences</td><td>62550-01</td><td>For embedding brains for cryosectioning and immunohistovhemistry</td></tr><tr><td>Advanced DMEM/F-12, no glutamine</td><td>Life Technologies</td><td>12634-010</td><td>For the culture of neurospheres</td></tr><tr><td>B-27&amp;uml; Supplement (50X), serum free&amp;nbsp;</td><td>Life Technologies</td><td>17504-044</td><td>Serum free supplement for the culture of neurospheres</td></tr><tr><td>N2 Supplement (100x)</td><td>Life Technologies</td><td>17502-048</td><td>Serum free supplement for the culture of neurospheres</td></tr><tr><td>Normocyn</td><td>InvivoGen</td><td>ant-nr-1</td><td>Anti-mycoplasma agent for the culture of neurospheres</td></tr><tr><td>Recombinant Human EGF</td><td>PEPROTECH</td><td>AF-100-15</td><td>Growth factor supplement for the culture of&amp;nbsp; neurospheres</td></tr><tr><td>Recombinant Human FGF-basic</td><td>PEPROTECH</td><td>100-18B</td><td>Growth factor supplement for the culture of&amp;nbsp; neurospheres</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>HyClone HyQTase&amp;nbsp; Cell Detachment Reagent&amp;nbsp;</td><td>Thermo Scientific</td><td>SV3003001</td><td>For the dissociation of neurospheres</td></tr><tr><td>Kimble-Chase Kontes Pellet Pestle</td><td>Fisher Scientific</td><td>K749510-0590</td><td>For the dissociation of freshly dissected tumors</td></tr><tr><td>Falcon Cell Strainers mesh size 70 um</td><td>Fisher Scientific</td><td>08-771-2</td><td>For generating single cell suspensions of dissociated neurospheres</td></tr><tr><td>Xylenes Histological grade</td><td>Fisher Scientific</td><td>C8H10</td><td></td></tr><tr><td>Protocol Harris Hematoxylin Mercury free (acidified)</td><td>Fisher Scientific</td><td>245-678</td><td></td></tr><tr><td>Protocol Eosyn Y Solution (intensified)</td><td>Fisher Scientific</td><td>314-631</td><td></td></tr></tbody></table>

transposon mediated integration of plasmid dna into the subventricular zone of neonatal mice to generate novel models of glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

为了表征的SB引起的胶质母细胞瘤的组织病理学特征，C57 / BL6新生小鼠注射在P1与质粒编码荧光素酶（pT2期/ SB100x吕克）联合的质粒编码座子用致癌基因DNA， 也就是说 ，NRAS（PT / CAGGS -NRASV12）和SV40 LGT（PT / CMVSV40-LGT）（ 图3c）或质粒编码的短发夹的p53与PDGFβ和GFP报告（pT2shp53 / GFP4 /mPDGFβ）结合NRAS（ 图3d）。对于生物发光一天注射（ 图2a）后的动物进行?...

Watch this Scientific Journal Video about Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma at JoVE.com

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

质粒DNA转座子介导整合到新生小鼠的脑室下区，以生成胶质母细胞瘤的新模型

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

transposon-mediated-integration-plasmid-dna-into-subventricular-zone

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

12.8K 次观看.  University of Michigan School of Medicine. Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

视频: Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

随后的三维颅内植入在体内</em生物发光成像鼠脑胶质瘤

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

科研

医学

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

遗传学

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

神经科学

通过基于电穿孔的质粒 DNA 递送进行体内脑肿瘤建模

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

作者聚焦:使用基于电穿孔的质粒 DNA 递送代表患者突变特征的体内脑肿瘤建模  

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

使用基于电穿孔的质粒DNA递送对 体内 脑肿瘤进行建模，以代表患者突变特征

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in ...

癌症研究

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

免疫活性动物模型胶质母细胞瘤的生物发光成像

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation. Additionally, we describe various adaptations to the in utero electroporation method that increase survivability and reproducibility. This method also involves establishing a titer for Cre-mediated recombination in a sparse or dense population of neurons. Histological preparations of labeled brain tissue do not require (but can be adapted to) immunohistochemistry. The constructs used guarantee that fluorescently labeled neurons carry the gene for Cre recombinase. Histological preparations allow morphological analysis of neurons through confocal imaging of dendritic and axonal arbors and dendritic spines. Because loss or gain of function is achieved in sparse mosaic tissue, this method permits the study of cell-autonomous necessity and sufficiency of gene products in vivo.

Inducing Cre-lox Recombination in Mouse Cerebral Cortex Through In Utero Electroporation

Cell-autonomous functions of genes in the brain can be studied by inducing loss or gain of function in sparse populations of cells. Here, we describe in utero electroporation to deliver Cre recombinase into sparse populations of developing cortical neurons with floxed genes to cause loss of function in vivo.

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To ...

质粒DNA转座子介导整合到新生小鼠的脑室下区，以生成胶质母细胞瘤的新模型

摘要

探索更多视频

此视频中的章节

随后的三维颅内植入在体内

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

使用基于电穿孔的质粒DNA递送对体内脑肿瘤进行建模，以代表患者突变特征

使用基于电穿孔的质粒DNA递送对体内脑肿瘤进行建模，以代表患者突变特征

使用基于电穿孔的质粒DNA递送对体内脑肿瘤进行建模，以代表患者突变特征

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

免疫活性动物模型胶质母细胞瘤的生物发光成像

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

质粒DNA转座子介导整合到新生小鼠的脑室下区，以生成胶质母细胞瘤的新模型

摘要

探索更多视频

此视频中的章节

随后的三维颅内植入在体内

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

原位胶质母细胞瘤小鼠模型的维护脑实质的物理约束，并适用于活体双光子显微镜

成神经细胞迁移的小鼠急性脑片在体内电穿孔产后和时间推移成像

使用基于电穿孔的质粒DNA递送对 体内 脑肿瘤进行建模，以代表患者突变特征

使用基于电穿孔的质粒DNA递送对 体内 脑肿瘤进行建模，以代表患者突变特征

使用基于电穿孔的质粒DNA递送对 体内 脑肿瘤进行建模，以代表患者突变特征

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

免疫活性动物模型胶质母细胞瘤的生物发光成像

宫内电穿孔诱导小鼠大脑皮质中氧合酶的重组

使用基于电穿孔的质粒DNA递送对体内脑肿瘤进行建模，以代表患者突变特征

使用基于电穿孔的质粒DNA递送对体内脑肿瘤进行建模，以代表患者突变特征

使用基于电穿孔的质粒DNA递送对体内脑肿瘤进行建模，以代表患者突变特征