Name: expression of fluorescent proteins in branchiostoma lanceolatum by mrna injection into unfertilized oocytes
Uploaded: 2015-01-12T12:45:00.000+00:00
Duration: 9 min 31 s
Description: Watch this Scientific Journal Video about Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes at JoVE.com

笔者想承认的"Animalerie中央去伊维特河畔吉夫"的支持畜牧业。这项工作是由ANR（ANR-09-BLAN-0262-02和ANR-11-JSV2-002-01）迈克尔·舒伯特支持基金，由欧盟FP6授予"Embryomics"，并通过了ANR授予"ANR- 10 BLAN-121801开发进程"让 - 弗朗索瓦·尼古拉和纳丁Peyriéras。若昂·伊曼纽尔卡瓦略通过FCT博士奖学金资助（SFRH / BD /二千零一十二分之八万六千八百七十八）。 请求对这里所描述的载体，可以直接给作者。

1.准备仪器及试剂<ol style="margin-left: 40px;"><li>转让巴斯德移液器<ol><li>通过拉动上述火焰以不同的速度230毫米长巴斯德移液器产生一系列转印巴斯德移液器具有不同尖端的直径。确保锥度为尽可能长的愿望光滑细腻的控制。 </li><li>用金刚石划线，划伤沿线吸管垂直于吸液管的长度。用双手拉平行的吸管其长度产生钝剪。快速火焰抛光无密封尖端的吸管。 </li><li>而变化的卵母细胞/胚胎的阶段的尖端的直径要吸:300-400微米为未受精的卵母细胞（约150微米的直径），600微米为受精卵（约在直径500微米），200-400微米对于阴影neurulae。用移液管带口监测的吸管从一个盘转移卵母细胞/胚胎到另一个。 </li></ol></li><li>注射针头<ol><li>操纵毛细管用手套，以确保无RNase的条件。用硼硅玻璃纤维毛细管的OD1.20毫米，ID0.94毫米，长10 mm尺寸。 </li><li>如果毛细管拉到加热丝针拔了材料清单描述的类型，使用以下设置:热火600，上拉50，速度80，时间60，压力200或300。否则，确保形状，这是对于成功的注射剂是至关重要的，则如图2具有以下属性:4-8微米外尖端直径，2厘米锥形长度。 注:针可在产卵季节前拉和整个赛季的使用。 </li></ol></li><li> 5％酚红储液（4×） <ol><li>准备每个产卵季节前的新鲜。 </li><li>在0.22微米的过滤管，掂量出了酚红粉25毫克。 </li><li>加入0.5毫升DNase-和无RNA酶的水，以含有粉末的管子。 </li><li>旋转3-5分钟，在18000×g离心，在室温下过滤，消毒溶液并除去晶体，可能堵塞注射针。 </li><li>储存在4°C或存储250微升等分在-20℃。 </li></ol></li><li> 0.25毫克/毫升聚 - 赖氨酸溶液<ol><li>准备每个产卵季节前的新鲜。 </li><li>溶解5毫克聚L-赖氨酸在20ml蒸馏水中。商场有5毫升分装于-20℃。 </li><li>立即使用解冻等分试样且仅一次，以确保该卵母细胞与聚赖氨酸包被的培养皿的可重复性和健壮的附着力。 </li></ol></li><li> mRNA合成<ol><li>准备每个产卵季节前的新鲜。 </li><li>线性化5微克的具有足够的兴趣酶的DNA（通常为2小时，在37℃）。检查消化的完整性，在消化混合物的体积上在TBE缓冲液中的1％琼脂糖TBE凝胶在150瓦20分钟运行2％。 </li><li>提取LINEA授权的DNA用25:24:1的苯酚（pH值为8.0）:氯仿:异戊醇。涡旋20秒，离心18000×g离心10分钟，并收集水（上部）相。 </li><li>带24再次萃取水相:1氯仿:异戊醇。涡旋20秒，离心18000×g离心10分钟，并收集水相。 </li><li>沉淀线性的DNA用100:10:3​​00的DNA线性化:3M乙酸钠（pH 5.2）:100％乙醇过夜，在​​-20℃下。 </li><li>离心20分钟18,000 XG在4℃。