作者希望在学习这种插管技术的过程中, 对参加过兔子处理和麻醉训练课程的员工和研究人员进行反馈。

下面概述的插管程序由梅奥诊所机构动物护理和使用委员会批准. 
 1. 准备工作 ( 图 1 ) <ol> <li> 获得5法国聚丙烯导管并切断 non-rounded 端, 因此总长度为8-10 英寸。标记油管的切口端, 以清楚地指示应将哪个端 不 插入气管 (箭头, 图 1 )。
	<li> 在喉镜上放置一个大小为0或1的米勒喉镜刀片, 并验证指示灯是否正常工作. </li>
	<li> 将0.25 毫升2% 利多卡因注射到1毫升注射器中. </li>
	<li> 在适当大小的 uncuffed 气管插管端使用无菌润滑剂 (通常为 2.5-4.0 mm). </li>
	<li> 准备在插入后固定气管插管的用品 ( 例如, 脐带胶带). </li>
	<li> 获取在程序结束时检测气流的设备, 以确认成功的插管 ( #5, 图 1 )。这是可选的. </li>
</ol> 2。麻醉 <ol> <li> 管理注射麻醉, 如35毫克/千克氯胺酮和5毫克/千克嗪注使用3毫升注射器与25测针在四头肌的后腿的约4月大, 3.0-3.5 公斤, 新西兰白色兔. 
	注: 这种麻醉方案是一种常用的注射剂组合在年轻, 健康, 特定的无病原体研究兔, 但根据临床情况和/或实验目标, 不同的协议可能更合适. </li>
	<li> 在兔被麻醉后, 将无菌的眼部润滑剂应用于眼睛以保护角膜组织. </li>
</ol> 3。气管插管 <ol> <li> 测量从门牙到胸廓入口的气管导管, 确定插入气管导管的适当长度。在胸廓入口的水平处放置管末端, 可以确保导管的开度超过喉部, 但颅至气管分岔处. </li>
	<li> 将兔 sternally 放在准备表上, 将其头部略微延伸到表的边缘 ( 图 2 )。应将兔的头部和脊柱置于正方形的直线上. </li>
	<li> 抬起头, 用纱布垫把兔子和 #39 的舌头侧向兔子和 #39; 右下门牙 (在纵), 注意避免门牙的外伤. </li>
	<li> 用右手握住喉镜和刀片, 然后把它放进门牙后面的嘴里, 从兔子和 #39 的左边进入. </li>
	<li> 沿尾端向口的顶部, 直到软腭可见. </li>
	<li> 用右手, 将喉镜向前 (弯曲手腕, 按住喉镜向下), 同时延长兔和 #39; s 头向后和颈部向前, 以保持一个开放的呼吸道和查看喉 ( 图 3 A ). 作为强制鼻呼吸, 会厌常高于软腭, 使用这种方法压缩舌头的底座以取代会厌并暴露声门。为了防止氧气饱和, 重要的是保持颈部延长, 以保持一个开放的气道在插管. </li>
	<li> 松开舌, 并将喉镜刀片切换到左手, 释放右手以放置导导管. </li>
	<li> 在持有喉镜和保持声门的视图时 ( 图 3 B ), 通过通过喉部的开口 ( 图 4 ) 推进导管的圆尖).导管会在气管分岔处自然停止. </li>
	<li> 在保持引导导管的同时, 抓住气管导管, 并将其穿线在导尿管上, 注意不要在气管导管连接器上抓住它的力量。推进管, 直到有阻力从声带 ( 图 5 ). </li>
	<li> 将0.25 毫升2% 利多卡因注入气管导管的开口。利多卡因会沿着气管插管到局部麻醉声带, 防止痉挛 22 ( 图 6 )。
	<li> 允许大约两分钟的局部麻醉生效, 并通过轻轻地扭转气管插管, 推进它通过声带 ( 图 7 )。一旦管子通过声带, 取下喉镜和导管, 注意不要把管子取出. </li>
	<li> 通过观察气流来验证气管插管的位置。有多种方法来实现这一点, 包括放置的组织或毛皮在年底的管, 以检测气流, 寻找雾的管或雾的玻璃/镜子在年底的管, 并听取气流。设计用于检测气流 23 或呼吸的设备也可以使用 ( 图 8 ) 来确定插管是否成功 (请参阅 材料表 选项)。使用优选的方法 (如脐带绑在管和头上) 固定气管插管。在呼吸时, 将麻醉回路和 auscult 两侧的胸腔连接起来, 以保证肺部两侧的通气. </li>
</ol>

