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Abstract
Introduction
Protocol
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Materials
References
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Developmental Biology

使用mRNA电穿孔快速有效地表达Avian胚胎中的多种蛋白质

Published: June 07, 2019
doi:
10.3791/59664
, ,
1Department of Biological Sciences,University of Southern California, Los Angeles, 2Department of Radiology and Developmental Neuroscience Program, Saban Research Institute,Children’s Hospital Los Angeles, 3Keck School of Medicine, Department of Radiology,University of Southern California, Los Angeles

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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 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…

Representative Results

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?…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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.

Materials

BamHI-HF New England Biolabs R3136L
BglII New England Biolabs R0144S
BsrG1-HF New England Biolabs R3575S
NotI-HF New England Biolabs R3189L
SalI-HF New England Biolabs R3138L
Phenol:Chloroform:Isoamyl Alcohol Thermo Fisher 15593031
SP6 mMessage Machine in vitro transcription kit Thermo Fisher AM1340
Fast Green FCF Sigma Aldrich F7252
Triton X-100 Sigma Aldrich 93443 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether
DAPI Sigma Aldrich D9542 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, 4′,6-Diamidino-2-phenylindole dihydrochloride, DAPI dihydrochloride
Whatman No.1 filter paper Sigma Aldrich WHA1001125
glycerol Sigma Aldrich G9012
Urea Sigma Aldrich 51457
pmTurquoise2-Golgi Addgene 36205 pmTurquoise2-Golgi was a gift from Dorus Gadella (Addgene plasmid # 36205 ; http://n2t.net/addgene:36205 ; RRID:Addgene_36205)
pmEGFP-N1-LifeAct Nat. Methods 2008;5:605-7. PubMed ID: 18536722
pCS2.Lifeact-mGFP Addgene This paper
pCS.H2B-citrine Addgene 53752 pCS-H2B-citrine was a gift from Sean Megason (Addgene plasmid # 53752 ; http://n2t.net/addgene:53752 ; RRID:Addgene_53752)
pCS.memb-mCherry Addgene #53750 pCS-memb-mCherry was a gift from Sean Megason (Addgene plasmid # 53750 ; http://n2t.net/addgene:53750 ; RRID:Addgene_53750)
Zeiss LSM-780 inverted microscope Carl Zeiss Microscopy GmbH 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 (>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.
CUY-21 EDIT in vivo electroporator Bex Co., Ltd.
Platinum flat square electrode, side length 5 mm Bex Co., Ltd. LF701P5E
Olympus MVX10 FL Stereo Microscope Olympus LifeScience
XM10 Monochrome camera Olympus LifeScience
Phosphate-Buffered Saline (PBS) for HCR (10×, pH 7.4) To prepare 1 L of a 10× 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).
1.37 M NaCl
27 mM KCl
80 mM Na2HPO4 20 mM KH2PO4
PBS/Triton Add 1 mL of Triton X-100 (Sigma Aldrich 93443) and 100 mL of 10× PBS to 890 mL of ultrapure distilled H2O. Filter the solution through a 0.2-μm filter and store it at 4 ̊C until use.
1× phosphate-buffered saline (PBS) (DEPC-treated; pH 7.4)
0.1% Triton X-100
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Tags

Keywords: MRNA ElectroporationAvian EmbryoProtein ExpressionFluorescent ProteinsTransient TransfectionDevelopmental StageElectroporation ChamberEpiblastVitelline MembraneFluorescent Imaging
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Tran, M., Dave, M., Lansford, R. Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation. J. Vis. Exp. (148), e59664, doi:10.3791/59664 (2019).
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