我们感谢 Dr. G. McGillivray, 公关. 美 Dobyns, 公关. Striano, 公关. 夏侯雅伯, 公关. 某地 Roberston 和公关. 顺 Berkovic 为患者提供服务。我们感谢 Dr. ·米歇尔和 d. 梅的技术咨询和帮助。这项工作得到了欧盟七框架方案的资助, 愿望项目, 合同编号: Health-F2-602531-2013, (共,., ar, 个和 cc), INSERM (ar 和 cc), ô勒琼 (R13083AA, 电 P 和 c。Région 普罗旺斯阿尔卑斯科特迪瓦蔚蓝海岸 (APO2014-通俗到 cc 和 d。检察官 K 是一个 EMBO 年轻的调查员, 并得到 FWF 赠款 (I914 和 P24367) 的支持。

伦理声明: 大鼠交配, 维护和使用的 INMED 动物设施, 与欧洲联盟和法国立法的协议。
1. 阵列全息和 WES 的 DNA 提取和定量
<ol><li>根据制造商的协议, 从使用自动 dna 隔离机器人的患者或商业上可用的人工 dna 提取试剂盒中提取人血白细胞的基因组 dna (gDNA)。用分光光度计对所有样品进行量化。</li>
</ol>2. 阵列全息协议
<ol><li>
gDNA 的限制性消化
	<ol>
<li>双消化 500 ng 纯化 gDNA 从一个病人和一个商业可用的人类控制 DNA 与 RsaI 和 AluI 2 小时在37° c。每个反应使用0.5 μ l 10 U/μ l 酶。在65° c 下孵育20分钟, 使酶钝化并将样品管移至冰层。</li>
	</ol>
</li>
	<li>
样品标签
	<ol>
<li>添加用于每个反应管的微阵列格式所需的适当数量的随机引物 (例如5 µl, 用于1包、2包和4包芯片)。移上下轻轻地混合均匀。</li>
		<li>将样品管转移到热循环。孵育在95° c 为3分钟, 然后在4° c 为 5 min。</li>
		<li>在冰上准备一个菁3和一个菁5标签主混合, 混合以下卷: 10.0 µl 5X 反应缓冲器, 5.0 µl 10x dNTPs, 3.0 µl 菁 3-dUTP 或青菁 5-dUTP 和1.0 µl 外 (-) 克莱诺酶, 2.0 µl 核酸-游离水。根据使用的微阵列格式选择每种试剂的体积。</li>
		<li>添加标签主混合到每个反应管包含 gDNA。通过轻轻地移上下混合好。</li>
		<li>将样品管转移到热循环, 在37° c 下孵育2小时, 65 ° c 为10分钟, 然后在4° c 下维持样品。</li>
	</ol>
</li>
	<li>
标记 gDNA 的纯化
	<ol>
<li>在每个反应管中加入430µl 的 1x TE (pH 8.0)。</li>
		<li>将净化柱放入2毫升的收集管中, 并适当地标注列。将每个标签的 gDNA 加载到净化柱上。</li>
		<li>在室温下, 在离心中离心10分钟, 1.4万 x g。丢弃流, 并将该列放回2毫升收集管中。</li>
		<li>每列添加480µL 1x TE (pH 8.0)。在室温下, 在离心中离心10分钟, 1.4万 x g。丢弃流。</li>
		<li>将柱形倒置到新鲜的2毫升收集管和离心机1分钟在 1000 x g 离心在室温下收集纯化样品。</li>
		<li>使用选矿厂或添加 1x TE (pH 值 8.0), 将样品量带到所需的微阵列格式。例如, 对于1包微阵列, 将该卷带到80.5 µL. 将样品在冰上孵育5分钟, 然后将溶液上下移10次。</li>
	</ol>
</li>
	<li>
杂交标记 gDNA 的制备
	<ol>
<li>结合测试和参考样品使用适当的菁5标记样品和菁3标记的新鲜1.5 毫升核糖核酸管样品。例如, 对于1个包装的微阵列, 确保最终的体积是158µL. 根据使用的微阵列格式选择每个样品的体积。</li>
		<li>使用分光光度计测量 A260 nm (DNA)、A550 nm (菁 3) 和 A650 nm (菁 5) 的吸光度, 以测量产量、标签度或特定活性。<ol>
<li>使用公式计算产量: [DNA 浓度 (ng/µl) x 样本体积 (µl)]/1000 (ng/µg)。使用公式计算特定的活动: pmol 每µl 染料/µg 每µl gDNA。 注: 例如, 对于0.5 µg 的输入 DNA, 其收率应为8至11µg, 而具体活动 (pmol/µg) 应为 Cyanine-3 标签样品的25至 40, 20 至35用于 Cyanine-5 标签的样品。</li>
		</ol>
</li>
		<li>要准备使用的微阵列格式所需的杂交主混合的数量, 混合以下试剂:50 µL Cot-1 DNA (1.0 毫克/毫升), 52 µL 10x aCGH 阻断剂和260µL 2x 高转速杂交缓冲。</li>
		<li>添加适当的杂交主混合的体积到1.5 毫升无核糖核酸管, 包含标签 gDNA, 以获得总体积所需的微阵列格式在使用。例如, 对于1包微阵列, 确保每个反应的最终体积为362µl。</li>
		<li>将样品由移上下混合, 然后快速离心 1.4万 x g, 在95° c 和37° c 下孵育3分钟, 30 分钟。</li>
	</ol>
</li>
	<li>
室组装和杂交
	<ol>
<li>将一个干净的垫圈滑动到会议厅底座上, 垫片标签朝上, 并与会议厅底座的矩形部分对齐。确保垫片滑轨与室底座齐平且不开半掩。</li>
		<li>将杂交试样混合物放在垫片上, 确保样品均匀分布。</li>
		<li>将微阵列滑动到垫片滑动上, 评估三明治对正确排列。然后, 组装反应室, 把盖子放在夹层的幻灯片和手收紧钳。</li>
		<li>通过垂直旋转组装室确保滑块湿润。确保气泡不存在于会议厅中。如有必要, 在坚硬的表面上敲击组件以移动固定气泡。</li>
		<li>将组装的滑动腔放在一个旋转架上, 设置为 20 rpm 转动。根据使用的微阵列格式, 将杂交室放在烤箱中, 并在65° c 下执行24或40小时的杂交。</li>
	</ol>
</li>
	<li>
微阵列清洗和扫描
	<ol>
<li>将杂交室从烤箱中取出, 浸入洗涤缓冲器1。继续进行其拆卸。</li>
		<li>取出微阵列滑动, 并迅速把它放入一个滑动机架在洗涤缓冲器1在室温。</li>
		<li>用搅拌 (使用跳蚤和磁力搅拌器) 清洗阵列幻灯片5分钟。将搅拌器的转速调整到 4 (中速), 得到好的但不太剧烈的搅拌。</li>
		<li>在洗涤缓冲器2洗涤阵列幻灯片九十年代搅动。轻轻地从缓冲器中恢复滑块 (它应该会出现干燥), 并借助滑动保护器将其加载到滑动支架上。</li>
		<li>将幻灯片加载到阵列扫描仪中, 并根据阵列制造商的说明执行扫描。</li>
		<li>根据制造商的说明使用扫描仪提供的抽取软件分析结果 (v 9.1. 3.1)。</li>
	</ol>
</li>
</ol>3. WES 议定书
<ol><li>目标富集
	<ol>
<li>使用 dsDNA BR 测定法, 根据仪器协议确定 gDNA 样品的浓度。</li>
		<li>用1x 低 TE 缓冲器在1.5 毫升低束缚管中稀释5µg 的高质量双链 DNA, 总容积为50µL。</li>
		<li>根据制造商的指示, 设置 sonicator 并将微放入装卸站。把帽子放在管子上。</li>
