作者也想感谢熊博士的佐治亚健康科学大学的研究小组制造和分享构建了neogenin的cDNA和shRNA载体。这项研究是由由韩国研究基金会资助的基础科学研究计划（2013R1A1A4A01012572）的赠款和由三育大学的研究资助。

注:动物养殖和关心所有程序均按照三育大学，首尔，韩国的IACUC规定进行。 1.超数排卵，育种和输卵管隔离<ol><li>保持在下列条件下的近交C57BL / 6小鼠:22±3℃，〜60％的湿度，12小时光照/黑暗循环，并在水和食物随意。 </li><li>由5 IU孕马血清促性腺激素（PMSG）的0.1毫升腹膜内注射诱导3-5周龄的雌性小鼠超排卵/头随后48小时后通过腹膜内注射5 IU人绒毛膜促性腺激素（HCG）以0.1ml /头。 </li><li>紧接着注射绒毛膜促性腺激素，通过使这些在同一个笼子隔夜交配一脉相承的性发育成熟的男性超数排卵引起的女性。 </li><li>第二天早晨，通过在女性生殖道的配合插头或阴道栓的存在确认成功交配。 </li><li>牺牲怀孕雌性小鼠通过CO 2中毒后注射HCG后颈椎脱位18-20小时。 </li><li>通过切割皮肤，然后用细剪刀腹膜层打开腹腔。 </li><li>拿起用细镊子子宫角，并从体腔轻轻扯远了子宫，输卵管，卵巢，和脂肪垫。 </li><li>切开输卵管和细剪刀卵巢之间的区域。重新定位钳切输卵管靠近子宫，留下至少1cm附着在子宫的上部。 </li><li>转移输卵管壶腹含M16培养基在室温下35毫米的培养皿。游泳池，距离几个小鼠输卵管在同一个菜。 </li><li>放置菜胚胎提取立体显微镜下。 </li></ol> 2.检索和2原核文化（2-PN）胚胎<ol><li>切断的30或32克皮下注射针的端部，它磨成一个钝头，并把它作为一个冲洗针。 </li><li>使用细镊子输卵管的末端滑入冲洗针头。轻轻按压菜举行到位底部的冲洗针尖。冲洗与输卵管〜0.1毫升M16培养基以释放2-PN胚胎成培养皿。 </li><li>收回2-PN胚胎用无菌玻璃吸管，并将它们传送到一个​​新的培养皿从体视显微镜下的刷新M16中周边群众积云一起。 </li><li>在37℃下在Dulbecco磷酸盐缓冲盐水（DPBS）1.0毫升0.1-0.5％透明质酸酶的解离从2-PN胚胎卵丘细胞通过酶消化5-10分钟。 </li><li>用玻璃吸管和物理去除卵丘细胞轻轻吹打剥夺胚胎。 </li><li>与每个胚胎30-50毫升的新鲜磷酸盐缓冲盐水（PBS）洗洁净胚胎三次。 </li><li>孵育在M16媒体35毫米塑料培养皿补充有10毫克/毫升的牛血清白蛋白（BSA，FRACT胚胎离子V），在37℃在5％CO 2在加湿培养箱直到显微注射制成，其通常小于4小时后。 </li></ol> 3.胚反转录（RT）和聚合酶链反应（PCR） <ol><li>收集来自每个阶段至少10胚胎至胚泡期，并在4.0微升5×逆转录含有逆转录酶，RNA酶抑制剂，寡dT引物，随机6聚体，dNTP混合液酶预混合物溶液池他们，并且在一个反应​​缓冲液PCR管。 </li><li>添加无RNase的蒸馏水2 O至20微升的总体积和超声处理汇集胚胎30秒，在200 W.立即启动RT反应在42℃进行1小时，接着用5分钟温育在95℃。 </li><li>对于每5.0微升产生cDNA溶液，进行正常的PCR扩增与neogenin引物，CDX2，SOX2，Tead4，Nanog的的Oct3 / 4，或β肌动蛋白（引物序列于表 </strong&gt;的1。 PCR包括15秒温育40个循环，在95℃，30秒退火温度（在表1中示出），并在72℃30秒。 </li><li>分离的PCR产物通过电泳在2％的琼脂糖凝胶和可视化在UV光下的凝胶用溴化乙锭染色后。 </li><li>正常化抗β肌动蛋白水平的靶基因的表达。 </li></ol> 4.免疫细胞化学胚胎<ol><li>从与在PBS中的4％多聚甲醛在室温下30分钟，随后简短洗涤3次用含0.01％BSA的PBS每个阶段固定的胚胎。 </li><li>由于1.0ml PBS中含有0.1％吐温20和0.1％的TritonX-100在室温下10分钟温育10胚通透细胞膜。 </li><li>以阻断非特异性结合孵育透胚胎于1.0ml PBS中补充了10％正常山羊血清，在室温下30分钟。 </li><li>孵育阻断胚胎机智在1小时的兔抗neogenin抗体:100稀释在PBS含有0.1％BSA在4℃下过夜。 </li><li>洗涤三次后，每30秒在PBS中滴，在1孵育与任一的Alexa 488缀合的山羊抗兔IgG或Alexa 568缀合的山羊抗兔IgG胚胎1:500稀释在PBS / BSA溶液在4℃过夜。 </li><li>为了显现微丝，孵育在1微克/毫升毒伞素胚在PBS中于室温下1小时。 </li><li>通过加入5毫升含有1微克/毫升的DAPI（4&#39;，6-二脒基-2-苯基二盐酸盐）在室温下5分钟的PBS染色细胞核。 </li><li>洗涤胚胎简要三次，用PBS。 </li><li>转移胚胎载玻片，并与矿物油的液滴覆盖它们。 </li><li>以在1000倍放大影像与适当波长的激发共聚焦显微镜。 </li></ol> 5.准备Neogenin-RFP和Neogenin-GFP的siRNA的质粒<ol><li>子克隆的cDNA编码小鼠neogenin成红色荧光蛋白（RFP）含载体，导致neogenin旗-RFP。 </li><li>亚克隆小发夹RNA靶向neogenin变成郁郁葱葱的绿色荧光蛋白（EmGFP）含载体，产生的pcDNA-EmGFP-MIR-neogenin（ 如图1所示）。 </li><li>既放大neogenin标志-RFP和的pcDNA-EmGFP-MIR-neogenin质粒和与传统方法净化他们。 </li><li>调整的质粒的终浓度至〜1.0微克/毫升的无菌的Tris-EDTA（TE）缓冲液。 </li></ol> 6.显微注射质粒的成2的PN胚胎<ol><li>洗洁净2-PN胚胎一次，简单地说，用新鲜的M16媒体。 </li><li>离心2-PN胚胎10分钟在一个台式离心机1000 xg离心以允许原核的观察。 </li><li>孵育在矿物油下20至30微升每胚胎的M16介质的微滴将胚在37℃下在5％CO 2的一个additiOnal地区1-2小时。 </li><li>拉硼硅玻璃毛细管制造注射移液器和移液器控股（OD = 1.2毫米; ID R = 0.94毫米），机械拉马。 </li><li>制造注射移液器拥有使用microgrinder和microforger钝头和&lt;500微米的尖端长度和1-2微米的尖端内径抛光开口。 </li><li>制造保​​持移液器使得它们具有钝头和抛光开口为20μm的内径为100微米的外径。 </li><li>在1.0微克/微升的负载注射移液器与足够量（&gt; 200 NL）的质粒溶液。 </li><li> Microinject〜10 PL的每个2-PN胚胎的质粒溶液成利用安装到机动显微一个微量的原核中的一个;在此过程中，通过施加负压力以保持吸管保持胚胎在适当的位置。 </li><li>显微注射后，洗2-PN胚胎3次，新鲜的M16媒体对于LESS比每次1分钟。 </li><li>文化2-PN胚胎新鲜M16媒体对由矿物油下降，以防止媒体蒸发覆盖塑料培养皿下降。 </li><li>在一个下微分干涉对比24小时的时间间隔（DIC）在200倍倒置显微镜下观察胚胎。 </li></ol>

