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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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. 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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 CRISPRDirect (https://crispr.dbcls.jp/) 或 CRISPRScan (http://www.crisprscan.org/) 是有效的, 当使用 250 pg/胚胎。但是, 在某些情况下, 可能需要使用更高的浓度 (例如, 由闪电和 al17发布的gsc sgRNA, 需要〜 1 ng/胚胎)。评估目标站点的诱变效果是必不可少的, 使用潮汐允许定量评估31。虽然这里强调的方法使用的直接桑格序列 PCR 增 (代表突变等位基因的人口在 F0胚胎), 许多其他方法已被用于基因组编辑文学为此目的。其他常用的低、中度成本技术包括片段长度多态性分析使用限制酶的损失33或 Cas9/TALEN 解理站点34, T7 切 135和测量器36化验, heteroduplex 移动性检测37, 高分辨率熔融测定38, 毛细管电泳片段分析39,40, 和基于 qPCR 的方法41,42。
第二个考虑因素是目标基因中目标位置的选择。为了允许在 F1生成中有效地恢复突变表型, 重要的是要最大限度地增加完全 LOF 等位基因的机会。当使用经典方法制作突变线时, 在编码区域的5末端附近的目标 Cas9 分裂是一个共同的策略, 以在蛋白质编码区域的开始处引出过早的翻译终止。F1子将被识别为 frameshift 突变, 这些动物将被选择作进一步的工作, 因为它们将携带空等位基因。由于跳跃策略使用 F0 intercrosses, 因此针对编码序列的5结尾的方法将是非常低效的。f0胚胎有马赛克 germlines, 交叉的结果是 f1变种的种群, 这些突变体主要是复合子17。由于大规模的分析证实, 平均来说, 双链断裂产生的突变的预期 ~ 1/3rd是在帧内的43, 大多数 F1的胚胎以这种方式产生至少有一个等位基因与一个框架内可能保留野生类型活动的突变。因此, 需要一个不同的策略, 以确保即使这些在帧突变位基因可能是空的。目标双链断裂在蛋白质折叠的领域, 是必不可少的正常蛋白质功能, 预计将导致更高的机会完全 LOF (空等位基因)17,44。仔细考虑蛋白质域的原子级结构 (s), 当可利用时, 能帮助辨认二级结构的区域, 即使包含单一氨基酸在框架删除, 可能被期望中断领域结构 (例如, 在gsc中的Δ3bp 变种, 由闪电战等17发布。如果 CRISPR/Cas9 目标站点可以在这些区域中被识别, 那么就有更高的几率成功地创建空等位基因。在蛋白质次级结构成分之间的溶剂暴露循环中的小的缺失是不可取的, 因为更灵活的区域的突变可能仍然会导致功能蛋白。
对跳跃的第三个考虑是从受体胚胎中去除 PGC 的效率。这一步必须是有效的, 以确保完整的系更换。目前尚不清楚所述方法所产生的所有移植受体胚胎是否完全清除了它们的生殖细胞. 整个装载原位杂交显示了囊胚阶段胚胎中 PGC 标记的局部化变化。在许多情况下, 所有或几乎所有的信号都在胚胎最末端的植物附近被发现, 因此将被这里概述的手术切除 (闪电战和 al.17和引文)。然而, 一些胚胎显示 PGC 标记表达在少数细胞接近囊胚地板。在受精后的植物极先将, 然后在早期分裂中沿沟沟清扫。一些种质可能会在植物半球中部的细胞中分布, 而不需要使用延伸到囊胚的移植来有效地去除。然而, 同样重要的是要注意, 这些含有生殖细胞标记的深细胞可能不会对系产生作用, 因为 rna 是已知的, 从体细胞中清除 PGC 基因45,46。这些深细胞染色阳性的 PGC 标记可能是体细胞正在进行这样的间隙。方法, 以确保完全 PGC 清除从受体胚胎目前正在发展。
最后, 同样重要的是要考虑多少 F0动物的跳跃产生。由于可变数量的动物将无法生存到性成熟, 它建议有超过20的胚胎中含有移植物的创建。这将有必要有足够的存活的动物, 以确保 (1) 充分的数量的两性将获得和 (2), 这些传播突变位基因在一个足够高的频率, 适合于研究员计划的分析。
使用这里描述的协议, 在一些实践中, 两栖动物学应该能够掌握的 PGC 移植技术的一些实践。跳跃应该是可能的, 不仅在其他两栖动物, 而且包含生殖细胞的其他有机体在个体之间容易地可移植在胚胎发生的早期阶段。