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作者没有什么可透露的。

在这里, 我们提供了一个兔子插管技术的详细描述, 可以很容易地掌握在任何临床设置。此技术类似于以前发布的直接可视化技术10,152025 , 还有一些额外的改进, 包括使用 3.5 mm uncuffed 气管插管和5法国导尿管作为我们选择的指南, 已被切断, 并在削减结束标记与永久标志。兔气管容易受伤, 因此, uncuffed 气管导管的使用通常建议当插管兔, 以防止损伤的气管粘膜从压力的袖口。26我们选择的5法国导尿管在喉部直接可视化过程中, 小到可以轻松地通过声带, 而不会阻碍视野。虽然小, 这个导管是僵硬的, 足以保持在地方作为指导的气管插管。远端的导管是四舍五入, 以帮助减少创伤时, 分歧达到。导管的近端被切断, 使导管在一个可行的长度 (不太长) 和删除的结束, 扩大为附件的注射器。切口末端标记以永久标记作为视觉指示不插入此末端入气管, 可能导致损伤对气管黏膜。
这项技术的主要限制是, 因为它确实需要使用喉镜刀片, 它可能是不可行的非常年幼的兔子或非常小的品种, 由于较小的嘴巴大小。此外, 一些设施可能没有这种规模的喉镜刀片现成的可用。在作者的机构, 这种方法是用于新西兰白 (≥6周) 和成年荷兰兔, 但我们没有尝试这种方法在年轻的动物或较小的品种。在兔子的病人很小的情况下, 或者如果一个喉镜与一个小米勒刀片是不可用的, 另一种选择是使用手持镜而不是喉镜。15,20使用带有 #5 耳窥器的镜, 将喉部可视化, 引导导管由直接可视显示, 并在按照上述步骤进行操作之前将窥镜移除。
当导管的末端进入气管并触及气管的分岔时, 使用导导管来避免造成外伤或支气管的可能性是非常重要的。这些并发症的风险可以减少或避免, 通过喂养导管与非常温和的动作, 一旦有阻力停止, 并避免头部和颈部的移动一旦导管到位。此外, 当在导管上推进气管插管时, 导管连接器上也有可能会发现导尿管。因此, 重要的是要确保导管通过连接器滑动而不捕获;或者, 连接器可以在固定气管插管或附着在麻醉回路时被移除和更换。
有任何插管方法, 有潜在的并发症和不良反应。使用喉镜直接可视化方法, 不正确插入喉镜刀片或积极放置气管插管可导致喉、舌或牙齿损伤。正确定位的动物是第一个关键的步骤, 最大限度地减少创伤的可视化喉。脊柱应伸直, 头部与脊柱直接对准;如果动物不是直的, 或者如果颈部被扭到左边或右边, 这将使喉部的可视化更加困难, 并可能导致喉镜刀片的过度创伤。使用这项技术成功的另一个关键关键是沿着口的顶部朝向喉部, 一旦软腭被可视化, 就会通过弯曲手腕将刀片移到适当的位置。这保持头到位, 而移动刀片的尖端到最佳的观察位置声带, 位于相当腹在兔子20,24。
