Name: identification of homologous recombination events in mouse embryonic stem cells using southern blotting and polymerase chain reaction
Uploaded: 2018-11-20T13:30:00
Description: Watch this Scientific Journal Video about Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction at JoVE.com

这项工作得到了国家自然科学基金总计划 (31571432)、中国省人类自然科学基金会 (赠款号 2015jc3097) 和湖南省教育局研究基金会的支持。中国省 (批准号: 15k054)。

1. pcr 的目标结构、南球探针和引物的设计
<ol><li>选择 myh9 基因的第一个编码外显子 (exon 2), 用于在此处报告的敲击/敲入应用中中断或插入。</li>
	<li>从genome.ucsc.edu网站上检索 myh9 外显子2周围的5-kb 上游和5-kb 下游 dna 序列。</li>
	<li>分析在这个10-kb 区域减少1-2 的酶 (res) 的限制性消化模式, 使用 pdraw 软件来确定合适的 re (s) 来消化用于南方印迹的基因组 dna。 注: dra i 满足此要求, 并?...

<ol>
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目前, 用于基因组编辑的设计核酸酶由于其目标外效应和插入 dna 片段30、31的困难问题, 仍不能取代基于 es 细胞的基因靶向技术。作为识别发生在小鼠 es 细胞中的 hr 事件的黄金方法, 本报告为该领域提供了一个详细的南方印迹和 pcr 协议。我们通过分析一系列构造所针对的鼠标 es 细胞中的单个克隆来验证这些方法的可靠性。这些方法确定的所需 es 克隆?...

hr 在小鼠 es 细胞中的基因靶向技术为剖析特定基因突变 1,2的细胞后果提供了有力的工具。2007年诺贝尔生理学或医学奖 3、4 对这一技术的重要性和意义反映在它的认可上;同时, 它也代表了现代基因工程时代的到来.通过 hr 进行基因靶向的基因可以用来设计小鼠 es 细胞 6,7基因组中几乎任何改变, 从点突变到大染色体重排。众所周知, 在所谓的基因组编辑工具出现之前, 基因敲除小鼠的产生需要在 es细胞8、9、10中应用基因靶向技术。在过去20年中, 这种方法产生了 5, 000多头基因靶向小鼠, 用于人类疾病建模或研究基因功能 11。建立了全基因组淘汰赛的工作, 将基因靶向载体、针对 es 细胞克隆和活鼠分配给科学界2、12、13、14,15. 毫无疑问, 基于 es 细胞的 hrc 介导的基因靶向技术极大地促进了我们对在生理或病理背景下发挥的基因功能的了解。
由于 hr 在哺乳动物细胞16,17中是一个相对较少的事件, 重要的和下一步的基因靶向在小鼠 es 细胞是分析许多 es 菌落, 以确定几个克隆与突变产生的hr 与目标向量 18。识别人力资源事件的黄金方法包括南方印迹和 pcr19,20。这些方法的优点包括南方印迹可以正确识别有针对性的 es 克隆, 并允许研究人员分析基因目标事件的结构, 例如验证构造的单个副本插入, 而基于 pcr 的战略允许更迅速地筛选21世纪、22日的人力资源活动。尽管这些方法有缺点, 例如它们很耗时, 可能存在误报, 但它们的组合用法在识别 hr 事件 (特别是 es 细胞) 以生成转基因产品时被广泛接受和应用动物。
哺乳动物中的非肌肉肌球蛋白 ii (nm ii) 有三种异型, 每种都由两个相同的 nmhc i i 由三个不同的基因 (名为 myosin、myosin 和 myosin) 和两对光链编码, 称为 nm-ii-a、ii-b 和 ii-c23。先前的研究表明, 至少 nm ii-a 和 ii-b 的异形对小鼠的发育至关重要, 因为这些同态的体内消融会导致胚胎性 24,25, 26。为了规避这一问题, 并在小鼠发育的后期阶段对 nm ii-a 和 ii-b 的特定于于等结构的功能有新的见解, 采用了基于 es 细胞的 hr 介导基因靶向技术的基因替代策略。生成一系列鼠标模型27。在确定所需 es 克隆体的过程中, 采用了南方印迹和 pcr 方法, 证明了这些方法的有效性和可靠性。
本文旨在提供南方印迹和 pcr 的详细描述, 包括靶向载体、探针和引物的设计, 实验的执行, 以及通过识别 hr 事件对结果进行分析。发生在 es 细胞中, 用于创建基因替代 nm ii 小鼠模型和代表性数据。这里介绍的这两种方法的方案也可以用来识别转基因细胞或动物的基因型。

