Name: microstructured devices for optimized microinjection and imaging of zebrafish larvae
Uploaded: 2017-12-08T13:00:00
Description: Watch this Scientific Journal Video about Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae at JoVE.com

作者要感谢大卫 Langenau 慷慨地提供水族馆空间;埃里克石头, 约翰 c. 摩尔和秦唐帮助与斑马鱼的维护和试剂, 和安妮罗伯逊和艾略特 Hagedorn 从伦纳德区的实验室购买的斑马鱼菌株在这里使用。他们还要感谢奥克塔维奥 Hurtado 关于光刻技术的建议。FE 的经费来自实力的儿童医院和美国澳大利亚协会的研究金。这项工作由 NIH 资助 GM92804。

根据《2011N000127 议定书》, 马萨诸塞州总医院研究动物保育小组委员会批准了微注射幼虫。
1. 设备制造
注: 此处描述的用于设计光刻掩模的所有计算机辅助绘图 (CAD) 文件 (图 1) 均可供下载。查看链接的材料表。
<ol><li>使用标准光刻技术7, 在1000级洁净室中制造主模具晶片。对于微结构阵列和通道, 按照制造商的说?...

<ol>
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在这里, 我们描述了使用的设备, 我们最近开发, 以方便 2 dpf 斑马鱼微注射5, 并介绍了一个简单的琼脂糖免费安装装置, 方便成像的胚胎。这些工具突出了光刻技术的效用, 用于制作适用于斑马鱼技术的设备。
我们发现 MSA 装置特别适用于注射针内的细胞或微粒, 如真菌孢子或人类癌细胞, 这需要使用更大的钻孔针来运送。注射到耳泡 (图 4</strong...

斑马鱼已经成为一个强大的模型, 许多领域, 从基础发展生物学研究到大规模的遗传和化学屏幕1,2。常规的基因操作, 如基因过度表达、击倒、CRISPR/Cas9 突变和转基因依赖于单细胞受精卵的微注射, 这导致了简单易用的商业用于定向和稳定注射的卵子的可用工具3。其他方法, 如移植和感染, 往往需要注射到后期胚胎和幼虫使用较大的规范毛细管针头4。然而, 使用更大的规格针提出了重大的技术挑战, 因为它是更难穿透目标组织不推或滚动的胚胎。在这些条件下, 获得稳定胚胎所需的适当的水张力, 同时避免在手术过程中干燥是困难的, 而且胚胎可能不是理想的定向注射到靶组织。
在注射后, 筛出的胚胎选择那些已成功注射的胚, 并捕捉初始时间点的图像, 通常是有用的。为了应对这些挑战, 我们开发了一系列的微设备, 帮助稳定 2 dpf 胚胎的各种方向, 既用于微注射5, 也用于快速基于图像的扫描后注射。
为了在这些装置中获得足够的结构分辨率, 我们利用了光刻技术。这些方法通常用于微电子工业, 并且最近被外推用于微流控芯片的制造, 因此可以实现垂直结构, 范围从1-1、000µm, 这是一种适合于斑马鱼胚胎和幼虫操作的鳞片。所有的设备都是用烷 (硅氧烷) 制造的, 它价格低廉, 身体健壮, 生物惰性, 而且透明。
微表面阵列 (协议) 被格式化成块的, 具有图案的顶面, 类似于简单的渠道琼脂糖块通常用于卵注射。对于后注射筛选, 6 成像设备可以排列在一个标准的玻璃底 6-井板。这些装置是为易于装载的胚胎而设计的, 而卸载程序方便地允许对特定的胚胎进行抢救, 比以前由毕比实验室6。

