这项工作是在国家儿童健康和人类发展研究所的一笔赠款5R21HD080684-02 的支持下进行的。作者希望感谢肯町持续的热情和支持。作者还要感谢布鲁斯布伦伯格使用他的相机, 利百利恰, 为关键的阅读手稿, 和肖恩麦克纳马拉和马辛 Wlizla 在国家爪资源 (RRID:SCR_013731), 为宝贵关于十酵母喂养和动物护理方案的对话。

这里描述的所有方法都得到了加州大学欧文分校动物保育和使用委员会的批准。
1. PGC 移植的准备
<ol><li>预先准备好解剖工具, 如前所述23。 注意: 眉毛刀是用来做切口的协助下的头发环, 以稳定的胚胎, 而执行手术。眉毛和头发圈被粘入硼硅酸盐玻璃管, 首先被绘制在一个火焰和破碎的部分 (参见图 1A, 箭头), 以创建一个更短, 更窄的提示 (图 1B), 以提高灵活性做手术的时候</li>
	<li>使用以前描述的24的过程生成 sgRNAs。 注: 简单地说, 短 DNA 模板编码为 sgRNAs 是创建使用重叠 deoxyoligonucleotides 含有 5 ' 噬菌体 T7 促进剂, 这是退火和 "填补" 的无差错, 耐热的 DNA 聚合酶。体外sgRNA 合成反应在一夜之间孵育数小时 (视所用试剂盒而定)。反应是 dnasei 处理删除模板。根据试剂盒制造商的说明24, 用苯酚/氯仿萃取和醋酸铵/异丙醇沉淀的标准方法纯化 sgRNAs。</li>
	<li>在执行 microinjections 前不久, 先在3µL 核糖核酸 (diethylpyrocarbonate 处理) H2O 在60-65 ° c 为 5 min, 然后在冰上快速冷却5分钟, 准备 Cas9-sgRNA 配合物的第一个变性〜 250 ng sgRNA。离心 sgRNA 几秒钟, 增加1µL 1 µg/µL Cas9 蛋白, 并在37° c 孵育10分钟, 以促进复杂的形成。 注意: 在这个孵化之后, 试管可以在准备胚胎的时候保持在冰上进行微注射。</li>
	<li>使用标准方法 (如前面描述的25), 通过体外获取x 酵母胚胎。De 果冻25,26胚胎在10分钟后受精。 注: 受精时间被认为是在精子悬液添加到卵子后的时间, 而盘子被1/9 的铃声 (MMR)26。对于 x酵母25, 与 x蟾不同, 执行 de jellying 包含3% 半胱氨酸释放基 (不HCl 盐), pH 值调整为 7.8-8.0, 然后在1/9 个 mmr 中进行多次洗涤。将胚胎移植到琼脂糖 (1%) 包覆的盘中, 室温下含有 1/9 x MMR。</li>
	<li>立即将胚胎移植到1% 个含有 1x MMR 的琼脂糖涂层盘中, 使用巴斯德吸管。</li>
	<li>
在1细胞阶段注射。有关爪微注射方法的详细说明, 请参阅参考26,27 。
	<ol>
<li>在动物极中的单个部位注入每个胚胎, 用 4 nL Cas9-sgRNA 复合物 (最后数量 = 1 ng Cas9 和 ~ 250 pg sgRNA; 请参见讨论)24。在10-20 分钟的注射部位愈合后, 将胚胎移植到含有 1/9 x MMR 的琼脂糖包衣皿中, 并在25° c 孵育。还创造了一碟 uninjected 的同胞胚胎, 将作为移植受体。</li>
	</ol>
</li>
	<li>
如果在x 酵母中进行移植, 或在x 蟾中进行 1x mmr, 则准备在 0.3x MMR 中制作的含有5毫米厚1% 琼脂糖的培养皿 (60 mm)。
	<ol>
<li>通过插入一个 3 x 4 井模 (通过切割 96-好的 PCR 板) 到熔融的琼脂糖浇注板 (见图 1C和 D), 创建凹陷〜3-4 毫米深。在培养皿底部的几毫米以上, 用一个固定在环架上的夹具, 直到琼脂糖硬化。</li>
		<li>此外, 通过在 1/9 x MMR 中涂上薄薄的1% 琼脂糖, 制作一个24孔的钢板来植入单个胚胎。 注: 添加到这些水井中的培养基为 1/9 x MMR, 50 µg 硫酸庆大霉素/毫升。</li>
	</ol>
</li>
</ol>2. 生殖细胞移植
<ol><li>当胚胎刚刚到达 Nieuwkoop 和费伯28囊胚阶段 9 (图 2A), 〜 4.5 h 后受精 (hpf) 为x 酵母, 把它们从25° c 孵化器, 并允许他们到平衡到室温的额外0.5 小时</li>
	<li>要开始移植在 5 hpf (图 2B), 使用巴斯德吸管转移一个 PGC 供体胚胎 (注入 Cas9-sgRNA) 到60毫米琼脂糖盘含有凹陷 (创建在步骤 1.7) 0.3x mmr (或 1x mmr, 如果使用x 蟾)。也转移一个 uninjected 同胞胚胎, 移植受体, 这道菜。 注意: 这是至关重要的, 每个胚胎的身份不混淆。</li>
	<li>使用镊子手动取出每个胚胎的卵黄信封, 并旋转两个胚胎, 使它们的植物极在视野内, 便于手术。 注: 关于人工胚胎 vitellination 的描述, 以前曾被描述为26。</li>
	<li>使用眉毛刀的尖锐尖端, 使四浅切口在一个正方形的形状在接受胚胎的植物极, 在区域里面未来瓶细胞标记 blastopore 将形成。通过将眉毛刀的尖端插入胚胎中, 就在表面下方, 使切口向上切片, 同时用发圈稳定胚胎。 注意:图 2显示了一个胚胎的植物性视图, 每隔半小时间隔从 4.5-7.0 hpf 显示细胞大小, 并提供一个指南, 用以估计瓶子细胞将形成的位置 (空心圆圈)。其目的是使切口, 在虚线黑框的指示。</li>
	<li>一旦正方形的四边被切口划定, 用类似的切削动作加深每个切口与眉毛刀, 达到大约1/3 到1/2 的距离到囊胚地板的深度。</li>
	<li>通过在深部植物区进行水平 (与植物表面平行) 切割, 从受体胚 (图 3A和 B) 中释放植物组织外植体。 注: 含 PGC 的植物外植体的大小约为每边 0.4-0.45 毫米, 深度为 0.25-0. 3 毫米。这种植物外植体从受体胚不再需要, 因此应预留放弃。</li>