		<li>缓慢转移50µl DNA 样本通过前分裂的隔膜, 而不引入气泡。</li>
		<li>根据制造商的指示, 按照 sonicator 制造商的建议, 对 DNA 进行剪切, 并使用 Bioanalyzer 进行质量评估。靶 DNA 片段的大小是150到 200 bp。</li>
	</ol></li>
	<li>DNA 末端的修复
	<ol>
<li>将端部修复酶混合40µL 与10µL 的末端修复寡糖混合, 制备最终修复反应混合料。在涡流混合器上混合均匀。</li>
		<li>将样品在20° c 下孵育30分钟, 在热循环, 然后在4° c 下举行。不要用加热的盖子。</li>
		<li>添加50µL 的年底修复反应混合准备在步骤3.2.1 每50µL 剪切 DNA 样本好。移上下或轻柔的涡流混合均匀。</li>
		<li>根据制造商的协议, 用一个基于珠子的净化套件来净化样品。</li>
	</ol></li>
	<li>多 3 ' 结束。
	<ol>
<li>添加20µl 的 dA 尾矿主混合到每个最终修复, 纯化的 DNA 样本 (约20µl)。</li>
		<li>移上下或轻柔的涡流混合均匀。</li>
		<li>在37° c 的热循环中孵育样品, 然后在4° c 下举行。不要用加热的盖子。</li>
	</ol></li>
	<li>预捕获索引适配器的结扎。
	<ol>
<li>在每尾 DNA 样本中加入5µL 的结扎主混合。</li>
		<li>将适当的预捕获索引解决方案的5µL 添加到每个示例中。</li>
		<li>密封井和混合彻底由涡流 5 s. 简要地离心样品。</li>
		<li>在20° c 下孵育样品15分钟, 在热循环, 然后举行在4° c。不要用加热的盖子。</li>
		<li>根据制造商的协议, 用一个基于珠子的净化套件来净化样品。</li>
	</ol></li>
	<li>索引库的放大。
	<ol>
<li>通过混合1µL 的引物混合剂和25µL 的 pcr 组合, 制备合适的预捕获 pcr 反应量。在涡流混合器上混合均匀。</li>
		<li>将26µL 的放大混合物分别放在 PCR 板的分离井和每个索引的 gDNA 库样本的24µL 中。</li>
		<li>运行一个 PCR 计划以以下步骤:98 ° c 为2分钟, 5 个周期在98° c 为三十年代, 60 ° c 为三十年代和72° c 为 1 min 和一个最后的引伸在72个° c 为 10 min. 举行样品在4° c。</li>
		<li>根据制造商的协议, 用一个基于珠子的净化套件来净化样品。</li>
		<li>根据制造商协议评估 Bioanalyzer 的质量。</li>
	</ol></li>
	<li>用于杂交的索引 DNA 样本的汇集
	<ol>
<li>对于每个捕获反应池, 将每个索引的 gDNA 库样本的适当体积与 PCR 板的一个井结合起来。例如, 对于人或鼠标全外显式捕获库, 使用每个索引库的 187.5 ng 将每个池中的8库组合在一起。确保每个最终捕获反应池包含 1500 ng 的索引 gDNA。</li>
		<li>减少每井的体积, 以 < 7 µL 使用真空浓缩器。避免样品完全干燥。</li>
		<li>在每个浓缩的 gDNA 池中加入足够的无核酸水, 将最后的井量带到7µL, 然后在三十年代剧烈地涡旋含有 gDNA 的板块. 离心机或微型平板纺纱机, 在井底收集液体。</li>
	</ol></li>
	<li>gDNA 库池与捕获库的杂交
	<ol>
<li>对每个7µL 索引的 gDNA 池, 添加9µL 的阻塞组合。吸管向上和向下混合。</li>
		<li>盖好井, 将含有 gDNA 的密封板转移到热循环, 在95° c 处孵育5分钟, 然后在65° c 处举行。确保板在65° c 以下至少5分钟。</li>
		<li>根据捕获库的大小, 准备适当稀释核糖核酸块 (库的10% 稀释 < 3.0 mb 和25% 稀释库 > 3.0 mb)。</li>
		<li>根据捕获库的大小, 将捕获库的适当数量与在冰上保存的 PCR 板中的稀释核糖核酸块溶液相结合。混合移。对于捕获库 < 3.0 Mb, 使用2µl 的库和5µl 的核糖核酸块在10% 稀释。对于捕获库 > 3.0 Mb, 使用5µl 的库和2µl 的核糖核酸块在25% 稀释。</li>
		<li>添加37µL 的杂交缓冲, 每个井包含7µL 捕获库/核糖核酸块混合。移和简单地将含有混合料的板材离心。</li>
		<li>保持 gDNA 池板在65° c, 而使用多通道吸管转移整个44µL 的捕获库混合物从步骤3.7.5 到每个样本井的 gDNA 池板。慢慢地移8到10次, 混合均匀。</li>
		<li>用圆顶带盖封住油井。确保所有井都完全密封。在热循环的 PCR 板上放置一个压缩垫。</li>
		<li>孵育杂交混合物为24小时在65° c。注: 样品可杂交为 72 h, 但必须验证扩展杂交不会导致样品井的大量蒸发。</li>
	</ol></li>
	<li>亲和磁性微珠的制备
	<ol>
<li>Prewarm 洗涤缓冲器2在65° c 在水浴或热块。</li>
		<li>大力重涡流搅拌器上的亲和磁珠。磁珠在贮存过程中沉淀。</li>
		<li>对于每个杂交样品, 添加50µL 的悬浮珠到一个 PCR 板的井。</li>
		<li>洗涤的珠子和重他们在200µL 的结合缓冲。</li>
	</ol></li>
	<li>利用亲和珠捕获杂交 DNA。
	<ol>
<li>估计并记录在 24 h 潜伏期后仍然存在的杂交溶液的体积。</li>
		<li>保持杂交板在65° c, 并使用多通道吸管转移整个体积 (约60µL) 的每个杂交混合物到含200µL 洗亲和珠的板块井。慢慢地移3到5次, 混合均匀。</li>
		<li>在室温下, 盖住井并在 Nutator 搅拌机上孵育捕捉板或相当于30分钟。确保样品在井中正确混合。</li>
		<li>简要地离心了抓住板材在离心机或微型板材微调机。</li>
		<li>将捕捉板放在磁选机中, 从悬浮液中收集珠子。除去并丢弃上清液。</li>
		<li>重200µL 洗涤缓冲器1。移上下混合, 直到珠子完全悬浮。</li>
		<li>简要地离心机在离心机或微型板材飞旋。</li>
		<li>把盘子放在磁选机里。</li>
		<li>等待解决方案清除, 然后清除并丢弃上清。</li>
	</ol></li>
	<li>多路复用排序的后捕获采样处理
	<ol>
<li>用以下混合法制备 PCR 反应混合物的适当体积: 9 µL 核酸水, 25 µL 主混合和1µL 底漆混合根据放大的数量。使用涡流混合器, 并保持冰。</li>
		<li>对于每一个放大反应, 将 pcr 反应混合物的35µL 3.10.1 在 pcr 板的井中。</li>
		<li>移液管的每个珠界捕获的库池样品上下, 以确保珠悬浮是均匀的。</li>
		<li>将每个捕获的库池珠子悬浮液中的15µL 添加到适当的 PCR 反应混合物中。由移彻底混合, 直到珠悬浮是均匀的。</li>
		<li>安置在点3.10.2 的板材在热循环并且跑以下 PCR 扩增节目:98 ° c 为 2 min, 8 到11个周期在98° c 为三十年代, 60 ° c 为三十年代和72° c 为1分钟, 跟随最后的引伸在72° c 为 10 min。将样品保存在4° c。</li>
