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>

<ol>
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L., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+achaete-scute+homolog+3+in+Xenopus+embryos+converts+ectodermal+cells+to+a+neural+fate.">Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate.</a> Genes Dev. 8, 1434-1447 (1994).</li><li>Nakagawa, S., Niimura, Y., Gojobori, T., Tanaka, H., Miura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+preferred+nucleotide+sequences+around+the+translation+initiation+codon+in+eukaryote+genomes.">Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes.</a> Nucleic Acids Res. 36, 861-871 (2008).</li><li>Nakamura, Y., Gojobori, T., Ikemura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Codon+usage+tabulated+from+international+DNA+sequence+databases:+status+for+the+year+2000.">Codon usage tabulated from international DNA sequence databases: status for the year 2000.</a> Nucleic Acids Res. 28, 292 (2000).</li><li>Deheyn, D. 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. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+retinoic+acid-Hox+hierarchy+controls+both+anterior/posterior+patterning+and+neuronal+specification+in+the+developing+central+nervous+system+of+the+cephalochordate+amphioxus.">A retinoic acid-Hox hierarchy controls both anterior/posterior patterning and neuronal specification in the developing central nervous system of the cephalochordate amphioxus.</a> Dev. Biol. 296, 190-202 (2006).</li><li>Yu, J. K., Holland, N. D., Holland, L. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+expression+of+FoxD+reporter+constructs+in+amphioxus+embryos.">Tissue-specific expression of FoxD reporter constructs in amphioxus embryos.</a> Dev. Biol. 274, 452-461 (2004).</li><li>Beaster-Jones, L., Schubert, M., Holland, L. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cis-regulation+of+the+amphioxus+engrailed+gene:+insights+into+evolution+of+a+muscle-specific+enhancer.">Cis-regulation of the amphioxus engrailed gene: insights into evolution of a muscle-specific enhancer.</a> Mech. Dev. 124, 532-542 (2007).</li><li>Holland, L. 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. V., Arendt, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+generating+year-round+access+to+amphioxus+in+the+laboratory.">Methods for generating year-round access to amphioxus in the laboratory.</a> PLoS One. 8, e71599 (1371).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1009">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Name of Material/ Equipment</td>
			<td style="width:156px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td style="width:413px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Consumables</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Petri dishes</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>culture-treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Filtration unit (Stericup 1L)</td>
			<td style="width:156px;">Fisher</td>
			<td style="width:143px;">W21719</td>
			<td>0.22 micron filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Spin-X tubes</td>
			<td style="width:156px;">Costar</td>
			<td style="width:143px;">8160</td>
			<td>0.22 micron filtration tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Needle storage jar for 1.2 mm diameter capillaries</td>
			<td style="width:156px;">WPI</td>
			<td style="width:143px;">E212</td>
			<td style="width:413px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Pasteur Pipettes</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td>230 mm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Aspiration tube</td>
			<td style="width:156px;">Dutscher</td>
			<td style="width:143px;">75056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Capillaries for injection needles</td>
			<td style="width:156px;">Sutter</td>
			<td style="width:143px;">BF 120-94-10</td>
			<td>Borosilicate glass with filament, OD 1.20 mm, ID 0.94 mm, length 10 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Reagents</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Low-melting agarose</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">A9414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phenol Red</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">114537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glycerol</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">G2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Poly-L-Lysine hydrobromide</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P9155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">H2O Dnase, Rnase-free</td>
			<td style="width:156px;">Gibco</td>
			<td style="width:143px;">10977-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">mMessage mMachine SP6 Transcription kit</td>
			<td style="width:156px;">Ambion</td>
			<td style="width:143px;">AM1340</td>
			<td>mRNA synthesis kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phenol pH8</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P4557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">24:1 chloroform:isoamylic alcohol</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">C0549</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">5:1 phenol pH4.7:chloroform</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P1944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Reef Crystal salts (200 kg)</td>
			<td style="width:156px;">Europrix&#160;</td>
			<td style="width:143px;"></td>
			<td>Commercial salts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Equipment</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Fluorescent dissecting scope with 200x magnification&#160;</td>
			<td style="width:156px;">Leica</td>
			<td style="width:143px;">MZ16F</td>
			<td>25x oculars, DSR and GFP2 filters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Micromanipulator</td>
			<td style="width:156px;">Marzhauzer</td>
			<td style="width:143px;">M-33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Injector</td>
			<td style="width:156px;">Picospritzer</td>
			<td style="width:143px;">model II or III</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Needle puller</td>
			<td style="width:156px;">Sutter</td>
			<td style="width:143px;">P97</td>
			<td>heating-filament needle puller</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">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

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

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

操作和<em&gt;在体外</em中&gt;成熟<em&gt;非洲爪蟾</em&gt;卵母细胞，其次是胞浆内单精子注射，研究胚胎发育

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

科研

发育生物学

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

通过注射心脏毒素急性骨骼肌再生的诱导

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

免疫荧光标记的微管和中心蛋白在体内肠道组织和 3D体外肠道 Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

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. ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls are composed of cellulose and non-cellulosic polysaccharides together with proteins, phenolic compounds and water. 90% of the cell wall is composed of&#160;polysaccharides (e.g., pectins) and arabinogalactan proteins (AGPs). The fluorescent immunolocalization of specific glycan epitopes in plant histological sections remains a key tool to uncover remodeling of wall polysaccharide networks, structure and components.
Here, we report an optimized fluorescent immunolocalization procedure to detect glycan epitopes from AGPs and pectins in plant tissues. Paraformaldehyde/glutaraldehyde fixation was used along with LR-White embedding of the plant samples, allowing for a better preservation of the tissue structure and composition. Thin sections of the embedded samples obtained with an ultra-microtome were used for immunolocalization with specific antibodies. This technique offers great resolution, high specificity, and the chance to detect multiple glycan epitopes in the same sample. This technique allows subcellular localization of glycans and detects their level of accumulation in the cell wall. It also permits the determination of spatio-temporal patterns of AGP and pectin distribution during developmental processes. The use of this tool may ultimately guide research directions and link glycans to specific functions in plants. Furthermore, the information obtained can complement biochemical and gene expression studies.

This protocol describes in detail how the plant material for immunolocalization of Arabinogalactan proteins and pectins is fixed, embedded in a hydrophilic acrylic resin, sectioned and mounted on glass slides. We show cell wall related epitopes will be detected with specific antibodies.

植物组织细胞壁中阿拉比诺格拉坦蛋白和皮克廷的荧光免疫化

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls ...

Minimum Volume Vitrification of Immature Feline Oocytes

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

未成熟费林卵母细胞的最小体积玻璃化

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in zebrafish.

斑马鱼卵巢卵巢中卵泡细胞和卵母细胞的分离

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

分离大鼠脂肪组织间充质干细胞以分化为产生胰岛素的细胞

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

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基因转移到鸡听觉器官通过