	<li>快速工作, 重复这个过程 (步骤 2.4-2.6) 的 PGC 捐赠胚胎, 以创建一个类似大小的植物组织片段的移植。 注意: 重要的是要进行这第二次解剖, 很少延迟, 以减少时间为受体胚胎愈合其开放性伤口。</li>
	<li>一旦含有生殖细胞的组织从供体胚胎中移除, 使用眉毛刀和毛发环将这种外植体移入在受体胚中创建的开口处。 注: 供体移植的内表面必须面对受体胚的内部。这是没有必要匹配的方向, 背腹和左右轴的移植与受体胚胎。</li>
	<li>使用眉毛刀轴的长边, 与嫁接的表面平行, 轻轻地将嫁接到受体胚的植物表面。</li>
	<li>一旦移植被放置, 使用 hairloop 或眉毛刀的轴轻轻滑动的 "胴体" 的施主胚胎在琼脂糖表面和琼脂糖抑郁症。确保胴体的开放性伤口朝向散装液体。如果没有, 用眉毛刀旋转胚胎以达到这个方向。</li>
	<li>滑动受体的胚胎, 随着移植愈合到位, 进入一个相邻的抑郁症, 同样确保嫁接的植物极组织面对散装液体。</li>
	<li>重复此过程 (步骤 2.1-2.11) 使用另一对捐赠者和受体胚胎, 以创建另一个移植/胴体对;将这些转换为空的凹陷。 注意: 这是至关重要的, 一个做记号, 以保持跟踪配对的胴体和移植轴承胚胎。在移植的生殖细胞中, 供体的胴体将被用作 Cas9-sgRNA-induced 诱变的效率的替代品, 在移植的动物达到性成熟之前不能轻易测定 (见下面的步骤 3.3, 和讨论).</li>
	<li>继续进行移植, 直到胚胎达到大约早肠阶段 10, 大约是 6.5 hpf 在x 酵母(参见图 2E)。</li>
</ol>3. 胚胎愈合后的护理
注: 移植物在30分钟内愈合到位, 观察从胚胎细胞裂解渗出的一些蛋黄碎片是正常的 (图 3C)。
<ol><li>
一旦痊愈, 用巴斯德吸管将胚胎从洼地移到琼脂糖涂层的24孔板的单个井中。在转移过程中, 渗出液会被液体混合去除 (图 3D)。
	<ol>
<li>旋转的胚胎, 使它们被放置在植物极, 面对散装解决方案。再次, 将供体尸体和接枝者安置在相邻的水井中, 以协助跟踪胚胎对;记录此信息。</li>
	</ol>
</li>
	<li>将含有胚胎的24个钢板移植到一个25° c 过夜培养的孵化箱中。 注意: 第二天, 胚胎将达到 tailbud 阶段。</li>
	<li>移接受赠者清洁琼脂糖涂层的6井板, 含 1/9 x MMR, 50 µg 硫酸庆大霉素/毫升, 每井1胚。保持与这些移植受体相匹配的捐赠者尸体的清晰记录。</li>
	<li>采用 pcr 增5、17、24或使用其他依赖于 pcr 增的方法 (参见讨论) 来评估诱变效率, 将单个胴体移动到0.2 毫升 pcr 条管。在100µL 裂解缓冲液中去除大多数培养基和质, 其中含有蛋白酶 K (PK), 通过重复上下移使用 P200 吸管.</li>
	<li>执行胚裂解24在56° c 为6小时到隔夜允许 PK 消化。将其加热至90-95 ° c 10 分钟, 然后快速冷却至4° c。使用裂解没有进一步清除步骤种子 PCR 反应获得短增直接桑格 DNA 测序24。在-20 ° c 下储存裂解。 注意: 在几天内, 蝌蚪将达到喂养阶段 (约45-46 阶段), 并可以被喂食悬浮的浮游粉末 (血清微米)。</li>
	<li>1周后, 随着日常培养基的变化, 保持清洁度和最小化微生物过度生长, 将蝌蚪移到循环水循环系统中的小罐中, 并滴流。利用测序数据对蝌蚪进行高效的 mutagenized 生殖细胞, 以降低诱变效率。一旦蜕变完成, 将 froglets 移到实验室中的成人水生系统。 注: 由国家爪资源开发的方案 (海洋生物实验室、伍兹洞、MA) 为蝌蚪喂养提供了指南29和成人青蛙维护30。</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+dissection+and+micromanipulation+tools.">Embryo dissection and micromanipulation tools.</a> CSH Protoc. , (2007).</li><li>Nakayama, T., 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=Cas9-based+genome+editing+in+Xenopus+tropicalis.">Cas9-based genome editing in Xenopus tropicalis.</a> Methods Enzymol. 546, 355-375 (2014).</li><li>Ogino, H., McConnell, W. B., Grainger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+transgenesis+in+Xenopus+using+I-SceI+meganuclease.">High-throughput transgenesis in Xenopus using I-SceI meganuclease.</a> Nat Protoc. 1 (4), 1703-1710 (2006).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early development of Xenopus laevis.: a laboratory manual. , (2000).</li><li>Kay, B. K., Kay, B. K., Peng, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injection+of+oocytes+and+embryos.">Injection of oocytes and embryos.