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:391px;">BAC CLONE</td>
			<td style="width:232px;">BACPAC Resources Center (BPRC)</td>
			<td style="width:113px;">bMQ-330E21</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAGEN Large-Construct Kit</td>
			<td style="width:232px;">QIAGEN</td>
			<td align="right">12462</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick Gel Extraction Kit</td>
			<td style="width:232px;">QIAGEN</td>
			<td align="right" style="width:113px;">28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification Kit</td>
			<td style="width:232px;">QIAGEN</td>
			<td align="right" style="width:113px;">28104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:232px;">QIAGEN</td>
			<td align="right" style="width:113px;">27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAGEN Plasmid Plus Maxi Kit</td>
			<td style="width:232px;">QIAGEN</td>
			<td align="right" style="width:113px;">12963</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">PfuUltra High-Fidelity DNA Polymerase</td>
			<td style="width:232px;">Agilent</td>
			<td align="right" style="width:113px;">600382</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:391px;">T-easy vector</td>
			<td style="width:232px;">Promega</td>
			<td style="width:113px;">A1360</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:391px;">Nuclei Lysis Solution</td>
			<td style="width:232px;">Promega</td>
			<td style="width:113px;">A7941</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Protein Precipitation Solution</td>
			<td style="width:232px;">Promega</td>
			<td>A7951</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DNA Denaturing Solution</td>
			<td style="width:232px;">VWR</td>
			<td>351-013-131</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">DNA Neutralizing Solution</td>
			<td style="width:232px;">VWR</td>
			<td>351-014-131</td>
			<td></td>
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			<td height="21" style="height:21px;">Ready-To-Go DNA Labeling Beads (-dCTP)</td>
			<td style="width:232px;">VWR</td>
			<td>27-9240-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">UltraPure SSC, 20X</td>
			<td style="width:232px;">Thermo Fisher</td>
			<td align="right">15557036</td>
			<td></td>
		</tr>
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			<td height="42" style="height:42px;">UltraPur Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)</td>
			<td style="width:232px;">Thermo Fisher</td>
			<td align="right">15593031</td>
			<td></td>
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			<td height="42" style="height:42px;">G418</td>
			<td style="width:232px;">Thermo Fisher</td>
			<td align="right">10131035</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">Salmon Sperm DNA Solution</td>
			<td style="width:232px;">Thermo Fisher</td>
			<td align="right">15632011</td>
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			<td height="42" style="height:42px;">Platinu Taq DNA Polymerase High Fidelity</td>
			<td style="width:232px;">Thermo Fisher</td>
			<td align="right">11304029</td>
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			<td height="21" style="height:21px;width:391px;">Not I</td>
			<td>Thermo Scientific</td>
			<td>ER0592</td>
			<td></td>
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			<td height="21" style="height:21px;width:391px;">Dra I</td>
			<td>Thermo Scientific</td>
			<td>ER0221</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:391px;">EcoR I</td>
			<td>Thermo Scientific</td>
			<td>ER0271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ganciclovir</td>
			<td style="width:232px;">Sigma</td>
			<td style="width:113px;">G2536</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Whatman&#160;TurboBlotter Transfer System, Large Kits</td>
			<td style="width:232px;">Fisher Scientific</td>
			<td>09-301-188</td>
			<td></td>
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		<tr height="24">
			<td height="24" style="height:24px;">[&#945;32P]dCTP</td>
			<td>PerkinElmer</td>
			<td>NEG013H100UC</td>
			<td></td>
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			<td height="63" style="height:63px;">ProbeQuan G-50 Micro Columns</td>
			<td style="width:232px;">GE Healthcare</td>
			<td>28-9034-08</td>
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			<td height="42" style="height:42px;">Hybrisol I Hybridization Solution</td>
			<td style="width:232px;">Millipore</td>
			<td style="width:113px;">S4040</td>
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			<td height="21" style="height:21px;">Kodak X-Ray Film</td>
			<td>Z&#38;Z Medical</td>
			<td>844 5702</td>
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	</tbody>
</table>

identification of homologous recombination events in mouse embryonic stem cells using southern blotting and polymerase chain reaction