<ol>
	<li>Yamanaka, Y., Ralston, A., Stephenson, R. O., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+molecular+regulation+of+the+mouse+blastocyst.">Cell and molecular regulation of the mouse blastocyst.</a> Dev. Dyn. 235 (9), 2301-2314 (2006).</li><li>Sasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+trophectoderm+fate+specification+in+preimplantation+mouse+development.">Mechanisms of trophectoderm fate specification in preimplantation mouse development.</a> Dev. Growth Differ. 52 (3), 263-273 (2010).</li><li>Nishioka, N., Yamamoto, S., Kiyonari, H., Sato, H., Sawada, A., Ota, 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=Tead4+is+required+for+specification+of+trophectoderm+in+pre-implantation+mouse.">Tead4 is required for specification of trophectoderm in pre-implantation mouse.</a> Mech. Dev. 125 (3-4), 270-283 (2008).</li><li>Ovitt, C. E., Sch&#246;ler, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+Oct-4+in+the+early+mouse+embryo.">The molecular biology of Oct-4 in the early mouse embryo.</a> Mol. Hum. Reprod. 4 (11), 1021-1031 (1998).</li><li>Stein, P., Schindler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Oocyte+Microinjection,+Maturation+and+Ploidy+Assessment.">Mouse Oocyte Microinjection, Maturation and Ploidy Assessment.</a> J. Vis. Exp. (53), e2851 (2011).</li><li>Shin, M. R., Cui, X. S., Jun, J. H., Jeong, Y. J., Kim, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mouse+blastocyst+genes+that+are+down+regulated+by+double-stranded+RNA-mediated+knockdown+of+Oct-4+expression.">Identification of mouse blastocyst genes that are down regulated by double-stranded RNA-mediated knockdown of Oct-4 expression.</a> Mol. Reprod. Dev. 70 (4), 390-396 (2005).</li><li>Khang, I., Sonn, S., Park, J. -. H., Rhee, K., Park, D., Kim, K. <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+epithin+in+mouse+preimplantation+development:+Its+role+in+compaction.">Expression of epithin in mouse preimplantation development: Its role in compaction.</a> Dev. Biol. 281, 134-144 (2005).</li><li>Prieto, D., Aparicio, G., Machado, M., Zolessi, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+DNA-Specific+Stain+Methyl+Green+in+the+Fluorescent+Labeling+of+Embryos.">Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos.</a> J. Vis. Exp. (99), e52769 (2015).</li><li>Lee, J. H., Choi, S. S., Kim, H. W., Xiong, W. C., Min, C. K., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neogenin+as+a+receptor+for+early+cell+fate+determination+in+preimplantation+mouse+embryos.">Neogenin as a receptor for early cell fate determination in preimplantation mouse embryos.</a> PLoS One. 9 (7), e101989 (2014).</li><li>Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A., Ruddle, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+mouse+embryos+by+microinjection+of+purified+DNA.">Genetic transformation of mouse embryos by microinjection of purified DNA.</a> Proc. Natl. Acad. Sci. USA. 77 (12), 7380-7384 (1980).</li><li>Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K., Palmiter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+efficiency+of+introducing+foreign+DNA+into+mice+by+microinjecting+eggs.">Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.</a> Proc. Natl. Acad. Sci. USA. 82, 4438-4442 (1985).</li></ol>