</a> Xenopus laevis: Practical Uses in Cell and Molecular Biology (Methods in Cell Biology Vol. 36). , (1991).</li><li>Nieuwkoop, P., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis. (Daudin): A Systematical and Chronological Survey of the Development from the Fertilized Egg till the End of Metomorphosis. , (1994).</li><li>Brinkman, E. K., Chen, T., Amendola, M., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easy+quantitative+assessment+of+genome+editing+by+sequence+trace+decomposition.">Easy quantitative assessment of genome editing by sequence trace decomposition.</a> Nucleic Acids Res. 42 (22), e168 (2014).</li><li>Sander, V., Reversade, B., De Robertis, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+opposing+homeobox+genes+Goosecoid+and+Vent1/2+self-regulate+Xenopus+patterning.">The opposing homeobox genes Goosecoid and Vent1/2 self-regulate Xenopus patterning.</a> EMBO J. 26 (12), 2955-2965 (2007).</li><li>Bedell, V. 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=In+vivo+genome+editing+using+a+high-efficiency+TALEN+system.">In vivo genome editing using a high-efficiency TALEN system.</a> Nature. 491 (7422), 114-118 (2012).</li><li>Kim, J. M., Kim, D., Kim, S., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+with+CRISPR-Cas-derived+RNA-guided+endonucleases.">Genotyping with CRISPR-Cas-derived RNA-guided endonucleases.</a> Nat Commun. 5, 3157 (2014).</li><li>Kim, H. J., Lee, H. J., Kim, H., Cho, S. W., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+genome+editing+in+human+cells+with+zinc+finger+nucleases+constructed+via+modular+assembly.">Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly.</a> Genome Res. 19 (7), 1279-1288 (2009).</li><li>Qiu, P., Shandilya, H., D'Alessio, J. M., O'Connor, K., Durocher, J., Gerard, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutation+detection+using+Surveyor+nuclease.">Mutation detection using Surveyor nuclease.</a> Biotechniques. 36 (4), 702-707 (2004).</li><li>Ota, S., 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=Efficient+identification+of+TALEN-mediated+genome+modifications+using+heteroduplex+mobility+assays.">Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays.</a> Genes Cells. 18 (6), 450-458 (2013).</li><li>Dahlem, T. 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=Simple+Methods+for+Generating+and+Detecting+Locus-+Specific+Mutations+Induced+with+TALENs+in+the+Zebrafish+Genome.">Simple Methods for Generating and Detecting Locus- Specific Mutations Induced with TALENs in the Zebrafish Genome.