Name: generation of genetically modified mice through the microinjection of oocytes
Uploaded: 2017-06-15T13:00:00
Description: Watch this Scientific Journal Video about Generation of Genetically Modified Mice through the Microinjection of Oocytes at JoVE.com

作者感谢动物设施（BRC）的工作人员不断的支持。这项工作由国家卫生和医学研究理事会和澳大利亚研究理事会资助。

所有程序已获得新南威尔士大学动物护理与伦理委员会的批准。 1.准备转基因（随机整合） <ol><li> 分析琼脂糖凝胶电泳。 <ol><li>根据制造商的建议，使用合适的酶（1小时孵育）或快速消化酶（15至30分钟孵育）在热循环仪中消化质粒以切割转基因（参见图2A及其图例）。 </li><li>加入1％三醋酸乙二胺四乙酸（EDTA）（TEA）琼脂糖凝胶，用0.5-1...

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协议中的关键步骤已知基因修饰的小鼠的产生在技术上是具有挑战性的。然而，这里提出的协议是一种优化和简化的方法，可以在记录时间内掌握和排除故障。成功完成技术需要两个步骤。首先，不用氯化镁（MgCl 2 ）可以实现线性DNA模板的合成（用于合成sgRNA）。然而，强烈建议系统地将MgCl 2加入到主混合物中，因为在没有MgCl 2的情况下经常防止正向引...

在脊椎动物和无脊椎动物中的动物模型已经有助于检查人类病情如阿尔茨海默病1,2的病理生理学。他们也是寻找疾病修饰剂的最宝贵的工具，并最终开发出新的治疗策略，希望得到治愈。虽然每个模型都有固有的局限性，但是使用动物作为整个系统模型对于生物医学研究至关重要。这是因为组织培养不能完全模拟代谢和复杂的生理环境。 迄今为止，鼠标仍然是用于遗传操作的最常见的哺乳动物物种，因为它具有几个优点。与疾病相关的生理过程和基因在小鼠和人之间是高度保守的。小鼠是第一个拥有全基因组测序的哺乳动物（2002年），人类基因组前一年我（2003）。除了丰富的遗传信息，小鼠具有良好的育种能力，快速的开发周期（从受精到断奶6周）和合理的大小。所有这些优点，加上生理指标，如独特的外套颜色（交叉策略所需），使得鼠标成为遗传操作的有吸引力的模型。值得注意的是，在现代遗传学的很早的时代，格雷戈尔·门德尔（Gregor Mendel）开始对老鼠进行植物移植3 。 基因转移技术导致在三十年前第一代转基因小鼠的产生4 ，最初使用病毒递送。然而，研究人员很快意识到，小鼠转基因的主要挑战之一是无法控制外源DNA的命运。因为转基因向小鼠卵母细胞的病毒递送导致随机地整合到基因组中的多个拷贝，这是可能的建立后续转基因株系的限制。 当Gordon 等人通过显微注射产生第一个转基因小鼠系列5,6 。这开始了重组DNA技术的时代，影响显微注射结局的参数已被广泛研究7 。虽然显微注射不允许控制转基因的整合位点（最终导致每个创始人小鼠的特异性表达水平），但是原核显微注射的主要优点仍然是形成连续体（ 即，转基因的多个拷贝的阵列，在基因组整合前5） 。多年来已经使用这种特征来建立数千个过表达感兴趣的基因的转基因小鼠品系。从那时起，转基因，a生物体基因组的人工修饰已广泛用于鉴定单一基因在疾病发生中的作用。 当马里奥·卡佩奇奇成功地破坏了小鼠中的单个基因时，达到了操纵小鼠基因组的另一个关键成果，打开了基因靶向时代8 。然而，基于ES细胞的基因靶向快速出现主要缺点，包括培养ES细胞的挑战，嵌合体的程度有所不同，以及进程的长度（ 即 12-18个月，最小化获得小鼠） 。 最近，已经出现了诸如工程内切核酸酶（ 例如锌指核酸酶（ZFN），转录激活物样效应核酸酶（TALEN））和聚集的定期交织的短回文重复序列（CRISPR / Cas9）的新技术的进展，作为替代方法加速麦克风中基因靶向过程e 9，10 。这些核酸内切酶可以通过显微注射容易地注入小鼠卵母细胞，从而在短短6周内产生基因靶向的小鼠。 自从关于使用CRISPR进行基因组编辑的第一份报告11以来，这种细菌适应性免疫系统由于具有许多优点而取代了ZFN和TALEN，包括易于合成和一次靶向多个基因座的能力（简称“复用” “）。 CRISPR首先用于小鼠12中的基因靶向，并已被应用于从植物到人类的无数种类13,14 。迄今为止，还没有关于抗CRISPR基因组编辑的单一物种的报道。 产生转基因小鼠的两个主要限制步骤是注射卵母细胞和再植入的这些卵母细胞变成假怀孕的雌性。尽管我们已经描述了这种技术15和其他16 ，但是近来在小鼠胚胎学和基因转移技术方面的技术改进已经改变了生产转基因小鼠的过程。这里将描述这些改进。