<table>
	<tbody>
		<tr>
			<td>Dow Corning Sylgard 184&#160; Polydimethylsiloxane (PDMS)&#160;</td>
			<td>Ellsworth Adhsives</td>
			<td>184 SIL ELAST KIT 0.5KG</td>
			<td>For casting the devices. Kit includes PDMS monomer and Initiator</td>
		</tr>
		<tr>
			<td>Low gelling temperature agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9414-10G</td>
			<td>For casting agarose devices</td>
		</tr>
		<tr>
			<td>PFDTS silane</td>
			<td>Sigma Aldrich</td>
			<td>448931-10G</td>
			<td>For casting of negative PDMS molds</td>
		</tr>
		<tr>
			<td>Tricaine (MS-222)</td>
			<td>Sigma Aldrich</td>
			<td>E10521-10G</td>
			<td>To anesthetize&#160; zebrafish&#160;</td>
		</tr>
		<tr>
			<td>Rhodamine Dextran 70,000 Da</td>
			<td>ThermoFisher</td>
			<td>D1818</td>
			<td>To trace microinjections</td>
		</tr>
		<tr>
			<td>Leukotriene B4 (LTB4)</td>
			<td>Cayman Chemicals</td>
			<td>20110</td>
			<td>Neutrophil chemoattractant</td>
		</tr>
		<tr>
			<td>N-Formylmethionine-leucyl-phenylalanine (fMLP)</td>
			<td>Sigma Aldrich</td>
			<td>F3506-50MG</td>
			<td>Neutrophil chemoattractant</td>
		</tr>
		<tr>
			<td>15 cm Petri dish</td>
			<td>Fisher scientific</td>
			<td>08-757-148</td>
			<td>For Casting from the master wafer</td>
		</tr>
		<tr>
			<td>Glass-bottom 6-well plates</td>
			<td>MatTek</td>
			<td>P06G-0-20-F</td>
			<td>For imaging devices</td>
		</tr>
		<tr>
			<td>Borosilicate glass microcapillaries</td>
			<td>World Scientific Instruments</td>
			<td>TW-100-4</td>
			<td>For microinjection needles</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Sigma Aldrich</td>
			<td>Z350796</td>
			<td>For transferring zebrafish embryos</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Fisher scientific</td>
			<td>E5242956003</td>
			<td>For loading the microinjection needles</td>
		</tr>
		<tr>
			<td>Harris Uni-Core 1.5 mm punch</td>
			<td>Ted Pella Inc.</td>
			<td>15111-15</td>
			<td>To punch ports in PDMS imaging devices</td>
		</tr>
		<tr>
			<td>No.&#160;11 Scalpel</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td>For cutting PDMS&#160;</td>
		</tr>
		<tr>
			<td>Dumont No. 5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-10</td>
			<td>For dechorionating embryos and breaking microinjection needle tips</td>
		</tr>
		<tr>
			<td>Marzhauser Micromanipulator</td>
			<td>ASI&#160;</td>
			<td>MM33-R</td>
			<td>For manipulating microinjection needle</td>
		</tr>
		<tr>
			<td>Magnetic stand</td>
			<td>MSC</td>
			<td>SPI - 87242624</td>
			<td>For mounting micromanipulator</td>
		</tr>
		<tr>
			<td>MPPI-3 Picopump controller</td>
			<td>ASI</td>
			<td>MPPI-3</td>
			<td>To control microinjection volume and timing</td>
		</tr>
		<tr>
			<td>EVOS inverted fluorescent microscope</td>
			<td>ThermoFisher</td>
			<td>EVOS FL</td>
			<td>To image injected embryos</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td>For visualizing microinjecion</td>
		</tr>
		<tr>
			<td>AutoCAD software</td>
			<td>Autodesk</td>
			<td></td>
			<td>Download AutoCAD files from: https://dx.doi.org/10.6084/m9.figshare.4282853 and on the ZFIN community protocols wiki page: https://wiki.zfin.org/display/prot/ZFIN+ Protocol+Wiki &#160;</td>
		</tr>
	</tbody>
</table>

microstructured devices for optimized microinjection and imaging of zebrafish larvae

斑马鱼已经成为各种人类疾病的强有力的模型, 并成为越来越多的实验研究的有用工具, 通过大规模的遗传和化学屏障跨越了基础发育生物学。然而, 许多实验, 特别是那些与感染和异种模型有关的试验, 依赖于胚胎和幼虫的显微注射和成像, 这些都是需要技能和专长的费力技术。为了提高目前显微注射技术的精确度和吞吐量, 我们开发了一系列微装置, 在2天后, 在腹侧、背部或侧面定向后, 对斑马鱼胚胎进行定位和稳定。程序.为了帮助对胚胎进行成像, 我们还设计了一个简单的装置, 使4只斑马鱼在平行的方向上与一个玻璃覆盖层滑动。在这里, 我们展示了光刻方法的有效性, 为斑马鱼技术的优化创造了有用的设备。

此处介绍的方法演示了设计 (图 1) 和用于 2 dpf 斑马鱼的设备的制造, 使用光刻 (图 2) 和软光刻 (图 3) 技术。此方法允许对许多设计迭代和修改进行快速测试, 并且在其他开发阶段与斑马鱼一起使用的微观结构尺寸的改变和优化可能会延长它们的应用范围。
我们...

Watch this Scientific Journal Video about Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae at JoVE.com

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

斑马鱼胚胎和幼虫的显微注射是许多斑马鱼模型中的关键但具有挑战性的技术。在这里, 我们提出了一系列的微型工具, 以帮助稳定和定位斑马鱼的显微注射和成像。

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