</a> PLoS Genet. 8 (8), e1002861 (2012).</li><li>Ramlee, M. K., Yan, T., Cheung, A. M., Chuah, C. T., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+genotyping+of+CRISPR/Cas9-mediated+mutants+using+fluorescent+PCR-capillary+gel+electrophoresis.">High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis.</a> Sci Rep. 5, 15587 (2015).</li><li>Bhattacharya, D., Marfo, C. A., Li, D., Lane, M., Khokha, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9:+An+inexpensive,+efficient+loss+of+function+tool+to+screen+human+disease+genes+in+Xenopus.">CRISPR/Cas9: An inexpensive, efficient loss of function tool to screen human disease genes in Xenopus.</a> Dev Biol. 408 (2), 196-204 (2015).</li><li>Yu, C., Zhang, Y., Yao, S., Wei, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+PCR+Based+Protocol+for+Detecting+Indel+Mutations+Induced+by+TALENs+and+CRISPR/Cas9+in+Zebrafish.">A PCR Based Protocol for Detecting Indel Mutations Induced by TALENs and CRISPR/Cas9 in Zebrafish.</a> PLoS ONE. 9 (6), e98282 (2014).</li><li>Mock, U., Hauber, I., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR+to+assess+gene-editing+frequencies+(GEF-dPCR)+mediated+by+designer+nucleases.">Digital PCR to assess gene-editing frequencies (GEF-dPCR) mediated by designer nucleases.</a> Nat Protoc. 11 (3), 598-615 (2016).</li><li>Varshney, G. K., 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=High-throughput+gene+targeting+and+phenotyping+in+zebrafish+using+CRISPR/Cas9.">High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9.</a> Genome Res. 25 (7), 1030-1042 (2015).</li><li>Shi, J., Wang, E., Milazzo, J. P., Wang, Z., Kinney, J. B., Vakoc, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+cancer+drug+targets+by+CRISPR-Cas9+screening+of+protein+domains.">Discovery of cancer drug targets by CRISPR-Cas9 screening of protein domains.</a> Nat Biotechnol. 33 (6), 661-667 (2015).</li><li>Koebernick, K., Loeber, J., Arthur, P. K., Tarbashevich, K., Pieler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elr-type+proteins+protect+Xenopus+Dead+end+mRNA+from+miR-18-mediated+clearance+in+the+soma.">Elr-type proteins protect Xenopus Dead end mRNA from miR-18-mediated clearance in the soma.</a> Proc Natl Acad Sci USA. 107, 16148-16153 (2010).</li><li>Yang, J., Aguero, T., King, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenopus+maternal-to-zygotic+transition+from+the+perspective+of+the+germline.">The Xenopus maternal-to-zygotic transition from the perspective of the germline.</a> Curr Top Dev Biol. 113, 271-303 (2015).</li></ol>