<table border="0" cellpadding="0" cellspacing="0" width="740">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Micropipette 0.1-2.5 ul</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">4920000016</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Micropipette 2-20 ul</td>
			<td style="width:147px;">Eppendorf</td>
			<td>4920000040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Micropipette 20-200 ul</td>
			<td style="width:147px;">Eppendorf</td>
			<td>4920000067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Micropipette 100-1000 ul</td>
			<td style="width:147px;">Eppendorf</td>
			<td>4920000083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular weight marker&#160;</td>
			<td>Bioline</td>
			<td>BIO-33025</td>
			<td>HyperLadder 1kb</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular weight marker&#160;</td>
			<td>Bioline</td>
			<td>BIO-33056</td>
			<td>HyperLadder 100 bp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Agarose</td>
			<td>Bioline</td>
			<td>BIO-41025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">EDTA buffer</td>
			<td>Sigma-Aldrich</td>
			<td>93296</td>
			<td>10x - Dilute to 1x</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Ethidium bromide</td>
			<td style="width:147px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15585011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR Safe gel stain</td>
			<td>Invitrogen</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Gel extraction kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:119px;">28706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">PCR purification kit (Qiaquick)</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:119px;">28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Vacuum system (Manifold)</td>
			<td style="width:147px;">Promega</td>
			<td style="width:119px;">A7231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease-free microinjection buffer&#160;</td>
			<td>Millipore</td>
			<td>MR-095-10F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrafree-MC microcentrifuge filter&#160;</td>
			<td>Millipore</td>
			<td>UFC30GV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cas9 mRNA</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:119px;">CAS9MRNA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">CRISPR expressing plasmid (px330)</td>
			<td style="width:147px;">Addgene</td>
			<td style="width:119px;">42230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Nuclease free water</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Phusion polymerase</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T7 Quick High Yield RNA kit</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>E2050S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">RNA purification spin columns (NucAway)</td>
			<td style="width:147px;">Thermo Fisher Scientific</td>
			<td>AM10070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">ssOligos</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>OLIGO STANDARD</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Donor plasmid</td>
			<td style="width:147px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">GeneArt</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Hyaluronidase</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">KSOMaa embryo culture medium</td>
			<td>Zenith Biotech&#160;</td>
			<td>ZEKS-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Mineral oil</td>
			<td>Zenith Biotech&#160;</td>
			<td>ZSCO-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">M2 Medium</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>M7167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cytochalasin B</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>C6762</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Mouthpiece</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Glass microcapillaries</td>
			<td style="width:147px;">Sutter Instrument</td>
			<td style="width:119px;">BF100-78-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Proteinase K</td>
			<td>Applichem</td>
			<td>A3830.0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Dumont #5 forceps</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">91150-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Iris scissors</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">91460-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Vessel clamp</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">18374-43</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Wound clips&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">12040-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Clips applier&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">12018-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-scissors&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cauterizer</td>
			<td>Fine Science Tools&#160;</td>
			<td>18000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-absorbable surgical sutures (Ethilon 3-0)</td>
			<td>Ethicon</td>
			<td>1691H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">5% CO2 incubator</td>
			<td style="width:147px;">MG Scientific</td>
			<td style="width:119px;">Galaxy 14S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Spectrophotometer</td>
			<td style="width:147px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Nanodrop 2000c</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Thermocycler</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">6321 000.515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Electrophoresis set up</td>
			<td style="width:147px;">BioRad</td>
			<td style="width:119px;">1640300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">UV Transilluminator</td>
			<td style="width:147px;">BioRad</td>
			<td style="width:119px;">1708110EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Thermocycler</td>
			<td style="width:147px;">Eppendorf</td>
			<td>6334000069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereoscopic microscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Inverted microscope</td>
			<td>Olympus</td>
			<td style="width:119px;">IX71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">2x Micromanipulators</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">5188000.012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Oocytes manipulator</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">5176000.025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Microinjector (Femtojet)</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">5247000.013</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Mice C57BL/6J strain</td>
			<td style="width:147px;">Australian BioResources</td>
			<td>C57BL/6JAusb&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of genetically modified mice through the microinjection of oocytes

使用转基因小鼠对生理和病理体内过程的研究有显着的贡献。将DNA表达构建体的原核注射到受精卵中仍然是产生用于过表达的转基因小鼠的最常用技术。随着用于基因靶向的CRISPR技术的引入，原核注入受精卵母细胞已经扩展到敲除和敲入小鼠的产生。这项工作描述了用于注射的DNA的制备和用于基因靶向的CRISPR指南的产生，特别强调质量控制。识别潜在创始人所需的基因分型程序至关重要。本文介绍了利用CRISPR“复用”能力的创新型基因分型策略。还概述了外科手术。一起，协议的步骤将允许生成gen以及随后建立的大量研究领域的小鼠集落，包括免疫学，神经科学，癌症，生理学，发育等。

下面描述了在随机整合和CRISPR介导的基因靶向的情况下显微注射的工作流程（ 图1 ）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55765/55765fig1v2.jpg" /> 图1：产生转基因小鼠的典型工作流程。对于随机整合，将纯化的转基因注射入受精卵母细胞的原核，然后输卵管转移到插入的寄养雌性中?...

Watch this Scientific Journal Video about Generation of Genetically Modified Mice through the Microinjection of Oocytes at JoVE.com

Generation of Genetically Modified Mice through the Microinjection of Oocytes

小鼠卵母细胞的显微注射通常用于经典转基因（ 即转基因的随机整合）和CRISPR介导的基因靶向。该协议审查显微注射的最新发展，特别强调质量控制和基因分型策略。

通过显微注射卵母细胞生成转基因小鼠

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Research

JoVE Journal

Developmental Biology

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

通过成熟的脂肪细胞在天花板文化去分化代人脂肪干细胞

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

科研

发育生物学

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

转基因器官皮肤培养使用灭活人真皮代

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

胚胎显微注射和电的脊索动物<em&gt;玻璃海鞘</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

小鼠肝外胆管有机体的产生

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

生成基于人类 iPSC 的血脑屏障芯片

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Minimum Volume Vitrification of Immature Feline Oocytes

未成熟费林卵母细胞的最小体积玻璃化

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

斑马鱼卵巢卵巢中卵泡细胞和卵母细胞的分离

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

患者外周血单核细胞中人心肌细胞的生成和扩增

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...