		<li>根据制造商的协议净化样品。</li>
		<li>根据制造商协议评估 Bioanalyzer 的质量。</li>
	</ol></li>
	<li>为复用测序准备样品 注意: 最后的富集示例包含8或16索引库的池, 基于捕获库的大小和生成的预捕获池策略。在单个测序车道中可复用的索引库总数由所用平台的输出规格以及捕获库所需的测序数据量决定。<ol>
<li>根据平台的容量计算每个车道可以组合的索引数。在单个测序车道中可复用的索引库总数由所用平台的输出规格以及捕获库所需的测序数据量决定。例如, 对于人类所有外显子 v5 库, 每个索引库的推荐测序数据为 4 gb, 每个预捕获池的推荐测序数据为 32 gb (8 索引池)。</li>
	</ol></li>
	<li>序列库。
	<ol>
<li>在选择的下一代测序平台上加载库, 并根据仪器的规格执行顺序运行。</li>
	</ol></li>
	<li>分析外测序数据。
	<ol>
<li>运行管道和基础调用的软件选择。例如, 使用 Stampy 映射读取20, 删除与皮卡德 (http://broadinstitute.github.io/picard/) 的重复, 并确定单核苷酸多态性和插入或删除基地 (indels) 与鸭嘴兽21。过滤同义变种和那些在等位基因频率 < 5% 观察和比较数据与那些从父母 exomes 辨认de 从头变形。</li>
	</ol></li>
</ol>4.子宫内电穿孔的质粒 DNA 制备
<ol><li>设计几个短发夹 rna (shRNAs) 的目标是编码序列或 3 ' UTR 的候选基因, 如前所述,22。</li>
	<li>通过使用标准方法对数据库进行爆炸搜索, 以防止目标效果测试 shRNAs 特异性。排除 shRNAs 显示超过50% 的互补性与其他大鼠的基因。</li>
	<li>克隆退火的寡核苷酸成一个 mU6-pro 的向量, 如前所述23。</li>
	<li>根据制造商的说明纯化质粒 DNA, 并将最终浓度定为3µg/µL。</li>
	<li>分20µl 的 DNA (0.5 毫克/毫升 pCAGGS-GFP 要么单独或与1.5 毫克/毫升的 shRNA 构造) 和添加2µl 的快速绿色染料 (2 毫克/毫升), 允许目视监测的注射。</li>
</ol>5.在子宫内电穿孔
<ol><li>
手术方法
	<ol>
<li>麻醉 E15.5 妊娠雌性大鼠 (E0 被定义为精子阳性阴道塞的确认日), 使用混合氯胺酮/嗪 (分别为0.1 和0.01 毫克), 这是通过腹腔注射麻醉诱导。</li>
		<li>通过观察脚趾捏反射的消失, 确保动物完全麻醉。</li>
		<li>用剪刀把动物的下腹部剃掉, 去掉毛皮。使用聚维酮碘和70% 酒精的磨砂对皮肤进行消毒。将兽医软膏涂抹在眼睛上, 防止麻醉时干燥。</li>
		<li>将动物放在热源上, 并在切口部位放置一个无菌的褶皱 (在中间有一个 4-5 厘米的开放窗口)。穿口罩, 实验室大衣, 帽子和无菌手术手套。保持不育直到手术结束。</li>
		<li>使用锋利的剪刀做垂直切口 (2-2.5 cm) 在皮肤沿中线在尾腹和然后做切口肌肉墙壁在皮肤之下-也 2.5 cm-沿中线。</li>
		<li>小心地从腹腔中露出一个子宫角。滴37° c 前温热生理盐水溶液, 以滋润胚胎。保持子宫湿润。</li>
	</ol>
</li>
	<li>
注射 DNA 和电穿孔
	<ol>
<li>用环形钳轻轻握住子宫角, 小心地将一个胚胎推向子宫壁。一只手握住胚胎, 另一只手将细玻璃毛细管插入1毫米, 从中线到左侧侧脑室 (2-3 毫米深), 并注入大约1µL 的 DNA 与快速绿色, 让视觉监测的注射使用微量.</li>
		<li>在 3 x 7 mm2电极表面上滴生理盐水溶液。将正无菌电极放在注射侧 (左侧脑室) 和右心室的阴性无菌电极上。用脚踏控制踏板 (4000 mF 电容器, 充电至40伏, 带 electroporator), 以950毫秒间隔提供5电脉冲。</li>
	</ol>
</li>
	<li>
后电穿孔程序
	<ol>
<li>将子宫角放回腹腔, 将生理盐水溶液滴入腔内, 使胚胎更自然地定位, 并考虑术中体液流失。</li>
		<li>用可吸收的外科缝合线关闭腹部肌肉壁, 然后用简单的间断缝合皮肤。</li>
		<li>把老鼠放回笼子里, 监视动物直到它从麻醉中出来。</li>
		<li>为治疗疼痛管理丁丙诺啡 (0.03 毫克/千克) 的动物由皮下注射每 8-12 小时, 最多48小时。</li>
	</ol>
</li>
</ol>6. 脑标本制备
<ol><li>
固定方法
	<ol>
<li>在手术后5天, 用气体麻醉 (异氟醚) 和快速颈椎脱位后, 牺牲孕鼠。</li>
		<li>做一个垂直切开, 重新打开腹腔。</li>
		<li>露出子宫角, 从子宫中取出胚胎, 用锋利的剪刀斩首它们。</li>
		<li>对组织的损伤最小的大脑: 使用锋利的剪刀, 做一个小切口沿后部 (小脑)/前 (嗅觉灯泡) 轴的头部刺穿皮肤和头骨, 然后用一副钳子剥去头骨暴露大脑和删除它使用一个小的刮刀。</li>
		<li>在4° c 的4% 甲醛, 在1x 磷酸缓冲盐水 (PBS) 中浸泡大脑过夜。</li>
	</ol>
</li>
	<li>
脑切片
	<ol>
<li>1x PBS 洗脑10分钟</li>
		<li>在塑料嵌入模具中嵌入4% 琼脂的大脑。<ol>
<li>在 1x PBS 中制备50毫升4% 琼脂, 用于植入5胚胎大脑。确保溶解的琼脂糖不超过50° c。</li>
		</ol>
</li>
		<li>保持在琼脂糖的大脑聚合至少1小时, 在4° c。清除大脑周围的过量琼脂糖。</li>
		<li>将大脑粘附在 vibratome 基板上, 使大脑的前/后轴与叶片垂直。等待几分钟的胶水干燥, 并把板与胶水的大脑进入 vibratome 室。</li>
		<li>用 1x PBS 填充 vibratome。切片100µm 厚剖面。</li>
		<li>将脑切片转移到幻灯片上, 去除多余的液体, 添加几滴安装介质, 并将片放在大脑的顶端</li>
	</ol>
</li>
</ol>7. 共焦成像和定量分析
<ol><li>让安装介质在成像前通宵干燥。图像剖面在10X 的激光扫描共聚焦显微镜。</li>
	<li>利用软件进行定量图像分析, 将图像转换为8位, 选择一个代表性的 GFP 阳性荧光细胞, 并手工定义其形状。为此, 请单击 "估计" 并使用手绘选择工具绘制单元格的边框。这样, 该软件将自动保留单元格的直径和强度阈值。</li>
	<li>单击 "查找单元格" 以允许软件将转染的细胞局部化到整个皮质。然后, 如果需要, 手动删除误报。</li>
	<li>将皮质的整个厚度划分为在各个部分中归一化的8感兴趣区域。要实现这一点, 点击 "区域工具", 选择 8 "区域条纹", 其中第一个 (1) 和最后 (8) 条纹分别对应于 VZ 和 CP 的顶部。单击 "应用" 以允许软件计算整个皮层中 GFP 转染细胞的相对位置。最后单击 "保存量化" 以导出 Excel 文件中的数据进行分析。</li>
</ol>