作者没有什么要透露的。

本报告提供了一个详细的协议, 移植的植物组织含有生殖细胞. 生殖细胞的移植与基因组编辑技术 (例如, CRISPR/Cas9) 一起使用, 以修改动物的系保持其几乎所有的体细胞组织为遗传野生类型。为了取得成功, 在执行移植手术之前有许多关键因素需要考虑。
为了确保完全取代系与突变等位基因, 建议第一个确定的体内效率的 sgRNAs。经验表明, 大多数 sgRNAs 设计的 CRISPRDi...

自孟德尔定律重新出现以来, 基因型编码表型在生物学中一直是一个重要的问题。理解基因的作用, 它们的调节和基因网络内的相互作用, 以及编码产品的功能, 有望为揭开新的生物学和改善疾病状态提供工具。在半个多世纪的1中, 非洲爪蛙,爪, 一直是研究基础生物学和生物医学领域的一个主要模型, 包括发展的遗传控制。历史上, 大多数关于爪的研究都使用了体蛙, x 蟾, 但最近由于它的二, x 酵母已经被开发为两栖动物遗传模型。完整的基因组序列已经从爪种2、3中进行了组装。"青蛙群落" 现在正处于一个转折点, 基本基因组修饰技术允许对基因功能的研究, 实际上是可以的。可编程 CRISPR/Cas9 酶已使基因的诱变高效, 与 biallelic 变异可能在大多数细胞的动物4,5,6,7, 8、9、10、11。b.b et al.强调了这些研究12和太et al.13表明, 在 F0马赛克动物中, 可以通过诱变研究许多基因的功能。这种方法有许多优点;然而, 由于功能不完全丧失 (LOF), Cas9-sgRNA-microinjected 胚胎往往显示可变的表型。更重要的是, 突变株的产生对某些应用非常有利, 特别是在研究具有母体 mRNA 贡献的基因时。母体的基因和蛋白质及其对胚胎遗传学的影响, 持续了一段长时间的发育过程14,15,16, 呈现了许多基因的早期发育贡献耐火到 F0分析。因此, 需要其他 LOF 方法。
当寻求创造突变株时, 获得合 LOF 胚胎的途径有几个障碍。首先, 有效的诱变产生 biallelic 突变可能是不利的, 因为基本基因功能的丧失导致无法生存到性成熟, 干扰生产的一个可行的线。一个常见的解决办法是仔细滴定 Cas9-sgRNA 的数量。在这里, 目标是达到减少杀伤力之间的平衡, 同时也最大化系的诱变效率。第二个问题来自标准育种方案, 其中表型分析被推迟到 F2代。使用标准方法, 性成熟的 f0动物通过系传播突变等位基因, outcrossed 产生 f1杂 "载体", 然后长成性成熟。然后, 两个 f1子交叉生成 f2突变胚胎, 其预期孟德尔频率为25%。因此, 两代育种是必要的分析突变表型。突变动物可以是纯或复合子 (即,包含两个不同的变种等位基因的后代, 这取决于在 F1交叉中使用的亲代动物的基因型)。
这些障碍可以通过限制可编程核酸介导的诱变到生殖细胞来克服, 这是一个新的方法称为跳跃17。跳跃有两个主要组成部分: (1) 将 Cas mRNA 与 sgRNA, 或核酸-sgRNA 配合物 (或在原则上, TALENs 或锌指核酸) 在单细胞阶段注射, 以有效诱变胚胎基因组, 其次是 (2)生殖细胞移植到野生的同胞胚胎, 其中内源性生殖细胞被移除。当两个步骤是有效的, 完全系替换与突变生殖细胞可以得到。Blackler 在二十世纪六十年代代初表明,爪生殖细胞可以移植到晚期 neurula 和早期 tailbud 阶段的胚胎之间,1819。对于跳跃, Blackler 的方法被修改了执行移植在囊胚阶段17, 当生殖细胞被本地化在胚胎的植物极的20,21。肠前移植有两个主要优点。首先, 当移植在囊胚阶段 (未发表的观察) 时, 被发现的移植的植入和随后正常发展是更加高效率的。第二, 通过在合转录开始后不久进行囊胚阶段的移植, 可以避免因发育基因突变而导致的致命性破坏肠, 否则会造成畸形的晚期 neurulae。PGC 移植-轴承 ("超越") 胚胎生长到性成熟, 和 intercrosses 之间的这些 f0动物已经证明, 在许多情况下, 100% 的 f1后裔显示 LOF 表型 (大多数是化合物子), 表明完全系替换与目标突变。
预计跳跃将加速在爪中的遗传方法。跳跃还提供了一种替代的 "主机传输" 方法22用于 LOF 分析母体效应基因 (未发表的观察).在当前出版物中, 对该方法的详细描述 (特别是针对 PGC 移植) 在x 酵母中介绍 (对x 蟾进行了较小的修改)。生殖细胞的移植在这里演示, 以促进这项技术更快地转移到其他实验室使用爪。这种方法的原则应该是成功的其他两栖动物 (例如, urodeles), 并且有机体特定的修改在方法学应该允许对许多其他动物的应用对高效率的诱变可以完成和生殖细胞很容易移植。