冲洗70％的乙醇。 </li><li>离心10分钟，在18000 XG 4℃。让干燥，重悬在无RNA酶的水以0.5微克/微升的最终浓度。 </li><li>转录1微克用mRNA合成试剂盒与适当的线性化聚合酶的DNA，根据生产商的说明。 </li><li>提取的mRNA以5:1的苯酚（pH值4.7）:随试剂盒提供氯仿和醋酸铵终止溶液。 </li><li>涡旋20秒，CEntrifuge在18000×g离心10分钟，并收集水相。 </li><li>带24再次萃取水相:1氯仿:异戊醇。涡旋20秒，离心18000×g离心10分钟，并收集水相。 </li><li>沉淀用100％异丙醇中的mRNA过夜，在-20℃下。 </li><li>离心20分钟18,000 XG在4℃。冲洗在80％的乙醇。 </li><li>离心10分钟，在18000 XG 4℃。让沉淀干燥，在室温下不超过5-10分钟，因为它随后将很难再悬浮。重悬在DNase-和无RNase水以至少2微克/微升的最终浓度，以确保在注入混合体面最终的mRNA浓度。 </li><li>检查转录产物的质量和尺寸，对在TBE缓冲液中的不含RNA酶的1％琼脂糖TBE凝胶上运行0.5微升mRNA的，在150瓦20分钟。店内2微升等分在-80℃。 </li></ol></li><li>多聚赖氨酸包被的菜 NOTE:聚赖氨酸COA泰德菜肴用过的注射过程中固定的卵母细胞。 <ol><li>对于每个35毫米细胞培养的培养皿（5个），覆盖培养皿用1ml解冻0.25毫克/毫升聚 - 赖氨酸溶液的底部。在室温下孵育5分钟。 </li><li>对于每个35毫米细胞培养的培养皿（5个），转移为0.25mg / ml聚赖氨酸溶液到另一个35毫米细胞培养的培养皿。在室温下孵育5分钟。 </li><li>丢弃0.25毫克/毫升聚 - 赖氨酸溶液。 </li><li>让培养皿干燥，在室温下颠倒2小时。 </li><li>存储包裹在保鲜膜的聚赖氨酸 - 包被的培养皿在4℃下，以避免污染一周最大。 </li></ol></li><li>琼脂糖涂层菜 注:它们被用来培养注射的胚胎。琼脂糖提供了一个缓冲用于注射的胚胎和防止它们粘到培养皿的底部。 <ol><li>让使用37-38克/升通讯人造海水（ASW）ercial盐+ 0.25毫米的NaHCO 3反渗透水。 </li><li>通过加热，在微波的溶液溶解琼脂糖至1％的浓度在0.22微米过滤ASW。 </li><li>迅速倾从一次35毫米的培养皿的温暖琼脂糖溶液成另一个，以便确保在盘子的一个非常薄的琼脂糖涂层。 </li><li>存储包裹在萨兰琼脂糖被覆菜裹，在4℃，以避免污染一周最大。 </li></ol></li><li>注射混合注射针和装载<ol><li>在开始注射前约2小时，使2微升注射混合在无RNA酶和DNA酶的水与终浓度为1-1.8微克/微升的mRNA，15％甘油，1.25％酚红。 注:酚红颜色的解决方案，它允许监测的注入效率，并成功地注入胚的鉴别。甘油有利于卵母细胞中mRNA的扩散。 </li><li>离心4分钟18000 XG到颗粒晶体。保持在冰上直到使用。 </li><li>用10微升移液管，收集0.5微升注射混合的，从而避免了管，其中该晶体已沉淀的底部。 </li><li>通过吸取注射混合物的0.5微升滴在大开口的针的回填的至少两个注射针的情况下（注射期间1时间）。 </li><li>在4℃下安装在存储罐用液体在底部的针，以防止注射混合的蒸发。让注射混合慢慢行进到注射针的前端为至少1小时，以尽量减少产生气泡。 </li><li>商店额外注射混合在-80℃下的最大的三个附加的用途，在这之后的mRNA品质恶化（数据未示出）。 </li></ol></li></ol> 2.收集生物材料，显微注射和胚胎培养的<ol style="margin-left: 40px;"><li>卵母细胞和精子采集 注:请参阅Theodosiou等 12诱导产卵和配子收集了详细的方案。 <ol><li>休克的男性和女性在ASW在23℃24小时。 </li><li>一到两个小时日落之前，在19℃，转移到成年人各个杯ASW，因为大多数成年人都会在日落之后产卵1-2小时。 </li><li>冲洗35毫米培养皿中过滤ASW，让他们干倒，防止卵母细胞粘在盘子的底部。 </li><li>产卵后，立即收集精子和卵母细胞有1000微升吸管。 <ol><li>保持精子和卵母细胞从成人文昌鱼分开，因为接触是有害的配子健康（数据未示出）。 </li><li>此外，避免惊心的成年人，这导致了消散的动作，因此稀释，既精子和卵母细胞。保持精子活性尽可能长，并优化受精率，收集精子尽可能地浓缩。 </li></ol></li><li>保持精子在冰上在1.5ml管中。 </li><li>转移的卵母细胞在过滤ASW到预漂洗35毫米培养皿。 </li><li>转印100-500与先前被拉300-400微米转印巴斯德吸管的卵母细胞，以另外35毫米的培养皿来执行注射。 </li><li>施肥离合器为精子和卵子的质量或其他实验控制的剩余部分。 </li></ol></li><li>卵母细胞注射<ol><li>在50°角相对于水平平面的安装注射针上的显微。 注:小于50°角将围绕卵母细胞的推盘，而超过50°的角度将不允许针位置相对于卵母细胞的的适当监视。 </li><li>下的荧光解剖范围与25X目镜，转移在含有过滤ASW聚赖氨酸包被的培养皿30的卵母细胞用300-400微米转印巴斯德吸管。 </li><li>存入的卵母细胞沿着线进行注射出在一个有序的方式，并区分非注入卵母细胞注射。注入少量（30卵母细胞），以最大限度地减少卵母细胞，以多聚赖氨酸，这往往会变形发育中胚胎的曝光时间（数据未显示）。 </li><li>使用暗场照明以使卵母细胞作为半透明越好。 </li><li>随着显微的粗动旋钮，将注射针接近卵母细胞。 </li><li>用细镊子，切割，其中尖端开始弯曲程度打开针。通过与喷油脉冲，确认红注射混合实际上是流出针。 </li><li>在200X放大倍数，并与微操作的微动旋钮，轻轻移动的卵母细胞的核内的注射针头。 注:如果插入过表面，注入的解决方案将不会留在卵母细胞内。如果插入太远，卵母细胞会被破坏。 </li><li>注射用1-3脉冲120毫秒durat离子与1-10磅的压力。如果针是足够细，注射用连续流在不断的压力。确保注射体积相当于1/5到单个卵母细胞的体积的1/3。 </li><li>注射后，拔出针头出迅速，以避免在卵母细胞的泄漏。 </li><li>验证注入的解决方案仍然是卵母细胞中，而少数秒钟后，注入的解决方案，在整个传播的卵母细胞。 </li><li>继续前进到线下一个卵母细胞。 </li><li>保持每个系列作为阴性对照在扫描注射的胚胎时估计背景荧光的一些未注射的胚胎。 </li></ol></li><li>施肥，选择注射的胚胎和胚胎培养的<ol><li>一旦一个序列已被注射受精的卵母细胞。随着卵母细胞的质量随着时间的推移下降，注入和产卵12后1小时内受精的卵母细胞。 </li><li>根据精子浓度，加1-5滴精子向卵母细胞和漩涡的菜。 注:后约1分钟的施肥信封应该成为胚胎明显。 </li><li>允许胚胎从多聚赖氨酸包被的培养皿分离，同时注入另一系列的卵母细胞。 </li><li>转移的600微米转移巴斯德吸管胚胎成琼脂糖涂层培养皿。如果在所有可能的2细胞期之前尽快除去从多聚赖氨酸包被的培养皿中的胚胎。 注意:如果长时间暴露在聚赖氨酸，胚胎往往成为密集的包装囊胚，扁平的一侧触及盘的底部。 </li><li>在2-细胞至4细胞期，选择用荧光解剖范围与DSR滤波器的成功注射的胚胎， 即，那些具有正常的形态，表现出一个酚红衍生的红色荧光信号。 </li><li>保持在培养的胚胎在过滤ASW琼脂糖包被的培养皿中在19℃，直到所要求的阶段体内成像。 </li></ol></li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+green+fluorescent+protein+(GFP)+in+amphioxus.">Endogenous green fluorescent protein (GFP) in amphioxus.</a> Biol. Bull. 213, 95-100 (2007).</li><li>Schubert, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+signaling+acts+via+Hox1+to+establish+the+posterior+limit+of+the+pharynx+in+the+chordate+amphioxus.">Retinoic acid signaling acts via Hox1 to establish the posterior limit of the pharynx in the chordate amphioxus.