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作者没有什么可透露的。

MCDs 是智力残疾的重要原因, 占20-40% 的耐药性儿童癫癎1,2。在过去十年中, 由于两个主要因素, 对 MCDs 的兴趣显著增加。第一个是大脑成像 (特别是 MRI) 的改善, 这使得医生和科学家能够想象出许多以前未被识别的大脑畸形。另一种是基因工具的进化, 使得许多新奇的致病基因得以鉴定。这大大提高了我们对大脑发育和功能的机制的认识, 并允许更准确的遗传咨询。
在过去, 研究人员主要致力于致病基因的鉴定, 留下了功能性检测的设计, 以阐明其在脑发育后期的作用。这已经变得更加困难, 因为从研究罕见的, 复发性的遗传性疾病, 以更常见的偶发性疾病, 传统的基因发现方法是不适合的。目前的方法, 以确定致病基因往往允许识别的基因组相对较大的区域含有众多的基因 (数组全息) 或几个变种的候选基因, 很难验证 (WES 或 WGS)。
阵列全息图和 WES (或 WGS) 方法拓宽了许多遗传性疾病的突变谱。然而, 一些关键的限制仍然存在。例如, 阵列全息计算需要高质量的 DNA, 无法识别平衡的易位或小的缺失/重复, 包括那些涉及一个或几个显的单一基因。为了克服这个问题, 阵列全息计算的分辨率可能比常规探头间距高 (例如使用1M 阵列全息包) 或用 SNP 阵列分析代替。WES 通常不覆盖大的内区域, 无法识别深子突变。此外, 有时覆盖面可能太低, 以确定原因突变 (特别是在突变的情况下, 低比例的嵌)。对于 WES 来说, 另一个关键的步骤是数据分析和过滤仍然需要相当大的努力。为了增加 WES 的覆盖面, 单个实验中的病人数量可能会减少, 或者在同一时间使用不同的采集工具。
井上靖是最合适的方法来分析基因击倒对神经元迁移的影响。然而, 研究其他步骤的皮层发育, 如神经再生和神经成熟, 受到一些技术上的限制。的确, 井上靖在 E13 之前的表现往往不成功, 而后期的调查则受到与此程序相关的高致死率的限制。另外, 基因击倒效率可能在 electroporated 胚胎之间不同导致可观的表型异质性。
总的来说, 本议定书有四重要的关键步骤。虽然这不是本文件中所述的议定书的一部分, 但我们必须指出, 要考虑的第一个基本步骤是选择接受这种研究的病人。事实上, 识别新的蛋白的过程需要临床和影像学调查在一组患者的表型应尽可能均匀。收集具有高度均质表型的患者增加了识别特定基因致病突变的几率。然而, 在这类研究中, 要获得成功的患者的最低数量很难预测, 因为这很大程度上取决于不同基因的变异率。例如, 对于像FLNA这样的基因, 在100% 的 X 连锁双边 PNH 的家族病例中发生突变, 而在26% 的零星患者中, 需要识别基因中多个命中的患者数量可能相对较低。反之, 对于低突变率的基因, 要筛查的患者数量更高。例如, 在 C6orf70 的情况下,仅在筛选64患者 (14 通过 WES 和50通过常规桑格测序)16时才能够识别出单一的致病突变, 估计该基因的突变率为约1.5%。第二个关键步骤是排除所有已知的蛋白突变基因, 以识别新的致病基因。由于新的下一代测序技术的问世, 对已知和候选基因的突变筛选现在可以在一个单一的实验中进行。但是, 应使用适当的变体筛选来避免存在过多的误报, 以进行实验验证, 并筛选出潜在的原因突变。事实上, 在 WES 实验中, 候选基因/变种的数量特别高。如果怀疑某一特定基因的参与, 还应通过法分析来补充突变筛查, 以排除微或 microduplications。第三个关键点是染色体重受到断点位置的强烈影响的事实。在这方面, 有必要尽可能排除不包括在删除/重复的基因之外的 cis 管理要素的破坏或移位。最后,在体内rna 干扰实验假设致病基因在胚胎期的神经细胞迁移中起直接作用。然而, 包括 PNH 在内的病原学是异质性的, 而子宫内方法可能无法检测到包括神经元增殖或细胞存活在内的发育步骤中所涉及的基因的影响。此外, 啮齿类动物大脑的低复杂性可以掩盖被击倒或过度表达的特定候选基因的影响, 这在人脑中可能更为明显。
我们认为, 基因组学和体内功能研究的整合将有助于开发新的诊断工具来鉴别新的致病基因。这项战略还可以提供新的动物模型来测试治疗靶点, 并了解糖尿病的病理生理学, 迄今为止, 由于缺乏实验模型和受影响患者对脑组织的访问有限, 这是有限的。破译与这些疾病相关的分子通路也将为一般的生理大脑发育提供有价值的新信息。