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>
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			<td>Eyebrow hair knife</td>
			<td></td>
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			<td>Homemade</td>
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			<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&#39;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&#39;s recommendations</td>
		</tr>
		<tr>
			<td>10X Marc&#39;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 PCR 增跨越基因组区域的目标。如果一个人希望从许多动物身上序列出大量的单独克隆的DNA 片段, 这将变得特别昂贵和效率低下。相反, 测序增在目标部位的异质性, 因为它们是从基因镶嵌胚胎中衍生出来的, 用来评估每个胚胎的诱变效率19。谱的可视化显示了目标序列的野类型序列 (峰值) 和峰值变为 "被打乱" (即,与出现在每个核苷酸位置上的多个碱相对应的峰值的 co 发生) 下游Cas9-catalyzed 双绞线断裂的部位。此类配置文件的示例可在闪电战et al的图 S1 中找到。17. 通过序列分解算法, 潮汐 (https://tide-calculator.nki.nl/)31, 可以精确地定量显示这些混乱峰所代表的突变程度。使用这种技术对单个胚胎进行筛选, 可以使每个胚胎都得到适当注射并成为诱变的目标。一个示踪剂, 如荧光标记的葡聚糖, 可以用来确认成功的注入24 , 也可以帮助建立一个蝌蚪确实携带移植的组织 (未公布的数据)。重要的是要注意, 正式的可能性, 共的荧光葡聚糖与 Cas9-sgRNA 可能改变 Cas9 诱变效率尚未探索。任何含有不良或 unmutagenized 捐献者的移植的 "超越" 胚胎都可以被丢弃。
通过允许在 f1胚胎中进行有效的表型分析, 跳跃绕过了标准遗传育种方案中的 f2代分析的需要。当使用标准的方法来变异的基本基因, 需要仔细滴定 Cas9-sgRNA 剂量, 以确保生存的创始人胚胎, 导致降低诱变效率。从这个标准方案幸存下来的 f0动物, 然后 outcrossed 与 wildtypes 获得可行的 f1胚胎, 这是筛选识别承运人。最后, 这些 f1子生长到性成熟和交叉获得纯合的 f2个体显示突变表型。相反, 因为跳跃产生的动物完全取代了他们的 germlines 与突变等位基因, 交叉这些 f0动物在 f1生成中产生突变的表型。闪电战et al.17报告说, 大多数 F0动物的目标是在酪氨酸酶(tyr) 轨迹, 这是黑色素细胞和视网膜色素沉着所需的, 显示100% 系传输 (用交测量tyr-白化动物) 的捐助者派生的突变基因组 (见图 4 a-b和表 1)。因此, 交叉两种由跳跃产生的动物将产生纯合或复合杂突变体 F1胚。
相似的结果得到了17由交叉 F0超越动物运载的变异在同源 (编码脱氧核糖核酸结合的同源) goosecoid (gsc) 基因 (参见图 4A, C-H)。用吗反义寡核苷酸 Knockdowns Gsc 表达的方法在x 蟾中建议Gsc基因对于前头部的完全发育是必要的32, 而 Cas9-sgRNA 注入的蝌蚪也显示胚胎致死型表型17。然而, 超越 f0动物, 体细胞野生类型, 是可行的和健壮的, 和 f1交叉生产的胚胎具有相同的 LOF 表型的那些发表在x 蟾knockdowns17。这些数据提供了吗表型的基因确证, 也证明了跳跃可以被用来克服胚胎的致死性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56035/56035fig1.jpg"> 图 1: 移植所需的材料(A) 硼硅酸盐玻璃管用于制作眉毛刀和头发环, 用于早期胚胎显微手术。使用本生燃烧器绘制出的玻璃, 使头发的插入更窄的一端。红色箭头标志着打破玻璃的大致位置。(B) 一个眉毛头发刀 (顶部) 和头发环 (底部) 的例子, 固定成管与快干胶。(C) 96 井 PCR 板的一部分用于在琼脂糖板上制造凹陷, 方法是允许1% 琼脂糖在霉菌周围凝固 (琼脂糖是在0.3x 或 1x MMR 中进行的, 这取决于移植是否在十酵母或x 蟾, 分别)。