</a> Development. 132, 61-73 (2005).</li><li>Schubert, M., Holland, N. D., Laudet, V., Holland, L. 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Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amphioxus+genome+illuminates+vertebrate+origins+and+cephalochordate+biology.">The amphioxus genome illuminates vertebrate origins and cephalochordate biology.</a> Genome Res. 18, 1100-1111 (2008).</li><li>Urasaki, A., Mito, T., Noji, S., Ueda, R., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposition+of+the+vertebrate+Tol2+transposable+element+in+Drosophila+melanogaster.">Transposition of the vertebrate Tol2 transposable element in Drosophila melanogaster.</a> Gene. 425, 64-68 (2008).</li><li>Li, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenesis+at+specific+genomic+loci+of+amphioxus+Branchiostoma+belcheri+using+TALEN+method.">Mutagenesis at specific genomic loci of amphioxus Branchiostoma belcheri using TALEN method.</a> J. Genet. Genomics. 41, 215-219 (2014).</li><li>Zhang, Q. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+culture+of+two+lancelets+and+production+of+the+second+filial+generations+in+the+laboratory.">Continuous culture of two lancelets and production of the second filial generations in the laboratory.</a> J. Exp. Zool. B. 308, 464-472 (2007).</li><li>Yasui, K., Igawa, T., Kaji, T., Henmi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+aquaculture+of+the+Japanese+lancelet+Branchiostoma+japonicum+for+7+years.">Stable aquaculture of the Japanese lancelet Branchiostoma japonicum for 7 years.</a> J. Exp. Zool. B. 320, 538-547 (2013).</li><li>Benito-Guti&#233;rrez, E., Weber, H., Bryant, D. 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The authors have nothing to disclose.