大脑皮层在认知和智力过程中起着关键的作用, 并且参与情绪控制以及学习和记忆。因此, 许多神经和精神疾病都是由皮质发育畸形引起的, 这并不奇怪。由于后天和遗传因素的影响, 其病因是复杂的。在大多数情况下, 遗传决定的肥胖率的累计患病比例约为 2%, 而且是零星的。例如, 据估计, 在人类人口中, 先天性脑发育的发病率高于 1%, 而且在14% 以上的癫痫患者和40% 的严重或顽固性癫痫中, 出现了某些形式的高血压,1,2。
脑室结节异 (PNH) 是最常见的 MCDs 之一, 是由心室区 (VZ) 神经元异常迁移到发育中的大脑皮层引起的。神经元迁移的失败会导致侧脑室壁的异位神经元聚集, 这通常可以用磁共振成像 (MRI) 进行可视化。PNH 的临床、解剖学和影像学特征均为异质性。结核的范围可以从小的和单边的到双边的和对称的。常见的临床后遗症包括癫痫和智力残疾3。在Filamin A (或FLNA) 基因中的突变, 在 Xq28 中映射, 在100% 的家庭中发现有 X 链接的双边 PNH 和26% 的散发性患者34。一个罕见的, 隐性形式的 PNH 引起的突变在ARFGEF2基因, 其中映射在 20q13, 已报告在两个近亲家庭5。最近, 在包括 PNH6在内的九例受断奶紊乱影响的患者中, 发现了受体配体粘蛋白对DCHS1和FAT4的基因 biallelic 突变。PNH 也已与脆性 X 综合征7, 威廉斯综合征8, 22q11 缺失综合征9, 复制在 5p1510, 删除在 1p3611, 5q14.3-q1512, 6p2513和6q 终端删除综合征14,15,16,17,18,19, 提示其他致病基因分散整个基因组。然而, 大约74% 的散发性 PNH 患者的遗传基础仍有待阐明17。
经典的基因映射方法, 如阵列比较基因组杂交 (数组-全息图) 已被证明是一个强大的工具, 检测亚显微染色体异常, 但是, 使用这种方法确定的基因组区域是通常大并且包含许多基因。
大规模并行测序技术 (即全外测序 (WES) 和全基因组测序 (WGS)) 的出现大大降低了整个人类外或基因组序列所需的成本和时间。然而, 在大多数情况下, 对 WES 和 WGS 数据的解释仍然具有挑战性, 因为对于每位病人来说, 从数据过滤中产生的变种到数百人 (甚至数千名患者) 都有不同的分析方法。
为了加快识别新的致病基因的过程, 设计了一种结合阵列全息、WES 和宫内电穿孔 (井上靖) 筛选候选基因的新系统策略。井上靖允许有选择地灭活 (或过度) 啮齿动物大脑中的特定基因或突变, 使其参与 corticogenesis18,19的快速评估。当基因与疾病的发展、神经元迁移和/或成熟的局部缺陷有关时, rna 介导-击倒或过度表达一个或多个候选基因将导致。在鉴定的基因的失活 (或过度表达) 再现的表型观察到啮齿动物的病人, 它成为一个杰出的候选者筛查的零星患者的。使用这种方法, 我们最近揭示了C6orf70基因 (也称为ERMARD) 在 PNH 发病机制中的重要贡献, 这些患者窝藏6q27 染色体删除16。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Picospritzer III</td>
			<td >Parker Hannifin Corp</td>
			<td >P/N 051-0500-900</td>
			<td >Intracellular Microinjection Dispense Systems</td>
		</tr>
		<tr >
			<td >Fast Green FCF</td>
			<td >Sigma-Aldrich</td>
			<td >F7252</td>
			<td>Fast green allow visual monitoring of the injection</td>
		</tr>
		<tr >
			<td >BTX ECM 830 electroporator</td>
			<td >BTX Harvard Apparatus</td>
			<td >45-0002</td>
			<td>The ECM 830 is a Square Wave Pulse generator designed for in vitro and in vivo applications</td>
		</tr>
		<tr >
			<td >Microtome HM 650 V</td>
			<td >Microm</td>
			<td>10076838</td>
			<td>Microtome HM 650 V vibratome 240V 50/60Hz with vibrating blade</td>
		</tr>
		<tr >
			<td >FluoView 300</td>
			<td >Olympus</td>
			<td ></td>
			<td>The Olympus FluoViewTM 300 is a point-scanning, point-detection, confocal laser scanning microscope designed for biology research application</td>
		</tr>
		<tr >
			<td >eCELLence software</td>
			<td >Glance Vision Technologies</td>
			<td ></td>
			<td>eCELLence is software designed for the quantitive analysis of cell migration</td>
		</tr>
		<tr >
			<td >Agilent Microarray Scanner Bundle</td>
			<td ></td>
			<td ></td>
			<td>Array slides</td>
		</tr>
		<tr >
			<td >for 1 x 244K, 2 x 105K, 4 x 44K or 8 x 15K</td>
			<td >Agilent</td>
			<td >Agilent p/n G4900DA, G2565CA or G2565BA</td>
			<td ></td>
		</tr>
		<tr >
			<td >for 1 x 1M, 2 x 400K, 4 x 180K or 8 x 60K</td>
			<td >Agilent</td>
			<td >Agilent p/n G4900DA or G2565CA</td>
			<td ></td>
		</tr>
		<tr >
			<td >Hybridization Chamber, stainless</td>
			<td >Agilent</td>
			<td>Agilent p/n G2534A</td>
			<td>Chamber for array CGH hybridization</td>
		</tr>
		<tr >
			<td >Hybridization Chamber gasket slides, 5-pack</td>
			<td></td>
			<td ></td>
			<td>Gasket for array CGH hybridization</td>
		</tr>
		<tr >
			<td >for 1-pack microarrays or</td>
			<td >Agilent</td>
			<td >Agilent p/n G2534-60003</td>
			<td></td>
		</tr>
		<tr >
			<td >for 2-pack microarrays or</td>
			<td >Agilent</td>
			<td >Agilent p/n G2534-60002</td>
			<td></td>
		</tr>
		<tr >
			<td >for 4-pack microarrays or</td>
			<td >Agilent</td>
			<td >Agilent p/n G2534-60011</td>
			<td></td>
		</tr>
		<tr >
			<td >for 8-pack microarrays</td>
			<td >Agilent</td>
			<td >Agilent p/n G2534-60014</td>
			<td></td>
		</tr>
		<tr >
			<td >Hybridization oven</td>
			<td >Agilent</td>
			<td >Agilent p/n G2545A</td>
			<td></td>
		</tr>
		<tr >
			<td >Hybridization oven rotator for Agilent Microarray Hybridization Chambers</td>
			<td >Agilent</td>
			<td >Agilent p/n G2530-60029</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermal cycler with heated lid</td>
			<td >Agilent</td>
			<td >Agilent p/n G8800A or equivalent</td>
			<td>Termal cycler for incubations</td>
		</tr>
		<tr >
			<td >1.5 mL RNase-free Microfuge Tube</td>
			<td >Ambion</td>
			<td >p/n AM12400 or equivalent</td>
			<td>Microcentrifuge</td>
		</tr>
		<tr >
			<td >Magnetic stir bar</td>
			<td >Corning</td>
			<td >p/n 401435 or equivalent</td>
			<td>Instrument for stirring</td>
		</tr>
		<tr >
			<td >Qubit Fluorometer</td>
			<td >Life Technologies</td>
			<td >p/n Q32857</td>
			<td>Instrument for DNA quantification</td>
		</tr>
		<tr >
			<td >Qubit dsDNA BR Assay Kit, for use with the Qubit fluorometer</td>
			<td >Invitrogen</td>
			<td >p/n Q32850</td>
			<td>Kit for Qubit fluorometer</td>
		</tr>
		<tr >
			<td >UV-VIS spectrophotometer</td>
			<td >Thermo Scientific</td>
			<td >NanoDrop 8000 or 2000, or equivalent</td>
			<td>Instrument for DNA quantification</td>
		</tr>
		<tr >
			<td >P10, P20, P200 and P1000 pipettes</td>
			<td >Pipetman or equivalent</td>
			<td ></td>
			<td>DNA dispensation</td>
		</tr>
		<tr >
			<td >Vacuum Concentrator</td>
			<td >Thermo Scientific</td>
			<td >p/n DNA120-115 or 
			equivalent</td>
			<td>Instrument to concentrate DNA</td>
		</tr>
		<tr >
			<td >SureTag Complete DNA Labeling Kit</td>
			<td >Agilent</td>
			<td >p/n 5190-4240</td>
			<td>DNA Labeling Kit (for Human Samples)</td>
		</tr>
		<tr >
			<td >Purification Columns (50 units)</td>
			<td >Agilent</td>
			<td >p/n 5190-3391</td>
			<td>DNA Labeling Kit (for Human Samples)</td>
		</tr>
		<tr >
			<td >AutoScreen A, 96-well plates</td>
			<td >GE Healthcare</td>
			<td >p/n 25-9005-98</td>
			<td>DNA Labeling Kit (for Human Samples)</td>
		</tr>
		<tr >
			<td >GenElute PCR Clean-Up Kit</td>
			<td >Sigma-Aldrich</td>
			<td >p/n NA1020</td>
