Name: transposon mediated integration of plasmid dna into the subventricular zone of neonatal mice to generate novel models of glioblastoma
Uploaded: 2015-02-22T14:00:00.000+00:00
Duration: 10 min 58 s
Description: 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

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所显示的，该模型确实产生与人类的GBM的显着特征包括:（1）伪栅栏状坏死，（2）血管增生，（3）核异型，（4）异常有丝分裂和肿瘤（ 5）血管周围弥漫浸润。利用新生小鼠是从后勤角度来看最优的，提供了一个以最小的设备和本身相对简单的技术流程dation /恢复时间，从而快速生成肿瘤的特定基因变异的大样​​本。这些肿瘤引起的动物屈服于肿瘤负荷可预测的中位生存期取决于诱发的基因改变。最后，对SB模型已被用作治疗干预屏幕用于治疗响应24。 有准备用于新生儿注射的解决方案时要考虑几个重要因素。 DNA的解决方案需要是无菌的，无内毒素，并浓缩（2-7微克/微升），以允许注射最小体积。氮残留量的聚乙烯亚胺（PEI）的磷酸盐残基中的DNA（N / P比）的最佳比例为7。这确保了正电荷的粒子复合物将结合阴离子部分在细胞表面，将被内吞的形成。一旦在细胞质中，渗透潮将导致颗粒破裂并释放encapsulat编辑质粒DNA。 在体内喷射的PEI溶液具有150mM的表示为氮残基的浓度。鉴于1微克DNA具有3纳摩尔阴离子磷酸盐，必要可以计算与式转染试剂的量: 体内喷射PEI =的微升[（微克DNA×3）×N / P比] / 150。 例如，为了制备40微升0.5微克/微升DNA溶液为7的N / P比，20的DNA微克是必要的。所需PEI量将是20 * 3 *一百五十○分之七= 2.8微升。 多达五个不同的质粒可以同时被注入。本文介绍的实验提供了睡美人转座反式上的质粒也编码荧光素酶（SBLuc）。如在引言中提到，转座分子与转座子的比率是很重要的，以确保最佳换位。所述SB100x质粒到运输的最佳比例（经验确定）oson DNA质粒为1:4。因此，若用两种不同的转座子的质粒，T1和T2例如，SBLuc的比例:T 1:T 2为1:2:2;或者，共20微克的DNA在40微升溶液4微克SBLuc将与8微克T2的T1和8微克混合。对三种不同的转座子质粒的比例将是SBLuc:T1:T2:T3 = 1:1.33:1.33:1.33和4转座子:1:1:1:1:1。 它由生物发光24-72小时注射后，验证质粒DNA的摄取是非常有用的。作为动物生长，皮毛，皮肤，颅骨和脑的增加的光密度，防止监控肿瘤进展直至肿瘤已经达到阈值的大小，大致1-2毫米3为暗色素C57BL6小鼠。光更好的传输有可能在这些小鼠，如果它们的皮毛被剃光，或使用鼠标有白色或无苔时。 若干限制需要使用该模型时，需要考虑。首先，该基因与注入的质粒引入抽动材料可在不同的位置和打印份数的宿主基因组中随机整合。这可能会导致变性的肿瘤细胞之间和肿瘤之间。第二，引入的DNA被插入到主要内含子的TA重复序列的DNA 25，然而，干扰与编码宿主基因组DNA的可能性仍然存在。第三，心室内注射可导致年轻小鼠小而持续速率脑积水，成为临床上可见的和有症状在生命的第3-4周。小鼠的一些菌株更容易积水比其他（ 即 ，C57 / BL6以上FVB）。为了尽量减少诱发脑积水的风险，建议注入尽可能小的体积尽可能（小于1微升在C57 / BL6小鼠），以确保该针是尖锐，所述溶液具有低盐度，并提供生长幼仔用丰富的食物，为了让及时骨化颅骨苯教上课。最后，晚期肿瘤常常发展坏死，其具有由生物发光监测小鼠时加以考虑。较低的读数可能表明一个先进的肿瘤大面积坏死，而不一定肿瘤作为治疗的结果的分辨率。最终，组织学分析仍然是黄金标准来评估肿瘤进展。 肿瘤全基因组测序，近年来的出现打开了在GBM反复地突变基因的作用探索。睡美人模式是最佳的验证潜在的癌基因或抑癌基因。如在本文中与配位体PDGFβ给出，克隆的候选基因的小鼠cDNA序列插入一个既定的SB质粒骨架的例子将导致转染细胞内的组成型表达。评价肿瘤抑制基因的作用，可通过克隆到的S​​B的质粒与候选序列中，骨干有效地进行（一vailable例如在RNAi的抄本数据库26），打造第二代的miR短发夹强劲表现。 在GBM研究领域的当前辩论涉及到细胞OF-出身的身份。近来已经证明，在小鼠中，诱发下调p53和NF1神经干细胞瘤，少突胶质细胞前体细胞27起源。使用睡美人座子模型，类似的研究可被设计，以测试是否有其他的遗传改变优先靶向干细胞/前体细胞的其他人群产生的GBM。知识从这些研究将大大提升我们的GBM病因的了解，并提供途径，新的治疗方法。