在这篇文章中，我们提出，第一次，详细的和可重复的协议，用于注射的B.卵披针叶 ，其中，经过B. floridae 13,14和B.文昌鱼 15，因此第三文昌鱼物种，其中这样的技术已被描述。重要的是，这里描述的协议也包括载体系统适合于荧光蛋白的B中生产的描述从体外产生的mRNA注入的披 （以下描述）。总之，这些新的工具允许在文昌?...

在开发过程中，单个细胞产生了一个完整的有机体中高度复杂的过程，涉及到两个细胞分裂和运动。为了更好地了解的细胞行为的动力学所依据的生物学原理，发育生物学家已经开始使用基于荧光的体内成像技术。细胞的特定区室，如细 ​​胞膜，可以通过处理来用荧光染料标记，通过组织穿透1的缺乏特异性和阻碍的方法，或由具体引入的编码荧光蛋白2外源性的mRNA胚胎。不同的技术可用于有效递送外源性化合物，如的mRNA。这些包括，但不限于，微注射，电穿孔，轰击与微粒，脂质转染和转导3,4。虽然所有这些方法可以用于引入外源性化合物成发育中的胚胎，只有微量注射允许预定义的和精确的批量应用到每个单元3。显微注射技术已被描述为所有主要发育模型系统4（ 例如 ，果蝇，线虫，斑马鱼，蛙，小鼠），以及对于某些替代模式4中 ，其中包括那些用于旨在了解发育机制的演化比较研究（ 例如 ，海葵，蠕虫环节动物，海胆，海鞘囊中，文昌鱼文昌鱼）。 Cephalochordates，连同被囊动物和脊椎动物建立脊索动物门，是特别适合的模型来研究脊索动物的演变和脊椎动物从脊椎动物的祖先5-8的多样化。在文昌鱼谱系很早脊索动物进化过程中分道扬镳;和现存cephalochordates，其被细分为三个属（ 麸chiostoma，Asymmetron和Epigonichthys），类似于脊椎动物无论是在整体解剖和基因组架构5-8的条款。已经描述到目前为止的约30种cephalochordates的五个可供胚胎和发育研究6,9:Asymmetron lucayanum（巴哈马文昌鱼）， 文昌鱼floridae（佛罗里达文昌鱼）， 文昌鱼披 （欧洲文昌鱼）， 文昌鱼 （中国的文昌鱼） 文昌鱼与日本血吸虫 （日本文昌鱼）。这三个品种的成熟的成年人（B.披针叶，B.文昌鱼和B.血吸虫 ）可诱导产卵按需在繁殖季节10,11。此外，至少对于B.披 ，高效产卵也可诱发在人造海水12，从而使这个特定文昌鱼物种laborator访问IES没有获得天然海水。的结合，在二披针叶 ，一个方便，可靠地获得胚胎具有高效的交付方法，如显微注射，至今在文昌鱼开发（在B. floridae和B.文昌鱼 ）13-15，只交付技术将使的发展新套件的操控技术，包括血统tracing-和动态细胞行为为基础的方法。 一种协议，它的mRNA的显微注射高效表达的B.荧光蛋白披针叶胚胎，因此开发。此外，提供一个基本的工具包B的实时成像披胚胎，载体系统被开发，使膜相关和荧光蛋白的细胞核表达。用于膜靶向，增强型绿色荧光蛋白（EGFP）融合到mCherry和EGFP的人的HRAS CAAX盒和核定位是由融合到斑马鱼组蛋白2B（H2B）外显子（ 图1中 ，补充文件1）中获得。此外，其目标是优化蛋白质的翻译，所述构建体含有Kozak序列和密码子已被修改，并适于使用在二披针叶 。两者合计，这里介绍的注射方法和表达载体将作为新的实验方法为cephalochordates产生的基础，特别是分析采用最新的基于荧光的体内成像技术。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Consumables&lt;/strong&gt;</td></tr><tr><td>35 mm Petri dishes</td><td>Falcon</td><td>353001</td><td>Culture-treated</td></tr><tr><td>Filtration unit (Stericup 1 L)</td><td>Fisher</td><td>W21719</td><td>0.22 &amp;#956;m</td></tr><tr><td>Spin-X tubes</td><td>Costar</td><td>8160</td><td>0.22 &amp;#956;m</td></tr><tr><td>Needle storage jar for 1.2 mm diameter capillaries</td><td>WPI</td><td>E212</td><td></td></tr><tr><td>Pasteur pipettes</td><td></td><td></td><td>230 mm long</td></tr><tr><td>Aspiration tube</td><td>Dutscher</td><td>75056</td><td></td></tr><tr><td>Capillaries for injection needles</td><td>Sutter</td><td>BF 120-94-10</td><td>Borosilicate glass with filament, OD 1.20 mm, ID 0.94 mm, length 10 mm</td></tr><tr><td colspan="4">&lt;strong&gt;Reagents&lt;/strong&gt;</td></tr><tr><td>Low-melting agarose</td><td>Sigma</td><td>A9414</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma</td><td>114537</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G2025</td><td></td></tr><tr><td>Poly-L-lysine hydrobromide</td><td>Sigma</td><td>P9155</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O, DNase/RNase-free</td><td>Gibco</td><td>10977-035</td><td></td></tr><tr><td>mMessage mMachine SP6 Transcription kit</td><td>Ambion</td><td>AM1340</td><td>mRNA synthesis kit</td></tr><tr><td>Phenol pH 8</td><td>Sigma</td><td>P4557</td><td></td></tr><tr><td>24:1 chloroform:isoamylic alcohol</td><td>Sigma</td><td>C0549</td><td></td></tr><tr><td>5:1 phenol pH 4.7:chloroform</td><td>Sigma</td><td>P1944</td><td></td></tr><tr><td>Reef Crystal salts (200 kg)</td><td>Europrix&amp;nbsp;</td><td></td><td>Commercial salts</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S6014</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>Fluorescent dissecting scope with 200X magnification</td><td>Leica</td><td>MZ16F</td><td>25X oculars, DSR and GFP2 filters</td></tr><tr><td>Micromanipulator</td><td>Marzhauzer</td><td>M-33</td><td></td></tr><tr><td>Injector</td><td>Picospritzer</td><td>model II or III</td><td></td></tr><tr><td>Needle puller</td><td>Sutter</td><td>P97</td><td>Heating-filament needle puller</td></tr><tr><td>Fine forceps</td><td>Fine Science Tools GmbH</td><td>11252-30</td><td>Dumont #5</td></tr></tbody></table>

