Name: primordial germ cell transplantation for crispr/cas9-based leapfrogging in xenopus
Uploaded: 2018-02-01T13:30:00
Description: Watch this Scientific Journal Video about Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus at JoVE.com

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?...

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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&#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...

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 ...

Research

JoVE Journal

Genetics

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

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

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

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

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 ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

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

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

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 ...

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

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 ...

Purification and Transplantation of Myogenic Progenitor Cell Derived Exosomes to Improve Cardiac Function in Duchenne Muscular Dystrophic Mice

肌源性祖细胞衍生外质细胞的纯化与移植, 改善杜氏肌营养不良性小鼠的心功能

Duchene Muscular Dystrophy (DMD) is an X-linked recessive genetic disease caused by a lack of functional dystrophin protein. The disease cannot be cured, ...

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Lhgra 介导的反激活在拟南芥诱导、细胞类型特异性表达中的表达

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

使用 Ever 设计自动化、高通量、连续细胞生长实验

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...