			<td>DNA Labeling Kit (for Human Samples)</td>
		</tr>
		<tr >
			<td >Human Genomic DNA</td>
			<td ></td>
			<td >p/n G1521</td>
			<td>DNA Labeling Kit (for Human Samples)</td>
		</tr>
		<tr >
			<td >For CGH microarrays:</td>
			<td >Promega</td>
			<td >(female) or p/n G1471 (male)</td>
			<td>Array CGH control DNA</td>
		</tr>
		<tr >
			<td >For CGH+SNP microarrays:</td>
			<td >Coriell</td>
			<td >p/n NA18507, NA18517, NA12891, NA12878, or NA18579</td>
			<td>Array CGH control DNA</td>
		</tr>
		<tr >
			<td >Oligo aCGH/ChIP-on-chip Wash Buffer Kit or</td>
			<td >Agilent</td>
			<td >p/n 5188-5226</td>
			<td>Array CGH hybridization and wash</td>
		</tr>
		<tr >
			<td >Oligo aCGH/ChIP-on-chip Wash Buffer 1 and</td>
			<td >Agilent</td>
			<td >p/n 5188-5221</td>
			<td>Array CGH hybridization and wash</td>
		</tr>
		<tr >
			<td >Oligo aCGH/ChIP-on-chip Wash Buffer 2</td>
			<td >Agilent</td>
			<td >p/n 5188-5222</td>
			<td>Array CGH hybridization and wash</td>
		</tr>
		<tr >
			<td >Stabilization and Drying Solution</td>
			<td >Agilent</td>
			<td >p/n 5185-5979</td>
			<td>Array CGH hybridization and wash</td>
		</tr>
		<tr >
			<td >Oligo aCGH/ChIP-on-chip Hybridization Kit</td>
			<td >Agilent</td>
			<td >p/n 5188-5220 (25) or p/n 5188-5380 (100)</td>
			<td>Array CGH hybridization and wash</td>
		</tr>
		<tr >
			<td >Human Cot-1 DNA</td>
			<td >Agilent</td>
			<td >p/n 5190-3393</td>
			<td>Array CGH hybridization and wash</td>
		</tr>
		<tr >
			<td >Agilent C scanner</td>
			<td >Agilent</td>
			<td ></td>
			<td>Scanner for array CGH slides</td>
		</tr>
		<tr >
			<td >SureSelect XT2 Reagent Kit</td>
			<td ></td>
			<td ></td>
			<td>Kit for target enrichment</td>
		</tr>
		<tr >
			<td >HiSeq platform (HSQ), 16 Samples</td>
			<td >Agilent</td>
			<td >p/n G9621A</td>
			<td></td>
		</tr>
		<tr >
			<td >HiSeq platform (HSQ), 96 Samples</td>
			<td >Agilent</td>
			<td >p/n G9621B</td>
			<td></td>
		</tr>
		<tr >
			<td >HiSeq platform (HSQ), 480 Samples</td>
			<td >Agilent</td>
			<td >p/n G9621C</td>
			<td></td>
		</tr>
		<tr >
			<td >MiSeq platform (MSQ), 16 Samples</td>
			<td >Agilent</td>
			<td >p/n G9622A</td>
			<td></td>
		</tr>
		<tr >
			<td >MiSeq platform (MSQ), 96 Samples</td>
			<td >Agilent</td>
			<td >p/n G9622B</td>
			<td></td>
		</tr>
		<tr >
			<td >MiSeq platform (MSQ), 480 Samples</td>
			<td >Agilent</td>
			<td >p/n G9622C</td>
			<td></td>
		</tr>
		<tr >
			<td >DNA 1000 Kit</td>
			<td >Agilent</td>
			<td >p/n 5067-1504</td>
			<td>Kit for the separation, sizing and quantification of dsDNA fragments from 25 to 1000 bp.</td>
		</tr>
		<tr >
			<td >High Sensitivity DNA Kit</td>
			<td >Agilent</td>
			<td >p/n 5067-4626</td>
			<td>Kit for analysis of fragmented DNA or DNA libraries.</td>
		</tr>
		<tr >
			<td >AMPure XP Kit</td>
			<td ></td>
			<td ></td>
			<td>Kit for automated PCR purification.</td>
		</tr>
		<tr >
			<td >5 mL</td>
			<td >Agencourt</td>
			<td >p/n A63880</td>
			<td></td>
		</tr>
		<tr >
			<td >60 mL</td>
			<td >Agencourt</td>
			<td >p/n A63881</td>
			<td></td>
		</tr>
		<tr >
			<td >450 mL</td>
			<td >Agencourt</td>
			<td >p/n A63882</td>
			<td></td>
		</tr>
		<tr >
			<td >Dynabeads MyOne Streptavidin T1</td>
			<td ></td>
			<td ></td>
			<td>Isolation and handling of biotinylated nucleic acids</td>
		</tr>
		<tr >
			<td >2 mL</td>
			<td >Life Technologies</td>
			<td >Cat #65601</td>
			<td></td>
		</tr>
		<tr >
			<td >10 mL</td>
			<td >Life Technologies</td>
			<td >Cat #65602</td>
			<td></td>
		</tr>
		<tr >
			<td >Quant-iT dsDNA BR Assay Kit, for the Qubit fluorometer</td>
			<td ></td>
			<td ></td>
			<td>DNA quantification</td>
		</tr>
		<tr >
			<td >100 assays, 2-1000 ng</td>
			<td >Life Technologies</td>
			<td >Cat #Q32850</td>
			<td></td>
		</tr>
		<tr >
			<td >500 assays, 2-1000 ng</td>
			<td >Life Technologies</td>
			<td >Cat #Q32853</td>
			<td></td>
		</tr>
		<tr >
			<td >Qubit assay tubes</td>
			<td >Life Technologies</td>
			<td >p/n Q32856</td>
			<td>DNA quantification</td>
		</tr>
		<tr >
			<td >SureSelec tXT2 Capture Libraries</td>
			<td >Agilent</td>
			<td >depending on the experiment</td>
			<td>Kit for libraries capture</td>
		</tr>
		<tr >
			<td >SureCycler 8800 Thermal Cycler</td>
			<td >Agilent</td>
			<td >p/n G8800A</td>
			<td>DNA amplification</td>
		</tr>
		<tr >
			<td >96 well plate module for SureCycler 8800 Thermal Cycler</td>
			<td >Agilent</td>
			<td >p/n G8810A</td>
			<td>DNA amplification</td>
		</tr>
		<tr >
			<td >SureCycler 8800-compatible 96-well plates</td>
			<td >Agilent</td>
			<td >p/n 410088</td>
			<td>DNA amplification</td>
		</tr>
		<tr >
			<td >Optical strip caps</td>
			<td >Agilent</td>
			<td >p/n 401425</td>
			<td>DNA amplification</td>
		</tr>
		<tr >
			<td >Tube cap strips, domed</td>
			<td >Agilent</td>
			<td >p/n 410096</td>
			<td>DNA amplification</td>
		</tr>
		<tr >
			<td >Compression mats</td>
			<td >Agilent</td>
			<td >p/n 410187</td>
			<td>DNA amplification</td>
		</tr>
		<tr >
			<td >2100 Bioanalyzer Laptop Bundle</td>
			<td >Agilent</td>
			<td >p/n G2943CA</td>
			<td>DNA amplification</td>
		</tr>
		<tr >
			<td >2100 Bioanalyzer Electrophoresis Set</td>
			<td >Agilent</td>
			<td >p/n G2947CA</td>
			<td>DNA amplification</td>
		</tr>
		<tr >
			<td >Covaris Sample Preparation System, E-series or S-series</td>
			<td >Covaris</td>
			<td ></td>
			<td>DNA shearing</td>
		</tr>
		<tr >
			<td >Covaris sample holders</td>
			<td ></td>
			<td >p/n 520078</td>
			<td>DNA shearing</td>
		</tr>
		<tr >
			<td >Nutator plate mixer</td>
			<td >BD Diagnostics</td>
			<td >p/n 421105 or equivalent</td>
			<td>Plate Mixer</td>
		</tr>
		<tr >
			<td >GaIIx</td>
			<td >Illumina</td>
			<td ></td>
			<td>next generation sequencing machine</td>
		</tr>
	</tbody>
</table>