由于直接可视化喉可能是棘手的, 如果精确的技术, 我们上面概述没有遵循, 使用替代插管策略, 不需要可视化喉可以吸引。例如, 盲技术是一种可能的选项, 不需要使用喉镜16,17,18,19,20。然而, 这项技术的使用依赖于通过气管插管检测气流的能力, 以便引导导管的位置, 如果动物的呼吸非常浅或被逮捕, 使用这种方法是不可靠的。声门设备也可以考虑, 因为放置这些设备不需要显着的喉, 他们通常很容易到位。这些设备不直接进入气管, 然而, 因此有潜在的缺乏直接流到气道, 无法通风, 如果痉挛发展, 缺乏密封足够的通风在开程序或紧急情况胃音的复苏和发展3,4,8。专门为兔子设计的新设备可能提供了一个很有前途的替代品, 但是需要更多的研究来评估这些设备, 而对于缺乏进入气管的担忧仍然存在。胃音也可能是一个潜在的并发症, 在插管动物, 如果气管插管是意外放置在食道;然而, 直接可视化技术的使用, 通过验证引导导管正确放置到喉部, 从而最大限度地减少了这种并发症的风险。
杓和/或气管粘膜的创伤是另一个潜在的问题, 当插入气管插管, 特别是当使用充气的手铐管26,27,28。家兔气管易受损伤, 可能导致皮下气肿、气胸等并发症, 甚至死亡10,27,28,29。在执行我们在这里描述的技术时, 应该注意预防气管损伤, 使用 uncuffed 气管插管, 确保引导导管的圆形尖端插入气管, 而不是将导导管强行注入声门。此外, 选择一个适当大小的气管插管是至关重要的。气管导管大小应考虑与动物的大小, 以及与开放的喉口, 因为它比气管直径小。气管导管直径过大可能导致杓粘膜或软骨的损伤。另一方面, 使用较小的气管导管会导致呼吸道阻力增加, 并增加因粘液或扭结而阻塞的几率。因此, 呼吸道监测在插管家兔中是非常重要的。除了脉冲氧, 气流的测量应 monitored 与二氧化碳;如果分析仪不可用, 气道监测器是一种可用于检测空气流量的替代品 (但不提供呼出的二氧化碳的测量)。此外, 提供辅助通风是有价值的, 特别是在使用较小直径管的情况下24,30。
除了已经提到的使用这项技术的好处之外, 这种方法的另一个主要优点是它可以由一个单独的个体来执行, 而无需助手, 这在繁忙的临床环境中可能是很重要的。在这种情况下, 重要的是要收集所有必要的用品, 并有他们在容易达到的插管过程中, 特别是当引导导管到位, 并应避免头部的移动。
我们在这里描述的技术是由我们机构的兽医和研究人员使用的所有兔插管, 这相当于每年大约200-400 插管。这是很容易教在一个典型的兔麻醉训练课程, 这需要大约30分钟。我们发现, 它是相对容易学习, 与学生学习它在他们的前3尝试, 一般能够成功地重复的过程中, 在下一个额外的尝试。在将来的研究中, 与其他插管技术相比, 将时间与熟练程度和保持熟练程度的能力进行比较是有价值的。虽然研究了盲技术的变体16, 但尚未对各种插管技术的学习曲线进行全面评估。另一个潜在的未来研究方向可能包括比较直接气管插管到较新的 species-specific 声门气道装置, 特别是在正压通气期间的功能。
总之, 我们在这里描述的家兔插管技术是无创的, 可靠的, 不需要使用专门的设备, 易于掌握, 并且可以由一个单独的个体来完成, 无需助手。所有的呼吸道接入技术都存在一定程度的风险, 但是通过直接的可视化技术, 并按照我们上面描述的步骤来减少并发症, 这些风险远远超过了在麻醉期间有足够气道的好处。