相对于非目标效应和在基因组编辑的设计师核酸酶应用中插入长 dna 片段的困难问题, 胚胎茎 (es) 细胞靶向技术并没有这些缺点, 而且广泛存在用于修改动物老鼠基因组, 从大的缺失插入到单核苷酸取代。值得注意的是, 确定获得所需的 es 克隆所需的同源重组 (hr) 事件相对较少, 是一个关键步骤, 需要准确和可靠的方法。南方印迹和/或常规 pcr 经常用于这一目的。在这里, 我们描述了使用这两种方法来识别发生在小鼠 es 细胞中的 hr 事件的详细过程, 在这些事件中, 内源性 myosin 基因的目的是被编码其他非肌肉肌球蛋白重链 i (nmhc iis) 的 cDNAs 所破坏和取代。南方印迹的整个过程包括靶向载体的构建、电穿孔、药物选择、es 细胞/克隆体的扩张和储存、基因组 dna (gdna) 的制备、消化和印迹、杂交和探头的清洗, 以及 x 射线胶片上自动成像的最后一步。pcr 可直接与制备的和稀释的 gdna 一起进行。为了获得理想的结果, 应仔细规划用于南方印迹的探针和限制性酶 (re) 切割位点和 pcr 引物。虽然执行南方印迹是耗时和劳动密集型的, pcr 结果有假阳性, 但南方印迹的正确识别和 pcr 的快速筛选允许这些方法的唯一或组合应用所描述的本文在 es 细胞和转基因动物基因型的鉴定中得到了大多数实验室的广泛应用和参考。

本文介绍了一种详细的南方印迹和 pcr 协议, 该协议利用基于 es 细胞的 hr 介导靶向技术, 用于识别小鼠 es 细胞中发生的 hr 事件, 用于生成 nm ii 基因替代小鼠模型。尽管南方印迹和 pcr 以及传统的基因靶向技术已经广泛使用了几十年, 但它们的成功应用需要仔细规划。至少需要考虑这些方面: 长臂和短臂的长度、探针的位置和长度、切割基因组 dna 的合适 res 以及 pcr 的引物, ?...

Watch this Scientific Journal Video about Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction at JoVE.com

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

在这里, 我们提出了一个详细的协议, 以确定同源重组事件发生在小鼠胚胎干细胞使用南方印迹和/或 pcr。利用传统的胚胎干细胞同源重组介导靶向技术生成的非肌肉肌球蛋白 ii 基因替代小鼠模型就是例证。