琼脂糖的厚度是〜5毫米, 有3-4 毫米深水井。(D) 一个用于移植的琼脂糖板的示例, 有12凹陷, 如面板C所示。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56035/56035fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56035/56035fig2.jpg"> 图 2: 植物极在晚期囊胚到早期肠阶段的形态学和解剖策略.(A-f)爪酵母胚胎在植物性视图中以半小时的时间间隔显示, 从囊胚阶段 9 (4.5 hpf) 开始到早期肠阶段 ~ 10.25 (7 hpf)。在 5 hpf 之前, 卵是大的, 更容易损坏。白色, 虚线圆圈标志着在肠的瓶子细胞形成的预期位置, 这可以看到形成从背侧 (顶部) 的面板 (E和F)。对于跳跃, 四切口是在一个正方形内, 由黑色虚线描绘, 最后削减与组织的表面平行 (没有说明), 以释放植物的外植体。缩放栏 = 400 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56035/56035fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56035/56035fig3.jpg"> 图 3: 移植.(a) 在去除植物性 PGC 组织后的 5.5 hpf 胚的例子。黑色虚线框和白色虚线圆圈与图 2中所示的相对维度相同。(B) 在面板 A 中从胚胎中取出的植物外植体, 面向观察者的内表面, 每边约 0.4-0.45 毫米, 深度为 0.25-0. 3 毫米。(C) 与移植的植物组织的胚胎。该图像在移植后10分钟获得。(D) 30 分钟后移植, 植物组织被视为已愈合到位 (温柔的旋转, 用眉毛刀在胚胎之上, 用来制造一个漩涡, 扫走了在面板C中看到的碎片)。在转移到 1/9 x MMR 和孵育在25° c, 多数胚胎完成肠正常。缩放栏 = 400 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56035/56035fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56035/56035fig4v2.jpg"> 图 4: 从跳跃产生的 F0动物的系传输.(A) 为跳跃, 两个基因的目标是 CRISPR/Cas9,酪氨酸酶(顶端) 和goosecoid (底部)。tyr基因的目标是4,5内的编码顺序 (蓝色阴影) 为 N 终端信号肽 (SP, 红色阴影), 而gsc基因是目标17内编码序列在同源的起点 (红色阴影), 在二个显之间被分裂。未翻译的 5 ' 和 3 ' exonic 地区是阴影。(B) 全部 160 F1蝌蚪从 (参见表 1) 交配的超越 F0男性 (LF1) 和一个白化 (tyr/-)女性是白化, 表明整个系从超越男性已替换为tyr突变生殖细胞。该插图显示了一种具有正常视网膜和黑色素沉着的野生型蝌蚪。(C-H)f1胚胎来自于两个 f0超越爪的交叉, 目标是同源基因gsc。大约 2/3rd的胚胎是型突变的gsc, 与表型是不同程度的前截断头部 (E H)。一半的突变体要么是 cyclopic 的, 要么是有严密间隔的眼睛 (F), 而另一半是眼 (H)。基因分型表现为纯或复方子。显示野生型表型 (C、D) 的胚胎的 1/3rd要么是纯合野生类型, 要么是子。原位杂交为otx2 (前脑、中脑和眼睛的标记), egr2 (后脑 rhombomeres 3 和5的标记, 和神经嵴流), 和hoxb9 (脊髓的标记) 显示, 失去了gsc函数只影响otx2域的前一部分。这些数据从闪电战et al.中重现17获得生物学家公司的许可。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56035/56035fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>跨</td>
			<td>男性</td>
			<td>女性</td>
			<td>白化</td>
			<td>野生型</td>
			<td>总</td>
			<td>% 白化</td>
		</tr>
<tr>
<td>1</td>
			<td>LF1</td>
			<td>tyr-</td>
			<td>160</td>
			<td>0</td>
			<td>160</td>
			<td>100</td>
		</tr>
<tr>
<td>2</td>
			<td>LF2</td>