expression of fluorescent proteins in branchiostoma lanceolatum by mrna injection into unfertilized oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

上文详述的协议提供了基础B的显微注射卵母细胞披针叶 ，因此引进来发展B. mRNA的披胚胎编码荧光蛋白用于体内成像。尽管该技术是肯定健壮和可靠的，使用此协议成功注射的速率保持变量（ 表1）。对于这样一个有趣的事实非常有可能的解释是卵母细胞离合器的极端变异性:不同蛋批次确实表现非常不同，当经受的注入压力。有些卵母细胞是相当有?...

Watch this Scientific Journal Video about Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes at JoVE.com

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

我们在这里报告的强大和高效表达荧光蛋白的mRNA注射后进入披文昌鱼的未受精的卵母细胞。显微注射技术在此基础脊索动物的发展铺平道路，在这个新兴的模型系统深远的技术创新，包括在体内成像和基因特异的操作。

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 ...

expression-fluorescent-proteins-branchiostoma-lanceolatum-mrna

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

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

11.0K 次观看.  Institut Pasteur. 我们在这里报告的强大和高效表达荧光蛋白的mRNA注射后进入披文昌鱼的未受精的卵母细胞。显微注射技术在此基础脊索动物的发展铺平道路，在这个新兴的模型系统深远的技术创新，包括在体内成像和基因特异的操作。 

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

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

产妇基因产品的官能化操作使用<em&gt;体外</em斑马鱼&gt;卵母细胞成熟

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

遗传学

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

光片为主的生活的荧光显微镜或固定和染色<em&gt;赤拟谷盗</em&gt;胚胎

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

生物学

<meta charset="utf8"></head><body>使用 CRISPR/Cas9 敲入在斑马鱼幼虫中进行荧光蛋白标记

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

<meta charset="utf8"></head><body>荧光标记以可视化斑马鱼中的低表达蛋白质

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

荧光标记以可视化斑马鱼中的低表达蛋白质

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

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

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. The high transfection efficiency permits at least 4 distinct mRNAs to be co-transfected with ~87% efficiency. Most of the electroporated mRNAs are degraded during the first 2 h post-electroporation, permitting time-sensitive experiments to be carried out in the developing embryo. Finally, we describe how to dynamically image live embryos electroporated with mRNAs that encode various subcellular targeted fluorescent proteins.

We report messenger RNA (mRNA) electroporation as a method that permits fast and efficient expression of multiple proteins in the quail embryo model system. This method can be used to fluorescently label cells and record their in vivo movements by time-lapse microscopy shortly after electroporation.

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. ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

荧光蛋白的表达

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测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

产妇基因产品的官能化操作使用

单分子荧光原位杂交 (SM-FISH) 在小鼠卵母细胞中 Mrna 的定量和定位

光片为主的生活的荧光显微镜或固定和染色

显微注射mRNA和斑马鱼的胚胎吗啉反义寡核苷酸。

荧光标记以可视化斑马鱼中的低表达蛋白质

荧光标记以可视化斑马鱼中的低表达蛋白质

荧光标记以可视化斑马鱼中的低表达蛋白质

使用mRNA电穿孔快速有效地表达Avian胚胎中的多种蛋白质

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像