Name: fast and efficient expression of multiple proteins in avian embryos using mrna electroporation
Uploaded: 2019-06-07T14:30:00
Description: Watch this Scientific Journal Video about Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation at JoVE.com

我们感谢大卫·胡斯对这项工作的有益见解。这项工作部分得到了玫瑰山基金会暑期研究奖学金(2016-2018年)和南加州大学教务长研究生研究奖学金、萨班研究所校内培养博士前博士奖和大学南加州本科生研究伙伴计划奖授予R.L.

所有动物程序均按照洛杉矶儿童医院和南加州大学机构动物护理和使用委员会批准的准则进行。
1. 生成基于 pCS2 的表达式矢量
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要克隆 pCS2.Lifeact-eGFP,请通过消化 2 μg 的 pCS2.CycB1-GFP(包含不同插入的构造)在适当的消化缓冲液中(参见材料表)在适当的消化缓冲液中(参见材料表)稀释到总反应中的 1 倍来制备载体主干在37°C下体积为50μL1小时(参见图1,</str...

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在此协议中,我们提供了关于如何精确微注射和电镀mRNA到胃化鹅胚胎细胞的逐步说明。我们证明,体外合成的mRNA电穿孔允许快速有效地表达荧光蛋白(FPs)在胃化鹅胚胎(图2和3)。 从电镀mRNA翻译的H2B-黄水晶蛋白的荧光可以在+20分钟内通过共聚焦显微镜检测,并在FP荧光中增加数小时(图3,补充电影1)。这是令人惊讶的快速和接近假定的...

电穿孔是一种物理转染方法,它使用电脉冲在等离子膜中产生瞬态孔隙,使核酸或化学物质等物质进入细胞醇。电穿孔被广泛用于将DNA输送到细菌、酵母、植物和哺乳动物细胞1、2、3。它通常用于将遗传有效载荷引入发育中的鸟类胚胎中的目标细胞和组织,以研究发育的遗传控制或标记细胞迁移种群4、5、6 7.然而,DNA电穿孔8也存在一些实验限制。例如,DNA电穿孔通常引入每个细胞表达载体数量高度可变,随后引入其编码的 mRNA 和蛋白质。这种变异性会导致相当大的细胞异质性,使图像分析和数据解释复杂化9,10。此外,来自DNA电穿孔的蛋白质在电穿孔后才开始表达±3小时,直到12小时才达到细胞数量和荧光强度的最高效率,这可能是因为需要时间转移到细胞核并完成转录和翻译在体内11。
相比之下,mRNA转染已有效地用于各种模型系统,包括通过微注射12、13、通过mRNA脂肪可敏转染14重新编程人类干细胞,在成年小鼠中电化顽固神经干细胞15。我们利用编码不同荧光蛋白(FPs)的体外合成mRNA,测试了mRNA电穿孔在早期鸟类胚胎发育过程中有效标记细胞的能力。在我们的研究中,我们使用pCS2+载体,一种多用途表达载体,通常用于在异种动物和斑马鱼胚胎中表达蛋白质。pCS2+ 中的 SP6 和 T7 RNA 聚合酶启动子允许在体外转录/翻译系统中使用时从任何克隆基因中合成 mRNA 和蛋白质。
在这里,我们证明了mRNA电穿孔允许快速有效地表达荧光蛋白(FPs)在胃化鹅胚胎。我们设计并生成了许多在这些研究中使用的表达式向量。例如,我们将 LifeAct-eGFP 基因16分克隆到 pCS2+ 载体17中,以从 CMV 启动子和 SP6 启动子表达。插入的基因位于SP6启动子的下游和SV40聚(A)尾18的上游。在与mRNA和DNA共电的胚胎中,从体外转录的mRNA编码的FPs在电穿孔后20分钟内首次被检测到,而DNA表达载体中的FPs仅在3小时后被检测到。膜蛋白可以同时电化到胚胎中,从而在单个细胞中快速有效地表达多种蛋白质。最后,使用光漂白(FRAP)测定后的体内荧光恢复,我们发现大多数电化mRNA在2小时内衰变。因此,快速的初始蛋白质生产与有限的新蛋白质转化相结合,使mRNA电穿孔成为一种有价值的技术,当需要时间控制表达时。