家兔有一个狭窄的, 深的口腔, 限制了气管插管的喉的能见度。它们还有非常大的肉质舌和颊齿, 当与狭窄的口腔结合时, 在插入喉镜和可视声门插入气管插管1时会引起问题。在尝试插管时, 必须非常小心避免造成口腔和呼吸道的损伤。喉组织是非常易碎和过度的创伤从反复尝试插管可能导致明显的炎症, 使插管更加困难2。
由于这些原因, 各种替代技术已经被描述, 特别是声门气道装置, 包括喉管3,4, 喉罩气道 (LMA)4,5,6和 species-specific 声门气道设备7。喉管包括一个管子与二个气球直接气流入气管: 一个更大的气球在管子中间形成封印在上部咽和一个更小的气球在远端封印食道入口3。该 LMA 由一个椭圆形的管, 在远端, 充气形成一个密封的密封在声门4,5,6。species-specific 装置是第一个兽医 species-specific 声门气道装置, 目前可用于猫和兔, 旨在适合每个物种的解剖结构, 以创建一个压力密封在咽7。然而, 由于这些设备不直接进入气管, 它们可能会出现诸如胃音等并发症的风险。气管内缺乏适当的密封, 这就引起了人们对使用这些装置用于呼吸支持、急诊或胸部手术的正压通气是否足够的担忧3,8。此外, 缺乏直接插管的气管可能导致人员暴露于废麻醉剂气体5。
许多方法的直接气管插管已被描述9,10,11,12,13,14,15。一种常用的方法是盲法, 因为喉镜的插入不需要16,17,18,19,20. 作为一种视觉盲技术, 该方法需要通过气管插管进行空气流动监测, 并进行细致的操作以引导放置。虽然这种方法是常用的研究和临床兽医实践, 在作者的经验, 这项技术可能是费时, 并不总是成功的, 因此更难教, 并可能需要额外剂量的麻醉药物9,10. 此外, 由于监测呼吸是用来指导适当的放置管, 这种方法将是更困难和不可靠的呼吸骤停。另一种流行的方法是使用传统的喉镜技术来插管, 就像做了较大的物种。然而, 传统的喉镜技术在家兔是困难的, 因为声带的看法是阻塞时, 气管插管插入到嘴里。因此, 使用这种方法可以导致反复插管尝试, 增加麻醉剂量, 并伤害口腔和气道10。
更昂贵和技术上具有挑战性的选择插管也存在。纤维喉镜可以方便地插入气管插管, 将喉镜插入管内, 并在屏幕上可视化插入气管。同样, 内窥镜可用于可视化喉区和引导放置的管。然而, 这两种方法都需要使用昂贵的设备, 在典型的临床设置9、1112中可能无法轻易获得。通过鼻腔插管是一种替代方法, 已被描述, 虽然这是一个风险引入机会病原体从鼻腔进入肺部, 特别是在兔子, 不是特定的-无病原体的21。最后, 更有侵入性的插管方法也已公布, 包括逆行插入导管通过针放置经到气管, 以及使用针 cricothyroidotomy 后, 引导导线的位置,容器扩张和鞘介绍人13,14。作为侵入性技术, 这些方法有潜在的并发症, 如 post-procedural 疼痛和感染。我们在这里描述的技术, 使用喉镜和导管, 可以教与短时间的训练, 以熟练, 是无创的, 可以执行由一个单一的个人不需要一个助手, 不需要使用专用设备。