			<td>tyr-</td>
			<td>148</td>
			<td>0</td>
			<td>148</td>
			<td>100</td>
		</tr>
<tr>
<td>3</td>
			<td>tyr-</td>
			<td>LF3</td>
			<td>409</td>
			<td>0</td>
			<td>409</td>
			<td>100</td>
		</tr>
<tr>
<td>4</td>
			<td>tyr-</td>
			<td>LF4</td>
			<td>0</td>
			<td>519</td>
			<td>519</td>
			<td>0</td>
		</tr>
<tr>
<td>5</td>
			<td>tyr-</td>
			<td>LF5</td>
			<td>223</td>
			<td>0</td>
			<td>223</td>
			<td>100</td>
		</tr>
<tr>
<td>6</td>
			<td>LF6</td>
			<td>tyr-</td>
			<td>493</td>
			<td>703</td>
			<td>1196</td>
			<td>41。2</td>
		</tr>
<tr>
<td>7</td>
			<td>tyr-</td>
			<td>LF7</td>
			<td>1</td>
			<td>94</td>
			<td>95</td>
			<td>1。1</td>
		</tr>
<tr>
<td>8</td>
			<td>tyr-</td>
			<td>LF8</td>
			<td>125</td>
			<td>0</td>
			<td>125</td>
			<td>100</td>
		</tr>
<tr>
<td>9</td>
			<td>tyr-</td>
			<td>LF9</td>
			<td>0</td>
			<td>417</td>
			<td>417</td>
			<td>0</td>
		</tr>
<tr>
<td>10</td>
			<td>tyr-</td>
			<td>LF10</td>
			<td>252</td>
			<td>0</td>
			<td>252</td>
			<td>100</td>
		</tr>
</tbody></table>表 1: 在酪氨酸酶中进行突变的跳跃式移植, 有效的系替换。靶向酪氨酸酶, 获得了两性的移植动物。这些动物被交叉与白化病, 这是纯合性的tyr-, 以测试的效率系传播突变等位基因, 和百分比的 F1胚胎显示白化在蝌蚪阶段。标记为 "雄性" 和 "雌性" 的列中的条目表明哪个父项是tyr--或来自跳跃 (LF) 的测试动物。在 "白化病" 下显示的一个十字架上的胚胎的百分比表明白化表型。请注意, 6 的10动物的跳跃式移植显示完成 (100%) 替换与突变等位基因。此表是从闪电战et al.中复制的17, 具有权限。

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 ...

primordial-germ-cell-transplantation-for-crisprcas9-based

Nanyang Technological University

Research

JoVE Journal

Genetics

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

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

科研

遗传学

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

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 ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

心脏细胞治疗中干细胞 dna 完整性的评估

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

利用 Crispr-ca 在哺乳动物细胞系中进行基因组编辑

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

工程神经前体宫内移植,用于Vivo神经结构研究

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...