<table>
	<tbody>
		<tr>
			<td>BamHI-HF</td>
			<td>New England Biolabs</td>
			<td>R3136L</td>
			<td></td>
		</tr>
		<tr>
			<td>BglII</td>
			<td>New England Biolabs</td>
			<td>R0144S</td>
			<td></td>
		</tr>
		<tr>
			<td>BsrG1-HF</td>
			<td>New England Biolabs</td>
			<td>R3575S</td>
			<td></td>
		</tr>
		<tr>
			<td>NotI-HF</td>
			<td>New England Biolabs</td>
			<td>R3189L</td>
			<td></td>
		</tr>
		<tr>
			<td>SalI-HF</td>
			<td>New England Biolabs</td>
			<td>R3138L</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl Alcohol</td>
			<td>Thermo Fisher</td>
			<td>15593031</td>
			<td></td>
		</tr>
		<tr>
			<td>SP6 mMessage Machine in vitro transcription kit</td>
			<td>Thermo Fisher</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green FCF</td>
			<td>Sigma Aldrich</td>
			<td>F7252</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>93443</td>
			<td>4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma Aldrich</td>
			<td>D9542</td>
			<td>2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, 4&#8242;,6-Diamidino-2-phenylindole dihydrochloride, DAPI dihydrochloride</td>
		</tr>
		<tr>
			<td>Whatman No.1 filter paper</td>
			<td>Sigma Aldrich</td>
			<td>WHA1001125</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol</td>
			<td>Sigma Aldrich</td>
			<td>G9012</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma Aldrich</td>
			<td>51457</td>
			<td></td>
		</tr>
		<tr>
			<td>pmTurquoise2-Golgi</td>
			<td>Addgene</td>
			<td>36205</td>
			<td>pmTurquoise2-Golgi was a gift from Dorus Gadella (Addgene plasmid # 36205 ; http://n2t.net/addgene:36205 ; RRID:Addgene_36205)</td>
		</tr>
		<tr>
			<td>pmEGFP-N1-LifeAct</td>
			<td></td>
			<td></td>
			<td>Nat. Methods 2008;5:605-7. PubMed ID: 18536722</td>
		</tr>
		<tr>
			<td>pCS2.Lifeact-mGFP</td>
			<td>Addgene</td>
			<td></td>
			<td>This paper</td>
		</tr>
		<tr>
			<td>pCS.H2B-citrine</td>
			<td>Addgene</td>
			<td>53752</td>
			<td>pCS-H2B-citrine was a gift from Sean Megason (Addgene plasmid # 53752 ; http://n2t.net/addgene:53752 ; RRID:Addgene_53752)</td>
		</tr>
		<tr>
			<td>pCS.memb-mCherry</td>
			<td>Addgene</td>
			<td>#53750</td>
			<td>pCS-memb-mCherry was a gift from Sean Megason (Addgene plasmid # 53750 ; http://n2t.net/addgene:53750 ; RRID:Addgene_53750)</td>
		</tr>
		<tr>
			<td>Zeiss LSM-780 inverted microscope</td>
			<td>Carl Zeiss Microscopy GmbH</td>
			<td></td>
			<td>The LSM-780 is a confocal and multi-photon microscope that offers the sensitivity required for vital imaging work. Equipped with a motorized stage, an autofocus device, and a full stage-top blackout incubator, the 780 is an excellent microscope for high-end live cell/embryo imaging. The high-sensitivity 32-channel Quasar detector allows for spectral imaging, linear unmixing, and high color count (&#62;4) image acquisition. Excitation can be performed with 6 lines single photon lasers (405, 458, 488, 514, 564 and 633 nm), Chameleon (Coherent) 2-photon laser (range from 690nm to 1000nm), and run with ZEN 2011 SP7 (Black) system software.</td>
		</tr>
		<tr>
			<td>CUY-21 EDIT in vivo electroporator</td>
			<td>Bex Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum flat square electrode, side length 5 mm</td>
			<td>Bex Co., Ltd.</td>
			<td>LF701P5E</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus MVX10 FL Stereo Microscope</td>
			<td>Olympus LifeScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>XM10 Monochrome camera</td>
			<td>Olympus LifeScience</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS) for HCR (10&#215;, pH 7.4) </td>
			<td></td>
			<td></td>
			<td>To prepare 1 L of a 10&#215; stock solution, combine 80 g of NaCl (Sigma-Aldrich S3014), 2 g of KCl (Sigma-Aldrich P9541), 11.4 g of Na2HPO4 (anhydrous; Sigma-Aldrich S3264), and 2.7 g of KH2PO4 (anhydrous; Sigma-Aldrich P9791). Adjust the pH to 7.4 with HCl, and bring the final volume to 1 L with ultrapure H2O. Avoid using CaCl2 and MgCl2 in PBS for HCR. It is important that the PBS for HCR is prepared as an RNase-free solution (e.g., via diethylpyrocarbonate [DEPC] treatment).</td>
		</tr>
		<tr>
			<td>1.37 M NaCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>27 mM KCl</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>80 mM Na2HPO4 20 mM KH2PO4</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS/Triton</td>
			<td></td>
			<td></td>
			<td>Add 1 mL of Triton X-100 (Sigma Aldrich 93443) and 100 mL of 10&#215; PBS to 890 mL of ultrapure distilled H2O. Filter the solution through a 0.2-&#956;m filter and store it at 4 &#778;C until use.</td>
		</tr>
		<tr>
			<td>1&#215; phosphate-buffered saline (PBS) (DEPC-treated; pH 7.4)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.1% Triton X-100</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

fast and efficient expression of multiple proteins in avian embryos using mrna electroporation