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

该方法有助于回答分子和细胞生物学领域的关键问题，如识别小鼠胚胎干细胞中的同源重组事件。该技术的主要优点是准确、可靠、应用广泛。因此，该方法可以深入了解小鼠胚胎干细胞中的同源重组事件。它也可以应用于其他系统，如转基因细胞或动物。通常，对此方法的个别新用户将难以操作，因为它需要很长时间并涉及多个步骤。对于每个 gDNA，混合 30 微升 10 x 缓冲液，用于 DRA 一个，3 微升 DRA 1 和 10 微克 gDNA。加入水，直到最终体积为30微升，并在37摄氏度孵育过夜消化gDNA。用溴化乙基准备1%的阿加罗斯电泳凝胶。将以前采集的样品以及一个 KB 梯子装载到凝胶上，并在一夜之间以 30 到 40 伏运行。第二天，将凝胶浸泡在含有0.2牛顿盐酸溶液的托盘中。在室温下，使用摇床轻轻摇动托盘 20 分钟。将凝胶转移到含有DNA变性溶液的托盘上。在室温下轻轻摇动20分钟。然后将凝胶转移到含有脱氧核糖核酸中和溶液的托盘中。在室温下轻轻摇动20分钟。使用快速向下传输系统，将DNA从凝胶转移到膜。接下来，按照制造商提供的说明中概述的，组装涡轮和印迹堆栈。之后，拿出膜，用两个X盐水柠檬酸钠洗涤一分钟。用组织吸收液体，然后使用UV交联器将DNA与膜交叉连接。要开始用放射性标记DNA探针，请将热变性探针DNA添加到含有随时可去DNA标记珠的管中。移液向上和向下混合。并添加五微升放射性标记脱氧核糖二苯二甲酸酯。在37摄氏度下孵育15分钟。根据制造商提供的说明，使用 G 50 微柱对标记探头进行净化。使用闪烁计数器测量放射性。要开始将膜与标记探头混合，请将膜放入杂交管中。添加混合预杂交解决方案。将管子放入杂交炉中，让预杂交在42摄氏度下进行30分钟。然后，从烤箱中取出管子，将预杂交溶液倒入 50 毫升管中。加入变性探头，轻轻混合。要去除非杂交探头，请将膜放入含有一个 X 盐水柠檬酸钠的托盘中，并在 55 至 60 摄氏度下轻轻摇动 10 分钟。在此之后，用塑料包装膜，并将其固定在曝光盒中。在黑暗的房间里，将膜暴露在两片 X 射线膜上。开发胶片以可视化结果。根据探测器检测到的DNA波段的大小，确定相应的ES克隆是否为具有重组目标的所需克隆。在这项研究中，利用南方印迹和PCR来识别小鼠ES细胞中发生的HR事件，以便使用基于ES细胞的基于HR的定位技术生成NM两个遗传替代小鼠模型。由于基因组DNA被切成许多不同长度的片段，它们在DNA凝胶上显示一种像涂片一样的状态。这表明基因组DNA的完全消化。作为南方印迹的最后一步，在胶片上显示标有探针的放射性信号与目标DNA片段混合。预期波段的出现反映了 ES 克隆中 HR 事件的发生。根据研究中的预设计，具有变异等位基因的ES克隆具有两个不同的大小带。虽然野生类型 ES 克隆只有一个波段，但表明所需的 ES 克隆是异质克隆。在PCR反应之后，PCR产品可以在DNA凝胶上进行分析。如果观察到正确大小的特定 NPCR 波段，则建议发生 HR 事件，可以通过对 PCR 频段进行测序来进一步确认。由于基础相同，该技术的含义扩展到识别点突变的疗法。尝试这个程序是重要的记住要有耐心和小心。按照这个程序，可以执行其他方法，如ES细胞的微注射到囊肿，以回答额外的问题，如有形成嵌合动物。随着技术的发展，这项技术为分子和细胞生物学领域的研究人员探索特定基因的功能或小鼠或外基因突变的结果铺平了道路。不要忘记，使用放射性标签 P32 DCTB 可能非常危险，执行此过程时应始终采取预防措施，例如使用保护板进行屏蔽。

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Unit 2

Studying DNA and RNA

SCIENTISTS IN ACTION

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

河豚有毒改进的聚合酶链反应 - 限制性片段长度多态性分型液相色谱/质谱法

An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by ...

科研

遗传学

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

人端粒酶逆转录蛋白 rna 依赖性 rna 聚合酶活性的半定量检测

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

CRISPR 的染色质环结构重组

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

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 ...

Culturing and Manipulation of O9-1 Neural Crest Cells

Neural crest cells (NCCs) are migrating multipotent stem cells that can differentiate into different cell types and give rise to multiple tissues and ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

一种通用嵌套逆转录酶链反应检测裂解病毒的评价

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

用于分离共转录RNA处理事件的人工RNA聚合酶II伸长复合物

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene

一种基于强健聚合酶链反应的测定法，用于定量易碎 X 智力迟钝-1 基因中的细胞氨酸-鸟-鸟-鸟- 三核苷酸重复

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; ...