作者有没有竞争经济利益透露。

在本协议中，我们证明遗传物​​质注射的新方法，无论是基因或shRNA，进入2-PN小鼠胚胎小鼠胚胎发育过程中，探索neogenin在早期细胞分化的作用。植入前胚胎的遗传修饰是在揭示的标的分子机制，例如，第一细胞谱系判定密钥信息的强大技术。遗传修饰是为确定基因功能最常用的方法之一。具有相对大的承受力的2-PN胚胎外源遗传物质和cDNA和shRNA的显微注射的可行性入2-PN胚胎的，这种胚胎操作技术可以用最少的物理破坏来实现对胚胎，更不用说本征与此遗传操作相关的互补性。 考虑到细胞质注入更容易，更有害生存比核注射，但尽管如此，较高的表达与核注射相关的外源DNA，一种改进应强调的是采用离心的显微注射前到原核定位到细胞核。技术上最复杂和苛刻的步骤是外源DNA显微注射到受精卵的原核。这一步很关键，需要大量的技术熟练程度和广泛的培训，以掌握显微注射过程。一旦这个技能是后天然而，技术的变化和错误变得微乎其微。显微注射胚胎的成活率常规在我们的工厂80％-90％之间。 与此不同的研究，其他研究人员握住吸管，使细胞膜附近的原核接近微针定位胚胎。然后微针插入原核10,11。我们遇到了一些困难，可视化的亲不正确的离心显微镜下细胞核。显然，离心使细胞核接近质膜为更好地识别。同时，应该有一定的提升空间的原核更好的可视化从而更好的定位。 相反，转基因小鼠的生产，我们的质粒显微注射成胚胎协议本质上是一个瞬时转染，因而适合于植入前胚胎发育过程中基因的解剖功能。因此，我们的一个损失 - 协议和增益的函数的技术可以进一步推广到识别信令参与早期胚胎发育的分子。 此外，neogenin信令可能是给定的胚胎干细胞系的开发非常有益的neogenin及其配体有可能提高干细胞特异性转录因子如的Oct3 / 4，Sox2和Nanog的。

植入前胚胎发育可以分成几个不同的阶段，从单细胞阶段到桑椹胚和胚泡。多的证据表明，极性和位置线索发挥早期细胞命运决定了作用到滋养外胚层（TE）和内细胞团（ICM）。然而，线索，它们是如何变换成蜂窝信号的性质，并在生理环境中，这样的信号是能够引发细胞谱系分化是不知道的。这些分化的细胞，然后进行专门化，开始采取所需的胎盘1-3的胚胎适当形成和生长特性结构和功能。 植入前胚胎是通过直接注射像siRNA或靶cDNA改性遗传物质特别适合于操纵，因此可用于研究特定的目标分子。人们越来越认识到遗传脊椎动物的胚胎的分析是推动我们的胚胎发育4的了解至关重要。精确引入野生型基因或突变形式的成胚胎及时上下文来完成GAIN-或失功能的基因的大大促进的发育调节的基因的研究。损失-并通过遗传物质为单独的早期非哺乳动物和哺乳动物胚胎显微注射获得性功能比较简单，因为其庞大的规模和巨大的接受5。显微注射，使用非病毒载体通常进行。特别的优点已采取便于使用非病毒载体在病毒载体和转基因的。在小鼠胚胎的快速损失-或增益的功能表达系统已经开发出来，让我们来剖析和理解脊椎动物胚胎6,7基因的功能。 胚胎的荧光标记将大大促进MICR的选择oinjected或遗传操作的胚胎，并在同一时间给予间接量化基于荧光强度8显微注射siRNA或cDNA的表达水平的装置。为了完成这个标签，荧光蛋白基因，GFP或RFP，是共注射如任一融合构建或单独。 GFP或RFP的荧光强度揭示siRNA或外源DNA的表达的程度，以确保损失 - 或增益的功能已经完成。 在这里，我们提出了损失 - 而获功能的技术，使我们能够确定neogenin作为继电器外线索早期细胞分化的重要植入前小鼠胚胎的受体的显微协议。