我们报告说,mRNA电穿孔允许荧光蛋白比DNA电穿孔更快地和广泛地标记活的鹅胚胎中的细胞。高转染效率允许至少4个不同的mRNA以±87%的效率进行共转染。大多数电穿孔的mRNA在电穿孔后的前2小时降解,允许在发育中的胚胎中进行时间敏感的实验。最后,我们描述了如何动态成像活胚胎电镀与mRNA编码各种亚细胞靶向荧光蛋白。

mRNA 电穿孔比 DNA 电穿孔更有效
我们使用 pCS2+。H2B-黄体素制备体外转录mRNA。由于DNA电穿孔通常在1-2μg/μL下进行,我们使用mRNA的等位浓度(计算为H2B-Citrine的约0.25-0.5微克/μL)进行mRNA电穿孔。我们首先测试了pCS2+的电穿孔效率。H2B-黄水晶DNA与H2B-黄水晶mRNA(从pCS2+的SP6启动子体转录的体外转录)。H2B-黄酸酯)通过将DNA或mRNA?...

Watch this Scientific Journal Video about Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation at JoVE.com

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

我们报告信使RNA(mRNA)电穿孔作为一种方法,允许快速有效地表达多个蛋白质在龟胚胎模型系统。该方法可用于荧光标记细胞,并在电穿孔后不久通过延时显微镜记录细胞体内运动。