a novel strategy combining array-cgh, whole-exome sequencing and in utero electroporation in rodents to identify causative genes for brain malformations

涉及大脑皮层的先天缺陷--也称为皮质发育畸形--是导致智力残疾的重要原因, 也是儿童时期20-40% 的耐药性癫痫的病因。高分辨率脑成像促进了在体内识别一大群的型式。尽管在脑部成像、基因组分析和动物模型的产生方面取得了进展, 但一个简单的工作流程, 系统地确定候选基因的优先顺序, 并测试假定突变的功能效应。为了解决这一问题, 开发并验证了一种能够识别新的致病基因的实验策略。这一策略是基于通过阵列全息或全外测序确定候选基因组区域或基因, 并通过宫内来描述它们的失活或在发展啮齿动物大脑中的特定突变的过度表达的影响。穿孔.这种方法导致识别的C6orf70基因, 编码为一个假定的水泡蛋白, 脑室结节性异的发病机制, 由缺陷的神经元迁移引起的。

在图 1中扼要了用于识别新的致病基因的实验策略。
通过在155例发育性脑异常变合并 PNH (图 2A)、胼胝体发育、colpocephaly、小脑发育不良和 polymicrogyria 与癫痫、共济失调和认知障碍, 我们确定了 1.2 Mb 的最小关键删除在6q27 共享的12患者 (图 2B)21。基因组区域包含四已知基因 (THBS2、 PHF10、 TCTE3和DLL1) 和两个预测基因 (WDR27和C6orf70) (图 2B)。
同时对14例孤立的双侧 PNH 和无拷贝数变异分析的患者进行了分析, 并在预测的C6orf70基因 (752 t > a: pIle250Asn) 中发现了一个患者, 并进行了de 从头突变 (基因库加入编号 NM_018341.1) (图 3)。
为了确认在C6orf70中识别出的突变在 PNH 中起到了致病作用, 并探讨了6q27 中的其他基因的 haploinsufficiency 是否可能影响表型, 探讨了它们在神经元迁移过程中的作用体内使用在子宫rna 介导的击倒方法23,24以沉默他们的表达在大鼠皮层神经祖细胞。只有在发育中的大鼠皮层、 PHF10、 C6orf70和DLL1中表达的基因才被测试 (图 4A)。不同的短发夹 rna (shRNAs) 是针对这三基因的编码序列生成的。然后, ShRNAs 被引入到大鼠大脑皮层的 neuroprogenitors 中, 由在子宫内在胚胎日 15 (E15) 的电穿孔, 当神经元对上部皮层层产生。绿色荧光蛋白被用作转染报道。电穿孔后5天检测 GFP 阳性细胞的相对分布。Dll1和Phf10的击倒导致了径向神经元迁移的轻微延迟 (数据未显示), 而C6orf70破坏了神经元迁移, 引起了异位结节的发展, 高度让人联想到在Flna击倒模型 (图 4B)24中观察到的那些。相反, 染与无效的C6orf70不匹配 shRNA 对神经元迁移没有明显影响 (图 4B)。
<img alt="Figure 1" src="/files/ftp_upload/53570/53570fig1.jpg"> 图 1:工作流用于鉴别新的致病基因的识别。不同方法的示意图表示, 可用于识别新的致病基因。黄色方框代表本文件所述的方法。<a href="http://cloudflare.jove.com/files/ftp_upload/53570/53570fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" src="/files/ftp_upload/53570/53570fig2.jpg"> 图 2:6q27 删除导致异常的大脑发育与 PNH.(A) 患者2。T2-weighed 冠状 (左面板) 和 T1-weighed 轴 (右面板) 剖面显示 PNH 内壁的左颞叶 (白色和黑色箭头) 下方的一个展开的岛屿皮层。(B) 患者11。T1-weighted 冠状部分, 显示两侧的异位结节沿颞角的墙壁 (白色箭头)。(C) 患者12。T2-weighed 冠部。在左侧, PNH 仍然可见 (黑色箭头) 和心室扩张。(D) PNH 患者使用阵列全息图 (上半部分) 确定的6q27 区域缺失的示意图表示。水平的虚线表示患者中发现的缺失。最小临界被删除区域的大小也被表明并且包含四已知的基因: THBS2、 PHF10、 TCTE3和DLL1和两个预测基因: WDR27和C6orf70 (上部部分)。<a href="http://cloudflare.jove.com/files/ftp_upload/53570/53570fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" src="/files/ftp_upload/53570/53570fig3.jpg"> 图 3:排序结果。() T2 加权轴向剖面, 显示 PNH 752 t > 突变的患者额角 (白色箭头) 在 C6orf70. (B) 桑格测序显示 WES 发现的突变发生在de 从头。突变的位置由黑色箭头表示。<a href="http://cloudflare.jove.com/files/ftp_upload/53570/53570fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" src="/files/ftp_upload/53570/53570fig4.jpg"> 图 4:6q27 极小临界区和 C6orf70-knockdown 的基因表达分析改变了皮层神经元的径向迁移.(A) 定量 rt-pcr 显示大鼠大脑皮层6q27 临界区的基因表达。亲用于正常化。(B) 代表皮层 E20 大鼠脑电穿孔后5天的冠状段, 要么是绿色荧光蛋白结构单独 (控制), 要么结合 shRNA 目标C6orf70编码序列 (C6orf70-shCDS)或与相对无效的不匹配构造 (C6orf70-shCDSmm)。Flna-击倒 (Flna-shCDS) 被用作控制受损的神经元迁移。在子宫电穿孔与 C6orf70-shCDS 或 FLNA-shCDS 诱导了在心室区 (VZ) (白色箭头) 内的 gfp 阳性细胞的逮捕, 而表达 gfp 单独或与不匹配结构结合的细胞没有改变神经元迁移。缩放栏 = 200 µm.<a href="http://cloudflare.jove.com/files/ftp_upload/53570/53570fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations at Jo