<table border="0" cellpadding="0" cellspacing="0" width="1507">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">M16 medium,</td>
			<td style="width:519px;">Gibco BRL (Grand Island, NY)</td>
			<td style="width:232px;">M7292 LOT# 11A832</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Goat serum,&#160;</td>
			<td style="width:519px;">Dako (Glostrup, Denmark)</td>
			<td style="width:232px;">X0907</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">PMSG</td>
			<td>Folligon, Intervet, Holland</td>
			<td style="width:232px;">G4877-1000IU LOT#SLBD0719V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Mineral olie</td>
			<td style="width:519px;">Sigma Aldrich</td>
			<td style="width:232px;">CG5-1VL LOT# SLBC6783V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">human chorionic gonadotropin and pregnant mare serum gonadotropin&#160;</td>
			<td style="width:519px;">(Intervet, Holland)</td>
			<td style="width:232px;">(invitrogen , 15596-026)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">SuperScript&#174; III Reverse Transcriptase</td>
			<td style="width:519px;">lifetechnologies</td>
			<td style="width:232px;">18080-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">PCR pre mixture</td>
			<td style="width:519px;">&#160;(Bioneer, Daejon, S. Korea)</td>
			<td style="width:232px;">GenDEPOT , A0224-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Agarose (molecular grade)</td>
			<td style="width:519px;">BioRad</td>
			<td style="width:232px;">161-3101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Paraformaldehyde Solution, 4% in PBS</td>
			<td style="width:519px;">Affymetrix USA</td>
			<td style="width:232px;">19943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">polyclonal rabbit anti-neogenin antibody</td>
			<td style="width:519px;">Santa Cruz Biotechnology, US</td>
			<td style="width:232px;">SC-15337</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Alexa fluor 488 labeled anti-rabbit antibody</td>
			<td style="width:519px;">Molecular Probes (Invitrogen, USA)</td>
			<td style="width:232px;">A-11094</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Alexa fluor 555 labeled anti-rabbit antibody</td>
			<td style="width:519px;">Molecular Probes (Invitrogen, USA)</td>
			<td style="width:232px;">A-21428</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Alexa Fluor&#174; 555 Phalloidin</td>
			<td style="width:519px;">Molecular Probes (Invitrogen, USA)</td>
			<td style="width:232px;">A34055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:519px;">Invitrogen, USA</td>
			<td style="width:232px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Recombinant Human RGM-C/Hemojuvelin</td>
			<td style="width:519px;">R&#38;D systems</td>
			<td style="width:232px;">3720-RG</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:496px;">all plasmid constructs&#160;</td>
			<td style="width:519px;">Prof. Wen Cheng Xiong at Georgia Health Sciences University (Agusta, GA).</td>
			<td style="width:232px;"></td>
			<td>the present studies were kindly provided by&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Neon&#174;&#160; Transfection System</td>
			<td style="width:519px;">lifetech</td>
			<td style="width:232px;">MPK10096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Stereo Microscrope</td>
			<td style="width:519px;">Nikon</td>
			<td style="width:232px;">SMZ1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Micromainpulation system with Nikon</td>
			<td style="width:519px;">Nikon</td>
			<td style="width:232px;">Narishige ONM-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">MicroInjector</td>
			<td style="width:519px;">Nikon Narishige</td>
			<td style="width:232px;">GASTIGHT #1750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Holder</td>
			<td style="width:519px;">Nikon Narishige</td>
			<td style="width:232px;">Narishige IM 16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Microfoge</td>
			<td style="width:519px;">Nikon Narishige</td>
			<td style="width:232px;">Narishige MF-900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">grinder</td>
			<td style="width:519px;">Narishige</td>
			<td style="width:232px;">EG400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Puller</td>
			<td style="width:519px;">Sutter Instrumnet company</td>
			<td style="width:232px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">CO2 incubator</td>
			<td style="width:519px;">Thermo Scientific Forma</td>
			<td style="width:232px;">EW-39320-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:496px;">Veriti&#174; 384-Well Thermal Cycler&#160;</td>
			<td style="width:519px;">lifetechnologies</td>
			<td style="width:232px;">4388444</td>
			<td></td>
		</tr>
	</tbody>
</table>

loss- and gain-of-function approach to investigate early cell fate determinants in preimplantation mouse embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