<table>
	<tbody>
		<tr>
			<td>Ketalar 
			Ketamine HCl Inj.</td>
			<td>JHP Pharmaceuticals</td>
			<td>NDC 42023-113-10</td>
			<td>35mg/kg IM in epaxial muscles or quadriceps muscle (preferred)</td>
		</tr>
		<tr>
			<td>Anased 
			Xylazine (100mg/ml)</td>
			<td>Lloyd Labs</td>
			<td>NADA #139-236</td>
			<td>5mg/kg IM in epaxial muscles or quadriceps muscle (preferred)</td>
		</tr>
		<tr>
			<td>Welch Allyn Laryngoscope Handle</td>
			<td>Welch Allyn</td>
			<td>60300</td>
			<td>Requires 2 size C alkaline batteries</td>
		</tr>
		<tr>
			<td>Size 0 or 1 Miller Laryngoscope Blade</td>
			<td>Welch Allyn</td>
			<td>68040</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 &#8211; 4.0 mm ID uncuffed endotracheal tube</td>
			<td>Jorgenson</td>
			<td>J0149V</td>
			<td>Suitable for 2.5-5 kg rabbits 
			3.5 mm most commonly used</td>
		</tr>
		<tr>
			<td rowspan="2">Sovereign polypropylene catheter, 5 Fr x 22&#8221;</td>
			<td rowspan="2">Medtronic</td>
			<td rowspan="2">8890703310</td>
			<td rowspan="2">Cut to 8-10 inches 
			Mark the cut end with permanent marker to indicate which end not to insert</td>
		</tr>
		<tr>
			<td>Lidocaine 2% HCL Inj</td>
			<td>VetOne</td>
			<td>L-2000-04</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Ply Nonsterile, Bulk Packed Versalon Sponges (Gauze pads)</td>
			<td>Covidien</td>
			<td>KC9024A</td>
			<td></td>
		</tr>
		<tr>
			<td>Tape, Twill, Bleached &#189;&#8221; x 72 yds</td>
			<td>Encompass Textiles</td>
			<td>48210-025</td>
			<td>Used for securing endotracheal tube</td>
		</tr>
		<tr>
			<td>HR Lubricating Jelly</td>
			<td>HR Pharmaceuticals</td>
			<td>201</td>
			<td>For lubricating endotracheal tube tip</td>
		</tr>
		<tr>
			<td>Great Plains Ballistics: BAAM (Beck Airway Airflow Monitor) 22</td>
			<td>BoundTree Medical</td>
			<td>21410</td>
			<td>For confirmation of successful intubation</td>
		</tr>
		<tr>
			<td>Ambu Pediatric Co2 Detector</td>
			<td>AMBU INC</td>
			<td>172713</td>
			<td>For confirmation of successful intubation</td>
		</tr>
	</tbody>
</table>