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

脑室结节性异 (PNH) 是成年期皮质发育畸形最常见的形式, 但在大多数偶发病例中, 其遗传基础仍然未知。我们最近制定了一项战略, 以确定新的候选基因的 MCDs, 并直接确认其致病作用在体内。

一种新的结合阵列全息、全外测序和宫内电穿孔的方法来识别脑畸形的致病基因

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

a-novel-strategy-combining-array-cgh-whole-exome-sequencing-utero

Nanyang Technological University

Research

JoVE Journal

Neuroscience

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

8.5K 次观看.  University of Florence. 脑室结节性异 (PNH) 是成年期皮质发育畸形最常见的形式, 但在大多数偶发病例中, 其遗传基础仍然未知。我们最近制定了一项战略, 以确定新的候选基因的 MCDs, 并直接确认其致病作用在体内。

视频: A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Combining Behavioral Endocrinology and Experimental Economics: Testosterone and Social Decision Making

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in rodents have shown a direct link between testosterone and aggressive behavior1 and folk wisdom adapts these findings to humans, suggesting that testosterone induces antisocial, egoistic or even aggressive behavior2. However, many researchers doubt a direct testosterone-aggression link in humans, arguing instead that testosterone is primarily involved in status-related behavior3,4. As a high status can also be achieved by aggressive and antisocial means it can be difficult to distinguish between anti-social and status seeking behavior.
We therefore set up an experimental environment, in which status can only be achieved by prosocial means. In a double-blind and placebo-controlled experiment, we administered a single sublingual dose of 0.5 mg of testosterone (with a hydroxypropyl-&beta;-cyclodextrin carrier) to 121 women and investigated their social interaction behavior in an economic bargaining paradigm. Real monetary incentives are at stake in this paradigm; every player A receives a certain amount of money and has to make an offer to another player B on how to share the money. If B accepts, she gets what was offered and player A keeps the rest. If B refuses the offer, nobody gets anything. A status seeking player A is expected to avoid being rejected by behaving in a prosocial way, i.e. by making higher offers.
The results show that if expectations about the hormone are controlled for, testosterone administration leads to a significant increase in fair bargaining offers compared to placebo. The role of expectations is reflected in the fact that subjects who report that they believe to have received testosterone make lower offers than those who say they believe that they were treated with a placebo. These findings suggest that the experimental economics approach is sensitive for detecting neurobiological effects as subtle as those achieved by administration of hormones. Moreover, the findings point towards the importance of both psychosocial as well as neuroendocrine factors in determining the influence of testosterone on human social behavior.

The procedure described in this protocol shows that testosterone administration and folk beliefs about testosterone may be associated with directly opposed social behaviors.

睾酮和社会决策，结合行为内分泌和实验经济学:

Behavioral endocrinological research in humans as well as in animals suggests that testosterone plays a key role in social interactions. Studies in ...

科研

神经科学

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

结合经颅磁刺激和功能磁共振成像检查默认模式网络

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

高效的基因传递到多个中枢神经系统使用新界在子宫内电穿孔

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔:鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the use of IUE for investigating behavior or neuropathology in the adult brain has been limited by insufficient methods for monitoring of IUE transfection success by non-invasive techniques in postnatal animals. 
 For the present study, E16 rats were used for IUE. After intraventricular injection of the nucleic acids into the embryos, positioning of the tweezer electrodes was critical for targeting either the developing cortex or the hippocampus. 
 Ventricular co-injection and electroporation of a luciferase gene allowed monitoring of the transfected cells postnatally after intraperitoneal luciferin injection in the anesthetized live P7 pup by in vivo bioluminescence, using an IVIS Spectrum device with 3D quantification software. 
 Area definition by bioluminescence could clearly differentiate between cortical and hippocampal electroporations and detect a signal longitudinally over time up to 5 weeks after birth. This imaging technique allowed us to select pups with a sufficient number of transfected cells assumed necessary for triggering biological effects and, subsequently, to perform behavioral investigations at 3 month of age. As an example, this study demonstrates that IUE with the human full length DISC1 gene into the rat cortex led to amphetamine hypersensitivity. Co-transfected GFP could be detected in neurons by post mortem fluorescence microscopy in cryosections indicating gene expression present at &ge;6 months after birth.
 We conclude that postnatal bioluminescence imaging allows evaluating the success of transient transfections with IUE in rats. Investigations on the influence of topical gene manipulations during neurodevelopment on the adult brain and its connectivity are greatly facilitated. For many scientific questions, this technique can supplement or even replace the use of transgenic rats and provide a novel technology for behavioral neuroscience.

Generation of Topically Transgenic Rats by In utero Electroporation and In vivo Bioluminescence Screening

Genes can be manipulated during development of the cortex or the hippocampus of the rat via in utero electroporation (IUE) at E16, to enable fast and targeted modifications in neuronal connectivity for later studies of behavior or neuropathology in adult animals. Postnatal in vivo imaging for control of IUE success is performed by bioluminescence of activating co-transfected luciferase.

外用转基因大鼠通过代<em&gt;在子宫内</em&gt;电穿孔和<em&gt;在体内</em&gt;生物发光筛选

 In utero electroporation (IUE) is a technique which allows genetic modification of cells in the brain for investigating neuronal development. So far, the ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

深部脑刺激与同步功能磁共振成像在鼠害

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Acrylic Resin Molding Based Head Fixation Technique in Rodents

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires surgical attachment of metallic frames on the skull. The attached frames are then clamped to a stationary platform resulting in immobilization of the head. However, metallic frames for head fixation have been technically difficult to design and implement in general laboratory environment. In this study, we provide a novel head fixation method. Using a custom-made head fixation bar, head mounter is constructed during implantation surgery. After the application of acrylic resin for affixing implants such as electrodes and cannula on the skull, additional resins applied on top of that to build a mold matching to the port of the fixation bar. The molded head mounter serves as a guide rails, investigators conveniently fixate the animal&#8217;s head by inserting the head mounter into the port of the fixation bar. This method could be easily applicable if implantation surgery using dental acrylics is necessary and might be useful for laboratories that cannot easily fabricate CNC machined metal head-posts.

Traditional head fixation techniques have used metallic frames attached on the skull as an interface for head fixation apparatus. This protocol demonstrates a frameless, acrylic resin molding based head fixation technique for behavioral tasks in rodents.

在鼠害丙烯酸树脂模塑基头固定技术

Head fixation is a technique of immobilizing animal's head by attaching a head-post on the skull for rigid clamping. Traditional head fixation requires ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

在子宫内电途径研究神经元亚群的兴奋性和单细胞连接

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...