我们发现，neogenin期间植入前的小鼠胚胎的早期发育阶段瞬时表达，早在2-细胞阶段和持久直到早期桑椹胚出现但成为缺陷在后期桑椹胚和胚泡阶段（ 图2A）。此外，neogenin的空间分布被限制主要是细胞外。 RT-PCR分析的结果与neogenin表达的早期和短暂性质相符，在4细胞期峰值，但在16个细胞阶段完全缺乏或更高（ 图2B）。与此相反，F-肌动蛋白没有发现这样的瞬态和偏振表达模式。 接下来我们调查是否neogenin水平既可以由neogenin基因载体（neogenin增益）或载体窝藏shRNA的目标neogenin的注射来操纵（neogenin洛杉矶多个）到2-PN受精卵。在视觉上区分neogenin损失，RFP neogenin增益和GFP共表达作为指标（ 图3），以及neogenin表达水平将所得物都通过免疫荧光和免疫印迹（ 图4）确认。 图5在2 PN阶段接收要么neogenin的shRNA或neogenin的cDNA后示出了在每一阶段的胚胎的代表性图像。有neogenin损失和neogenin增益胚胎，并至少直到胚泡阶段（ 表2）没有发育电位差之间没有明显的大体形态学差异。实际上，在每一个阶段存活的胚胎的数量和被捕胚胎数没有显著不同。 但是，ICM发展neogenin损失不太明显比neogenin增益为胚胎由ICM-特定的Oct3 / 4在neogenin增益胚胎（ 图6A）的较高的表达和由每胚泡的Oct3 / 4阳性的ICM细胞neogenin增益比neogenin损失胚胎（ 图6B）的数字较高证明的。此外，RT-PCR分析揭示了三种转录因子的表达水平以及neogenin的之间有很强的相关性。在neogenin损失胚泡，小的Oct3 / 4，Sox2和Nanog的检测，这是在形成鲜明对比，在neogenin增益超过对照胚胎（ 图6C）所有三种转录因子的表达的显著增加。出乎意料的是，CDX2和Tead4的表达水平，在TE分化/维护6,7-牵连转录因子，分别少由neogenin的表达水平的影响，证实其上调neogenin的导致特异于ICM转录调节而激活不是为TE建立。所有显示的数据分别为m<html>从参考9 odified。 <img alt="图1" src="/files/ftp_upload/53696/53696fig1.jpg" /> 图1:结构的pcDNA-EmGFP-MIR-neogenin。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53696/53696fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53696/53696fig2.jpg" /> 图2:Neogenin的表达在小鼠胚胎发育 （A）在植入前胚胎不同发育阶段neogenin表达的共聚焦显微图像。 （B）中的反转录聚合酶链neogenin mRNA在植入前胚胎在不同发育阶段的反应。 β肌动蛋白被用来作为内部对照。96fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图3" src="/files/ftp_upload/53696/53696fig3.jpg" /> 图 3: 绿色荧光蛋白（GFP）和红色荧光蛋白（RFP）表达作为指标分别Neogenin损耗和Neogenin增益，窝藏neogenin基因载体窝藏RFP成共轭GFP或注射一neogenin靶向的shRNA载体显微注射后2-PN合子，GFP和RFP的表达在2细胞和4-细胞阶段在荧光显微镜下观察。左侧面板中，相衬图像;中间和右侧面板，荧光图像。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53696/53696fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4"SRC ="/文件/ ftp_upload / 53696 / 53696fig4.jpg"/&gt; 图4:Neogenin的在囊胚表达的任shRNA的靶向Neogenin或Neogenin的cDNA微注射后 （A）一种neogenin靶向shRNA的载体的显微注射到2-PN受精卵后，在个人胚泡细胞neogenin的表达水平通过评价用抗Flag抗体的免疫染色。在左侧面板，炒neogenin的shRNA载体显微注射（控制）。在右侧面板中，neogenin靶向的shRNA载体显微注射。 DAPI，DAPI（蓝色）被用来染色核;反旗，上neogenin国旗标记的可视化（红色）;绿色荧光蛋白，绿色荧光蛋白（绿色）;合并，DAPI，抗旗，和GFP的叠加。 （B）的囊胚全细胞裂解物与抗旗抗体免疫印迹。 GFP用作上样对照。炒的shRNA，炒neogenin的shRNA注射液;吴的shRNA，neogenin靶向的shRNA injec<html>化。 （C）中neogenin cDNA的载体或neogenin靶向shRNA的载体注射到2-PN合子，所得囊胚经受用抗neogenin抗体免疫染色来测量neogenin表达水平。在上面板，neogenin靶向shRNA的注入。囊胚用抗neogenin抗体（红色）免疫染色。绿色荧光蛋白，绿色荧光蛋白（绿色）;合并，DAPI，抗neogenin，和GFP的叠加。在下部面板，neogenin cDNA的载体显微注射。囊胚用抗neogenin抗体（绿色）进行免疫染色。 DAPI，DAPI核染色（蓝色）; RFP，红色荧光蛋白（红色）;合并后，DAPI，RFP，反neogenin的叠加。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53696/53696fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> PLOAD / 53696 / 53696fig5.jpg"/&gt; 图5:Neogenin损失或收益胚胎发育的影响 。 2-PN小鼠胚胎显微注射或者与小发夹RNA（shRNA）靶向neogenin（neogenin损失），或与载体窝藏neogenin的cDNA（neogenin增益）并且是直到达到胚泡期进行培养。区分neogenin增益胚胎neogenin损失，GFP和RFP被共表达，分别。相差在不同发育阶段的胚胎的图像显示， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53696/53696fig5large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/53696/53696fig6.jpg" /> 图6:Neogenin表达费沃斯ICM分化 （A）ICM细胞囊胚被免疫染色的Oct3 / 4观察。 DAPI用于站在细胞核中。 （B）中的Oct3 / 4阳性的ICM细胞的对照，neogenin损耗和neogenin增益胚胎的胚泡的数量被表示为平均值±SEM来自三个独立实验，在每个实验中所用的至少10胚胎。 *由学生t检验与对照的显着差异在P &lt;0.05。从neogenin损失，neogenin增益和对照胚胎的囊胚的Oct3 / 4，SOX2，Nanog的，CDX2和Tead4的mRNA（C）的RT-PCR分析。 β-actin作为内部控制。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53696/53696fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/53696/53696table1.jpg" /> 表1:用于RT-PCR的引物和PCR条件的序列。 <img alt="表2"SRC ="/文件/ ftp_upload / 53696 / 53696table2.jpg"/&gt; 表2:生存小鼠胚胎在每个发展阶段接收Neogenin的shRNA或Neogenin基因在2-PN阶段之后的数目。