endotracheal intubation of rabbits using a polypropylene guide catheter

由于其解剖异常, 家兔的气管插管可能具有挑战性。在麻醉期间实现一个专利气道对于避免气道阻塞、预防胃音和允许通气支持至关重要。由于插管困难, 探讨了使用喉罩气管或喉管等替代方法。然而, 这些方法并不导致直接进入气管, 因此可能出现并发症的发展风险。此外, 缺乏直接插管的气管可能导致人员暴露于废弃的麻醉气体。许多方法的气管插管已被描述, 包括盲目放置, 使用纤维喉镜或内窥镜, 和环状软骨放置。尽管有这么多的出版物, 许多人仍在努力取得成功。在这里, 我们提供了一个插管技术的详细描述, 可以教与短时间的训练, 以熟练程度最低。简要地, 经过注射麻醉和适当的定位兔, 聚丙烯导管被放置到气管直接可视化使用喉镜。导管被用作引导气管插管进入气管的指南。 这种方法可以在不需要昂贵设备的情况下进行插管, 并且可由单个个人执行, 而无需助手。 总之, 这项技术可以很容易地教授和执行在任何临床或研究设置很少的成本。

在作者的机构, 这项技术通常教授给实验室和兽医人员在训练期间的兔子处理和麻醉技术。教学课程是用专门的训练动物进行的。培训师在培训师的指导下, 演示了技术, 然后受训者完成了这项技术。受训者通常在1-3 次尝试中取得成功。在他们成功地对兔子进行了一次插管后, 个体被要求第二次进行插管, 并且通常能够在第一次额外的尝试中再次成功地给兔子插管。这些培训期间的反馈, 特别是以前受过替代技术训练的个人, 一贯包括对这项技术如何容易学习和成功完成的评论。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56369/56369fig1.jpg"> 
图 1。用于气管插管的供应: 1) 喉镜与大小0或1米勒喉镜刀片, 2) 脐带 (或其他供应的安全气管插管), 3) 0.25 毫升2% 利多卡因, 4) 5-法国聚丙烯导管切割到8-10 英寸non-rounded 端标记有一个永久性的标志, 明确指出不应插入气管 (箭头), 5) 空气流动检测装置, 6) 无菌润滑剂, 适用于远端的管, 7) uncuffed 气管插管。<a href="//ecsource.jove.com/files/ftp_upload/56369/56369fig1large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56369/56369fig2.jpg"> 
图 2: 胸骨定位用于气管插管.兔子被定位 sternally, 前肢延伸到桌子的末端。后肢向后伸展, 兔头与脊柱对齐。在准备插入喉镜时, 用左手将头部抬高至伸展。<a href="//ecsource.jove.com/files/ftp_upload/56369/56369fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56369/56369fig3.jpg"> 
图 3: 喉镜插入.A) 喉镜用右手插入左纵, 在口的顶部跟随刀片的尖端, 直到软腭可见。一旦软腭是可见的, 喉镜是向前倾斜, 同时延长兔的头部背部和颈部向前, 以可视化喉。B) 喉的可视化。<a href="//ecsource.jove.com/files/ftp_upload/56369/56369fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56369/56369fig4.jpg"> 
图 4: 导导管的推进.A) 在将喉镜刀片切换到左手后, 右手通过喉部的开口向前推进导管的圆尖。B) 导导管通过声带通过直接可视化。<a href="//ecsource.jove.com/files/ftp_upload/56369/56369fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56369/56369fig5.jpg"> 
图 5: 气管导管的推进.气管内管是螺纹在导导管上, 并先进, 直到有阻力从声带。<a href="//ecsource.jove.com/files/ftp_upload/56369/56369fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56369/56369fig6.jpg"> 
图 6: 利多卡因灌输.利多卡因 (2%、0.25 毫升) 被注入气管导管的开口。<a href="//ecsource.jove.com/files/ftp_upload/56369/56369fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56369/56369fig7.jpg"> 
图 7: 气管插管的进展.利多卡因注入两分钟后, 气管导管通过轻柔的扭动, 通过声带而推进。<a href="//ecsource.jove.com/files/ftp_upload/56369/56369fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56369/56369fig8.jpg"> 
图 8: 通过气管插管验证气流.气流检测装置可以用来验证气管导管内的气流, 也可以使用牙科镜来检测呼吸指示成功插管的冷凝。<a href="//ecsource.jove.com/files/ftp_upload/56369/56369fig8large.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Endotracheal Intubation of Rabbits Using a Polypropylene Guide Catheter at JoVE.com

Endotracheal Intubation of Rabbits Using a Polypropylene Guide Catheter

家兔的气管插管由于其解剖异常而具有挑战性。在这里, 我们提出了一种技术, 直接插管的气管使用聚丙烯导管作为指导。这种方法使用相对低廉的用品, 需要最少的培训, 并且可以很容易地在任何临床环境中执行。

用聚丙烯导管引导家兔气管插管

Endotracheal intubation in rabbits can be challenging due to their unusual anatomy. Achieving a patent airway during anesthesia is critical for the ...

endotracheal-intubation-rabbits-using-polypropylene-guide

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

33.3K 次观看.  Mayo Clinic. 家兔的气管插管由于其解剖异常而具有挑战性。在这里, 我们提出了一种技术, 直接插管的气管使用聚丙烯导管作为指导。这种方法使用相对低廉的用品, 需要最少的培训, 并且可以很容易地在任何临床环境中执行。

视频: Endotracheal Intubation of Rabbits Using a Polypropylene Guide Catheter

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

快速PCR热循环利用微型热对流

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

A Practical Guide to Phylogenetics for Nonexperts

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this topic and so it presents inherent problems. Here we compile a practical introduction to phylogenetics for nonexperts.&#160;We outline in a step-by-step manner, a pipeline for generating reliable phylogenies from gene sequence datasets. We begin with a user-guide for similarity search tools via online interfaces as well as local executables. Next, we explore programs for generating multiple sequence alignments followed by protocols for using software to determine best-fit models of evolution. We then outline protocols for reconstructing phylogenetic relationships via maximum likelihood and Bayesian criteria&#160;and finally describe tools for visualizing phylogenetic trees. While this is not by any means an exhaustive description of phylogenetic approaches, it does provide the reader with practical starting information on key software applications commonly utilized by phylogeneticists. The vision for this article would be that it could serve as a practical training tool for researchers embarking on phylogenetic studies&#160;and also serve as an educational resource that could be incorporated into a classroom or teaching-lab.