<table><tbody><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11252-20 or 11252-30</td><td></td></tr><tr><td>Eyebrow hair knife</td><td></td><td></td><td>Homemade</td></tr><tr><td>Hair loop</td><td></td><td></td><td>Homemade</td></tr><tr><td>Pasteur pipettes, borosilicate glass</td><td>Fisher Scientific</td><td>13-678-20A</td><td></td></tr><tr><td>Krazy Glue (Cyanoacrylate-based)</td><td>Elmer&#039;s Products, Inc.</td><td>KG581</td><td>To affix eyebrow hair and hair loops into Pasteur pipettes, other similar glues can be used.</td></tr><tr><td>Oligodeoxynucleotides, custom ordered</td><td>Integrated DNA Technologies</td><td>Custom ordered</td><td>Template oligos for sgRNA synthesis, see Nakayama et al., 2014 for design details.</td></tr><tr><td>Megascript T7 kit</td><td>Ambion/ThermoFisher</td><td>AM1334</td><td>Or use Megashortscript T7 (AM1354) kit</td></tr><tr><td>Phenol, Tris buffered</td><td></td><td></td><td>High quality distilled phenol from any commercial supplier</td></tr><tr><td>Chloroform</td><td></td><td></td><td>High quality chloroform from any commercial supplier</td></tr><tr><td>Ethanol</td><td></td><td></td><td>High quality ethanol from any commercial supplier</td></tr><tr><td>Diethylpyrocarbonate (DEPC)</td><td>Sigma Chemical Co.</td><td>D5758-50ML</td><td></td></tr><tr><td>Cas9 protein, with nuclear localization signal</td><td>PNA Bio, Inc.</td><td>CP01</td><td>Reconstituted using DEPC-treated water according to manufacturer&#039;s recommendations</td></tr><tr><td>10X Marc&#039;s Modified Ringers solution</td><td></td><td></td><td>Homemade. Recipe (ref 26) for 1X MMR (lacking EDTA) is 100mM NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 5 mM HEPES. Solution is pH adjusted to 7.4.</td></tr><tr><td>Agarose</td><td></td><td></td><td>Any Molecular Biology grade agarose is sufficient</td></tr><tr><td>60 X15 mm Petri plates</td><td>Falcon</td><td>351007</td><td></td></tr><tr><td>24-well plates</td><td>Falcon</td><td>3047</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma Chemical Co.</td><td>C7352-100G</td><td>Free base, not HCl salt</td></tr><tr><td>Proteinase K</td><td>Roche</td><td>03 115 828 001</td><td>Typically ~20mg/ml from Roche</td></tr><tr><td>sera Micron</td><td>sera</td><td></td><td>Tadpole food. Resuspend in growth medium. Can be purchased from a variety of online retailers</td></tr></tbody></table>

primordial germ cell transplantation for crispr/cas9-based leapfrogging in xenopus