Watch this Scientific Journal Video about Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos at JoVE.com

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

该协议的目的是描述一个损失 - 和增益的是适用于确定neogenin作为通向滋养外胚层和中植入前小鼠胚胎内细胞团的分化阶段特异性受体功能的方法。

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

loss-gain-function-approach-to-investigate-early-cell-fate

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.7K 次观看.  Dankook University. 该协议的目的是描述一个损失 - 和增益的是适用于确定neogenin作为通向滋养外胚层和中植入前小鼠胚胎内细胞团的分化阶段特异性受体功能的方法。 

视频: Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

方法细胞或DNA的精确本地化转移到着床后早期小鼠胚胎

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

科研

发育生物学

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

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

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 ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

三维高频超声对小鼠早期妊娠特征的新应用

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result in a series of bone metabolic diseases, including osteoporosis. To develop pharmaceutical targets for the prevention of pathological bone mass loss, the mechanisms by which osteoclasts differentiate from precursors must be understood. The ability to isolate and culture a large number of osteoclasts in vitro is critical in order to determine the role of specific genes in osteoclast differentiation. Inactivation of the mammalian/mechanistic target of rapamycin complex 1 (TORC1) in osteoclasts can decrease osteoclast number and increase bone mass; however, the underlying mechanisms require further study. In the present study, a RANKL-based protocol to isolate and culture osteoclasts from mouse bone marrow and to study the influence of mTORC1 inactivation on osteoclast formation is described. This protocol successfully resulted in a large number of giant osteoclasts, typically within one week. Deletion of Raptor impaired osteoclast formation and decreased the activity of secretory tartrate-resistant acid phosphatase, indicating that mTORC1 is critical for osteoclast formation.

This manuscript describes a protocol to isolate and culture osteoclasts in vitro from mouse bone marrow, and to study the role of the mammalian/mechanistic target of rapamycin complex 1 in osteoclast formation.

RANKL 小鼠骨髓破骨细胞培养法研究 mTORC1 在破骨细胞形成中的作用

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

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

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

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 ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...