使用mRNA电穿孔快速有效地表达Avian胚胎中的多种蛋白质

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

mRNA电穿孔提供了鸟类模型系统中多种蛋白质的快速、高效、在空间和时间上控制的表达。与标准DNA电穿孔获得的标签，此方法允许更广泛、更快速地表达荧光蛋白。电穿孔有助于血浆膜中毛孔的瞬态开口，作为将RNA和DNA等遗传有效载荷转移到许多模型生物体的细胞的导管。对于最初的实验工作与超过一天的胚胎，因为他们更硬，更耐外部操作，如电穿孔。在适当的胚胎发育阶段轻轻地打破和倒鹅蛋内容到10厘米的培养皿。使用转移移液器去除大部分厚白条。使用实验室组织轻轻擦拭束缚的表面，取出胚胎周围剩余的厚白化细胞，确保胚胎紧紧粘在纸环上。将预切滤纸放在胚胎上，用剪刀在胚胎周围平滑切割。使用巴斯德移液器将PPS层下胚胎，使用温和的溪流，以腾出任何粘在组织上束缚的束缚。慢慢地将胚胎纸环以斜角向上和从束缚上拉下。将纸张放入装满 PBS 的培养皿中，以进行进一步清洁。一旦大部分的束缚被移除，将胚胎腹侧放在一个35毫米的培养皿中，上面覆盖着半固体的阿加和白细胞的混合物。通过将黑色电极与 PBS 连接并将胚胎放入内，准备电穿孔室。使用玻璃微胶囊将 200 纳米的 mRNA 注入覆盖所需区域的表膜和葡萄球菌膜之间的腔中。然后用红色电极对胚胎进行电分。将电胚胚胎放回阿加-白蛋白混合物中，并在38摄氏度下孵育，直到达到所需的发育阶段。对于电穿孔编码荧光蛋白的成像，在荧光解剖立体镜下观察所有电穿孔胚胎，为动态成像实验选择最健康、最好的电穿孔胚胎。继续在单独的培养箱中孵育其他电镀胚胎和非电镀胚胎。用 PBS 短暂冲洗所选胚胎，以去除在电穿孔期间在胚胎的后侧可能形成的任何气泡。将清洁的胚胎直接放入一个成像盘中，内含一层约150微升的白化素，注意不要在胚胎的背表面产生任何气泡。沿着成像盘的内边缘添加一小块湿润卷起的纸巾。用石蜡膜密封盘子，以尽量减少成像和孵育过程中蒸发。快速将培养皿移动到共体显微镜的预预热阶段，并使用亮场通道定位胚胎中的彩色染料。将成像软件设置为所需的目标、二色镜和发射光谱，并打开适当的激光器。注意整个影片的单元格将保留在图像视觉中。使用足够高的成像分辨率，并确保成像条件不会引起光毒性。单击成像软件中的"实时"，根据每种显微镜激光功率将激光功率调整为适合荧光强度的设置。开始使用 1%激光功率和 800 增益对胚胎进行成像，将激光功率缓慢增加 1%，直到看到饱和像素。当观察到饱和像素时，激光功率会略有降低，直到无法可视化饱和像素。每三到五分钟对胚胎进行成像，使用整个电镀区域的五微米 Z 堆栈检查单个细胞跨不同时间点的迁移，并针对 Z 堆栈的底部增加一些空间，以防胚胎在成像过程中沉入阿加罗斯床。要观察单元格的移动速度，请检查第一部电影的正几个时间点。如果单元格以快速的速度移动，请考虑展开图像区域的缩放或成像不同的区域。当胚胎的整个电镀区域被成像后，图像在相同胚胎的未电镀区域中确定自荧光水平。要确定转染mRNA在立体显微镜上确认感兴趣的基因的电穿孔后可以转化为荧光蛋白多长时间，将胚胎置于倒置共体显微镜的预热阶段，并使用具有70%激光功率、100次迭代的405纳米激光，以及从电镀基因的4到光漂白大部分细胞荧光的扫描速度， 在不同时间点。在光漂白后，在36摄氏度的温度下继续在舞台上孵育胚胎，注意注意光漂白区域内通常只见健康胚胎的主动分裂细胞，从而表明电穿孔没有伤害胚胎细胞。定期以三到五分钟的时间间隔获取电镀区域的漂白后 Z 堆栈图像，时间长度达到所需时间。确保成像条件不致影响胚胎存活同时孵育未光漂白的电镀胚胎，以控制成像。为了量化mRNA衰减后电穿孔的光漂白结果，使用 ImageJ 测量每个细胞电镀区域中心7.5微米圆的荧光强度，以跟踪每三到五分钟间隔的细胞荧光。当光漂白区域内所有未经历线粒化且已完全光漂白的细胞都经过测量后，在胚胎光漂白的每个时间点绘制荧光强度。虽然DNA电穿孔导致某些电镀细胞的荧光更亮，但与广泛表达的mRNA编码荧光蛋白相比，DNA电穿孔的效率明显较低。当比较同一胚胎中的DNA和mRNA共电时，表达荧光蛋白效率的DNA效率也明显且始终低于表达荧光蛋白的mRNA。所有胚胎的定量仅使用mRNA、DNA或两者的组合进行电镀，发现mRNA在给定区域中转染约75%的细胞，而DNA只转染约25%的细胞。与转染的DNA相比，转染mRNA应能更快地产生蛋白质，因为细胞转化机制可以立即识别mRNA。虽然多个DNA的共电化以前已被证明是相对低效的，但本实验中4个MRNA的共电化导致所有电镀区域内所有四个MRNA的转染效率达到87%。虽然光漂白最初将光漂白细胞的荧光强度降低了约95%，但一些细胞在电穿孔后不久仍能够恢复分裂和表达荧光蛋白的能力。然而，这种能力在电穿孔后五小时内会降低。花时间优化最佳成像条件，甚至在开始拍摄电影后，也要继续进行修补，如果需要，可以进行任何调整。

Research

JoVE Journal

Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

在小鸡视网膜有效的基因转移的原代细胞培养研究：<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

科研

发育生物学

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

荧光蛋白的表达<em&gt;披文昌鱼</em&gt;通过基因注射到未受精的卵母细胞

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Whole Retinal Explants from Chicken Embryos for Electroporation and Chemical Reagent Treatments

鸡胚全视网膜外植体电穿孔和化学试剂处理

The retina is a good model for the developing central nervous system. The large size of the eye and most importantly the accessibility for experimental ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

Tracing Gene Expression Through Detection of ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â²-galactosidase Activity in Whole Mouse Embryos

通过检测全小鼠胚胎β乳糖酶活性追踪基因表达

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

使用光遗传TAEL/C120系统在斑马鱼胚胎中的光诱导GFP表达

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...

In Ovo Intravascular Injection in Chicken Embryos

In Ovo Intravascular Injection in Chicken Embryos

在 奥沃 鸡胚血管内注射

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

Rapid and Efficient Spatiotemporal Monitoring of Normal and Aberrant Cytosine Methylation within Intact Zebrafish Embryos

快速有效地监测完整斑马鱼胚胎中正常和异常胞嘧啶甲基化的时空监测

Cytosine methylation is highly conserved across vertebrate species and, as a key driver of epigenetic programming and chromatin state, plays a critical ...