基因组编辑的突变株系的建立加速了许多生物体的遗传分析。CRISPR/Cas9 方法已被改编为使用在非洲爪蛙,爪, 一个长期的生物医学研究模型生物。传统的 CRISPR/Cas9-targeted 诱变品系的选育方案具有耗时费力的几个步骤。为了促进突变胚胎的产生, 特别是为了克服与敲出胚胎发育所必需的基因相关的障碍, 一种称为跳跃的新方法被开发出来。此技术利用爪胚胎的健壮性来 "剪切和粘贴" 胚胎方法。跳跃利用原始生殖细胞 (生殖细胞) 从有效的 mutagenized 供体胚胎转移到 PGC 消融的野生兄弟姐妹。这种方法可以通过创建具有突变系的野生体细胞的嵌合体动物来有效地突变基本基因。当携带 "跳跃式移植" (即突变生殖细胞) 的两个 F0动物被交叉时, 它们产生纯合物, 或复合杂, 空 F1胚胎, 从而节省了整整一代的时间来获取表型数据。跳跃也提供了一种新的方法来分析孕产妇效应基因, 这是耐火到 F0表型分析后 CRISPR/Cas9 突变。本手稿详细介绍了跳跃的方法, 特别强调如何成功地执行 PGC 移植。

在移植后, 应先进行 CRISPR/Cas9 诱变效果的定性测定, 然后再将动物饲养的努力扩大并维持动物的性成熟。由于携带跳跃式移植的动物是体细胞野生, 系难以获得直接测量, 因此保存供体胚胎的尸体变得非常重要。DNA 分析从胴体作为一个代理的范围内发生的突变, 在移植系17。
一种评估突变成功的方法是直接序列...

Watch this Scientific Journal Video about Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus at JoVE.com

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

生存所必需的基因构成了创造突变株系的技术障碍。通过将基因组编辑与原始生殖细胞移植结合在一起, 创造出具有系突变的野生型动物, 从而绕过致命性。跳跃也允许在 F1生成中高效生成纯合 null 突变体。在这里, 移植步骤被证明。

CRISPR/Cas9-based 跳跃的原始生殖细胞移植在爪

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

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Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

8.3K 次观看.  University of California, Irvine. 生存所必需的基因构成了创造突变株系的技术障碍。通过将基因组编辑与原始生殖细胞移植结合在一起, 创造出具有系突变的野生型动物, 从而绕过致命性。跳跃也允许在 F1生成中高效生成纯合 null 突变体。在这里, 移植步骤被证明。

视频: Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

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

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

干细胞样<em&gt;爪</em&gt;胚胎外植体，研究早期神经发育特点<em&gt;体外</em&gt;和<em&gt;在体内</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

功能性克隆使用<em&gt;爪</em&gt;卵母细胞表达系统

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

遗传学

Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as axolotls, to regenerate entire limbs as adults. Here, we outline techniques used to generate chimeric axolotls with Cas9-mutagenized haploid forelimbs that can be used for exploring gene function and the fidelity of limb regeneration. We combine several embryological and genetic techniques, including haploid generation via in vitro activation, CRISPR/Cas9 mutagenesis, and tissue grafting into one protocol to produce a unique system for haploid genetic screening in a model organism of regeneration. This strategy reduces the number of animals, space, and time required for the functional analysis of genes in limb regeneration. This also permits the investigation of regeneration-specific functions of genes that may be required for other essential processes, such as organogenesis, tissue morphogenesis, and other essential embryonic processes. The method described here is a unique platform for conducting haploid genetic screening in a vertebrate model system.

This goal of this protocol is to produce chimeric axolotls with haploid forelimbs derived from Cas9-mutagenized donor tissue using embryonic tissue grafting techniques.

通过胚胎移植，生成具有突变倍体肢体的奇美体轴球

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

移植嵌合斑马鱼胚胎生成

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

生物学

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

施肥爪蟾卵母细胞使用主机传输方法

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

应用小鼠部分遗传类胚胎干细胞作为精子的替代品

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

一种简单有效的斑马鱼胚胎移植装置

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...

CRISPR/Cas9-based 跳跃的原始生殖细胞移植在爪

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