Here we describe a step-by-step pipeline for generating reliable phylogenies from nucleotide or amino acid sequence datasets. This guide aims to serve researchers or students new to phylogenetic analysis.&#160;

实用指南系统发育的非专家

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.

Differential scanning fluorimetry is a widely used method for screening libraries of small molecules for interactions with proteins. Here, we present a straightforward method to extend these analyses to provide an estimate of the dissociation constant between a small molecule and its protein partner.

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

指南现代荧光定量免疫印迹与故障排除策略

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do not allow for accurate interpretation of this interplay. Using a quantitative imaging-based approach, we present a high-content protocol for characterizing the dynamic phenotypic responses (i.e. morphology changes, proliferation, apoptosis) of heterogeneous cell populations to changes in environmental stimuli. We highlight our ability to distinguish between cell types based upon either fluorescence intensity or inherent morphology features depending on the application. This platform allows for a more comprehensive characterization of subpopulation response to perturbation while utilizing shorter time, smaller amounts of reagents, and lower likelihood of error than traditional cell biology assays. However, in some cases, cell populations may be difficult to identify and quantitate based on complex cellular features and will require additional troubleshooting; we highlight some of these circumstances in the protocol. We demonstrate this application using response to drug in a cancer model; however, it can easily be applied more broadly to other physiological processes. This protocol allows one to identify subpopulations within a co-culture system and characterize the particular response of each to external stimuli.

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

使用定量成像技术鉴定和表征异源细胞群体

Cellular processes are complex and result from the interplay between multiple cell types and their environment. Existing cell biology techniques often do ...

A Versatile Model of Hard Tick Infestation on Laboratory Rabbits

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the laboratory. In ticks, all developmental stages (except egg) are hematophagous, and acquiring a blood-meal when attached to their vertebrate hosts is essential for the successful completion of their life cycle. Here we demonstrate a simple method that uses easily openable capsules for feeding of hard ticks on rabbits. The advantages of the proposed method include its simplicity, short duration and most importantly versatile adjustment to the needs of specific experimental requirements. The method makes possible the use of multiple chambers (of various sizes) on the same animal, which permits feeding of multiple stages or different experimental groups while reducing the overall animal requirement. The non-irritating and easily accessible materials used minimizes discomfort to the animals, which can be easily recovered from an experiment and offered for adoption or reused if the ethical protocol allows it.

We have developed a simple and versatile system to feed hard ticks on laboratory rabbits. Our non-laborious protocol uses easily accessible materials and can be adjusted depending on the requirements of the various experimental settings. The method allows comfortable monitoring and/or sampling of ticks during the entire feeding period.

实验家兔硬蜱的多用途模型

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the ...

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

同步加速器微CT高通量皮质骨样品采购和分析的剖面、科林和图像处理指南

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

蚊子电图分步指南

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...

用聚丙烯导管引导家兔气管插管

摘要

探索更多视频

此视频中的章节

产生稳定的转基因C.使用微量注射线虫

一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内

快速PCR热循环利用微型热对流

实用指南系统发育的非专家

蛋白质 - 配体相互作用采用差示扫描荧光法测定

指南现代荧光定量免疫印迹与故障排除策略

使用定量成像技术鉴定和表征异源细胞群体

实验家兔硬蜱的多用途模型

同步加速器微CT高通量皮质骨样品采购和分析的剖面、科林和图像处理指南

蚊子电图分步指南

用聚丙烯导管引导家兔气管插管

摘要

探索更多视频

此视频中的章节

产生稳定的转基因C.使用微量注射线虫

一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内

快速PCR热循环利用微型热对流

实用指南系统发育的非专家

蛋白质 - 配体相互作用采用差示扫描荧​​光法测定

指南现代荧光定量免疫印迹与故障排除策略

使用定量成像技术鉴定和表征异源细胞群体

实验家兔硬蜱的多用途模型

同步加速器微CT高通量皮质骨样品采购和分析的剖面、科林和图像处理指南

蚊子电图分步指南

蛋白质 - 配体相互作用采用差示扫描荧光法测定