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）后的动物进行了监测，并定期直到到达垂死阶段，当他们被安乐死。垂死阶段被定义为当动物变成症状表现削弱流动性，弯腰驼背的姿势，褴褛的皮毛和减肥的临床分期。有时候，动物制定发作或运动的异常模式，就像走在圈子里和突然的跳跃。在实验动物的端s麻醉。将脑灌注，包埋在石蜡中，并处理用于苏木精和伊红染色。数据显示肿瘤出血（ 图3c），伪pallisading坏死（ 图3E）和血管周围及弥漫性浸润至脑实质显示人类GBM（IV级WHO）的标志（ 图3g，F）。 pseudopallisades的形成之前的大肾小球样血管与漏内皮（ 图3I），其导致出血与单核细胞的大量渗入（ 图3H）的区域的破裂。非典型有丝分裂（ 图3J）和巨大的多核肿瘤细胞（ 图3K）也特异病征性人类GBM的。 使用睡美人转座系统可以在整个肿瘤的生长与生物发光的进展进行监控，生成的胶质母细胞瘤如果注入质粒编码荧光素酶。一个示例实验示于图4a。动物通常死于肿瘤负荷时发光达到10 7 -10 9光子/秒/厘米2 / SR强度。动物的平均存活是可预见的依赖注入新生儿脑致癌转座子的组合，如图中在图4b中显示的存活曲线。需要注意的是最积极的肿瘤诱导NRAS和SV40 LGT抗原（30天中位存活），而诱导shp53 NRAS和PDGF动物GBM的中位生存期为62.5天，动物注射shp53和NRAS83天。 从头形成的肿瘤可以通过它们的分子神经胶质肿瘤的特征的表达来表征免疫组织化学。 图5示出了一个新生肿瘤（22 dpi的，生物发光2×10 5光子/秒/厘米2 / SR），诱导shp53和NRAS。注入shp53质粒编码绿色荧光蛋白（GFP），以允许转染细胞的鉴定和其后代（pT2期/ shp53 / GFP）。 GFP +的肿瘤细胞表达神经干细胞标记物巢蛋白和一些的GFP +细胞也表达胶质纤维酸性蛋白（GFAP）。 图6示出了在56 dpi的，肿瘤将其还诱导shp53 / GFP和NRAS从垂死动物肿瘤。许多GFAP +星形胶质细胞是肿瘤周围。 GFP +的肿瘤细胞表达巢蛋白，但不GFAP。 使用这种技术来诱导的GBM当一大优点是通过喷射出的转座子的装置，以产生新的GBM细胞系具有独特的遗传改变的能力。此外，定制的细胞系可以使用转基因动物具有特定遗传组成产生。这些细胞系都有助于询问使用生化分析很多机械问题。它们非常适合用于新型CH毒性研究emotherapeutic代理商。 图7显示了从睡美人肿瘤诱导shp53，PDGF和NRAS，显示GFP，被编码在注入质粒之一的表达的神经球。注意，表达式不是在所有细胞同样强烈，表明这些原发性GBM细胞的异质性。 <img alt="图1" src="/files/ftp_upload/52443/52443fig1highres.jpg" /> 图1:（一）示意图说明使用的睡美人转座到转座子整合到宿主染色体DNA的供体转座子质粒描述与由反向重复/直接重复两侧目的基因的"剪切和粘贴"机制。 （IR / DR;红色箭头框）序列。在SB转座（绿色）结合到IR / DR，切除转座子和重新整合它在宿主染色体DNA的随机的TA二核苷酸的碱基对之间。 克隆（b）的实施例感兴趣（mPDGFβ）的基因导入到SB载体 （PKT-IRES-Katushka） 的骨干 。在SB矢量包含两个红外/ DR重复序列（红色）和一个基因表达盒，其包括启动子和增强子序列，多克隆位点（MCS;蓝色），内部核糖体进入位点（IRES），荧光报道标记（Katushka:黄），和一个聚腺苷酸化位点（多聚A）。感兴趣（mPDGFβ;橙色）的原癌基因被包含在一个克隆载体PGEMT。的癌基因，由特定的限制性位点（NcoI位/ SacI位）侧接被释放通过酶消化，并减弱由钝化酶。载体线性化，并减弱为好。最后，mPDGFβ基因插入到供体载体由平末端连接反应，生成新的质粒转座子载体的手段:PKT-mPDGFβ-IRES-Katushka <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52443/52443fig1large.jpg" target="_blank">请点击此处查看大图Ø</a>F该数字。 <img alt="图2" src="/files/ftp_upload/52443/52443fig2highres.jpg" /> 图2:试验设置和引导帧内心室注射新生小鼠（a）一个立体框架（2）与千分表配备有一个自动注射器保持一个10微升注射器（4）。在U框架内，新生儿适配器框架牢固（3）。自动喷射器（1）的控制面板允许精确选择的注射器，体积和流量率（b）一种新生小鼠（P1）与在所需的注射至侧脑室坐标插入的针的照片:1.5毫米腹侧和0.8毫米横向于拉姆达。（c）通过一个新生小鼠（P1）的脑插图一冠状部分的突出尺寸和心室的位置相对。 图3:诱导睡美人转座系统肿瘤表现出新生儿小狗24小时的人GBM（一）生物发光图像的脑室注射质粒编码NRAS，SV40 LGT后的组织学特点（二）生物发光的成年动物的图像（。 50 dpi）的具有大肿瘤诱导shp53 NRAS和PDGF。（三）苏木和从濒死小鼠诱导NRAS和LGT（28 dpi）的肿瘤的大脑冠状部的曙红染色。（四）冠状部从垂死的老鼠与肿瘤的诱导大脑与shp53 NRAS和PDGF。（五）Pseudopalisading坏死，人类GBM的组织学特点从头产生肿瘤的观察。箭头指向细胞排列栅栏从坏死（N）中心区迁移走（f）和（克） 的从头产生肿瘤是高度侵入性，表现出侵袭沿血管（克）和漫（F）进入正常脑实质。 （H）出血进行大规模浸润单核细胞（箭头），pseudopalisading坏死的区域的初始阶段的区域。（i）与漏内皮（箭头）大型肾小球样容器中于扩散出血诱导NRAS肿瘤内的原点和SV40 -LgT。（J）在肿瘤细胞有丝分裂不典型（箭头）。（K）与多个大型核（箭头）巨型肿瘤细胞。 <img alt="图4" src="/files/ftp_upload/52443/52443fig4highres.jpg" /> 图4:荷瘤动物可与生物发光整个疾病进展的时间进行监控。动物的平均存活通过组合预测致癌DNA注入。 （一）实施例的从7 C57 / BL6小鼠队列生物发光痕迹。需要注意的是小鼠屈服于大约一个星期后，生物发光达到10 8光子/秒/平方厘米/ SR。（二）比较动物的中位生存期与睡眠有不同质粒组合，产生美容的肿瘤样品生存曲线。诱导NRAS和SV40 LGT肿瘤的中位生存期为30 dpi的动物与诱导shp53 NRAS和PDGF或shp53和NRAS肿瘤，而分别是62.5 dpi或83 dpi的。 DPI:天注射后。 <img alt="图5" src="/files/ftp_upload/52443/52443fig5highres.jpg" /> 图5:初生宏观GBM（22 dpi）的诱导shp53和NRAS表明巢蛋白和GFAP的GFP +肿瘤细胞（共焦显微照片）的表达图（a）示出了一个新生肿瘤表达GFP。图（b）代表同场展示巢蛋白表达。面板<str翁&gt;（c）是一个覆盖层（A）和（b）表示共定位巢和GFP的。 （四）在肿瘤细胞中的GFP表达。在某些肿瘤细胞和周围新生肿瘤细胞（E）GFAP的表达。（F）的（D）覆盖投影和（e）示出了一些肿瘤细胞（白）共表达GFAP和GFP。的（a）和（d）比例尺代表75微米。 <img alt="图6" src="/files/ftp_upload/52443/52443fig6highres.jpg" /> 图6:GBM诱导shp53，NRAS和PDGF从濒死动物（56 dpi）的表示GFP和巢蛋白表达在肿瘤细胞和丰富染色肿瘤周围的神经胶质成熟标志物GFAP面板（a）表示acoronal部的尼氏染色。通过一个垂死的动物与肿瘤脑（强烈的蓝色染色˚FROM增加了细胞结构）诱导使用shp53，PDGFΒ和NRAS睡美人转座子体系。分图（b），冠状相邻部分，以在面板中所示的一种:（a）染色与核染色剂DAPI，显示在肿瘤细胞中的GFP表达。面板（c）示肿瘤边界示出了在肿瘤GFP表达的共焦显微照片，确定了高密度的核用DAPI。面板（d）表示的同一视场中（a）中，表示在相邻的肿瘤细胞强烈GFAP的表达。面板（E）表示板的覆盖层（c）和（d）所示。面板（f）是肿瘤边界的共焦显微照片，显示出在肿瘤细胞中的GFP表达。面板（g）表示的同一视场中（f）中 ，示出在肿瘤细胞中巢蛋白的表达。面板（h）为面板的覆盖投影（F）和（g） <str翁&gt;。（c）中比例尺条为150微米，在（f）表示75微米。 <img alt="图7" src="/files/ftp_upload/52443/52443fig7highres.jpg" /> 图7:神经球从睡美人肿瘤诱导shp53，PDGF和NRAS后5天培养，通道2将细胞表达GFP编码上shp53 PDGF质粒（pT2shp53 / GFP4 / mPDGFβ）面板（a）是一个神经球的明显微照片。面板（b）表示同一视图的外延荧光显微镜照片如（a）中示出了在神经球的细胞的GFP表达。面板（c）示面板的覆盖层（a）和（b）中。

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 San Sebastian

Research

JoVE Journal

Medicine

12.9K 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

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 ...

科研

遗传学

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

新生儿脑室下区电穿孔

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

神经科学

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 ...

医学

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 ...

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

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

皮质的interneurons的亚型选择性电穿孔

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

免疫与感染

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 ...

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

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此视频中的章节

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

新生儿脑室下区电穿孔

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

皮质的interneurons的亚型选择性电穿孔

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

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

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

新生儿脑室下区电穿孔

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

皮质的interneurons的亚型选择性电穿孔

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

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