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0.5-1.0μg/ mL溴化乙锭（EtBr）染色。 注意:使用EtBr时要小心;它是一种有效的诱变剂。请使用适当的个人防护装备（参见材料安全数据表）。 </li><li>加载1 kb分子量标记。 </li><li>加载线性化片段（ 即转基因和骨架）。 </l我&gt; <li>在100V下运行电泳45分钟。 </li><li>可视化紫外线（UV）透射仪上的凝胶，以检查消化是否完整并确认转基因的正确大小（ 即 7,338 bp; 见图2A ）。 </li></ol></li><li> 制备琼脂糖凝胶电泳。 <ol><li>注射1％TAE琼脂糖凝胶，用低毒凝胶染色剂染色。加载不含分子量标记物的8等份（每个1μg）线性化转基因，并在100V下运行45分钟。使用手术刀切除对应于线性化转基因的所有8个带。 </li><li>根据制造商的建议，使用凝胶提取试剂盒提取DNA（参见材料表 ）。 <ol><li>将凝胶碎片在50℃在1.5-mL管中熔化至少15分钟。用异丙醇沉淀，然后加入结合缓冲液。 </li><li>结合整个DNA将其全部移入一列。洗脱到不含核酸酶的显微注射缓冲液（8mM Tris-HCl和0.15mM EDTA）中。通过0.22微米离心过滤器过滤并以12,000 x g离心60秒。 </li></ol></li><li>使用分光光度计测量DNA的浓度和质量。检查A260 / A280的比例在1.8左右（ 即没有蛋白质污染），A260 / A230的比例至少为2.0（ 即没有有机溶剂污染）。在无核酸酶的显微注射缓冲液中稀释至3ng /μL，等分（20-50μL），并在-20℃下冷冻。 </li></ol></li></ol> 2. CRISPR成分的合成（基因靶向） <ol><li> Cas9 mRNA。 <ol><li>在无核酸酶的显微注射缓冲液中，将Cas9 mRNA（从商业来源获得，参见材料表）稀释至1μg/μL的浓度。 </li><li>等份在2μL和免费<html>在-80°C。 </li></ol></li><li> 单向导向RNA（SgRNA）。 <ol><li>使用计算设计工具识别所需的双靶基因组序列（指南），允许最小的潜在脱靶活动17 。 <ol><li>对于基因敲除，系统地选择位于相反方向上几百个碱基对（bp）的两个指南，并且包括感兴趣的基因的起始密码子。对于同源性直接修复，选择两个重叠的引导（尽可能），在相反的方向。 注意:例如，以下指南最近已成功地共同注射以破坏TPM4.2基因的第一个外显子: 指南1:5&#39;CGCCATCCAGTTCGCGCTGC 3&#39;; 指南2:5&#39;CAGAACGATTGAGCTATGGC 3&#39; </li></ol></li><li>订购一组如表1所示的引物。 </li><li>合成线性DNA模板。 <ol><li>稀释px330不含核酸酶的水中的质粒为10ng /μL。 </li><li>准备主混合物，如表1所示 。最后添加聚合酶，并将主混合物保持在冰上。 </li><li>混合并分成8个PCR管（19μL/管）。每管加入1μL的px330，并在以下条件下进行PCR:98℃1分钟; 98℃10秒，64℃30秒和72℃15秒的40个循环;最后在72℃延伸5分钟。保持在4°C。 </li><li>使用PCR纯化试剂盒（参见材料表 ），歧管和真空源（用于更快的处理）来纯化PCR产物。应用最大真空强度（ 即 8毫巴）。 <ol><li>向1体积的PCR样品中加入5体积（100μL）的结合缓冲液并混合。 </li><li>将（提供的）二氧化硅膜旋转柱放置在（提供的）2-mL收集管中用于离心，或在歧管上进行真空处理ING。为了结合DNA，将所有8个样品连续施加到柱上，并对每个负载以12,000 xg进行真空或离心60秒。 </li><li>为了洗涤，向柱中加入0.75mL洗涤缓冲液（缓冲液PE），真空或以12,000xg离心60秒。以12,000 xg离心柱60 s，以消除残留的乙醇。 </li><li>将色谱柱置于干净的1.5 mL微量离心管中。为了洗脱DNA，将30μL无核酸酶的水加入到膜的中心，让柱静置1分钟，并以12,000g离心60秒。 </li></ol></li><li>使用分光光度计测量浓度;通常浓度应为100ng /μL或更高。检查A260 / A280的比例在1.8左右（ 即没有蛋白质污染），A260 / A230的比例至少为2.0（ 即没有有机溶剂污染）。 </li></ol></li><li>使用T7 RNA合成试剂盒进行体外转录。 <ol><li>准备t他的主要混合物，如表1所示 ，并在37℃下在热循环仪中孵育3小时。加入28μL无核酸酶水和2μLDNA酶I，并在37℃下在热循环仪中孵育另外15分钟。 </li></ol></li><li>使用旋转柱进行RNA纯化。 <ol><li>将所提供的旋转柱中包含的粉末溶解在650μL不含核酸酶的显微注射缓冲液中，小心地清除所有气泡。在室温下盖上管子并水化5-15分钟。 </li><li>取出底部的蓝色帽子，并将色谱柱放入2-mL管中。以750 xg和室温离心2分钟。将色谱柱放入新鲜的1.5 mL管中，并将50μLRNA溶液逐滴加入中心，而不要接触柱壁。在750 x g下旋转色谱柱2分钟。 </li><li>使用分光光度计测量RNA浓度（一个反应的典型产率为30 - 50μg）。检查A260 / A280比率是否为（ 即没有蛋白污染），A260 / A230比例至少为2.0（ 即没有有机溶剂污染）。将sgRNA存储在-80°C直到使用。 </li><li>使用常规用于DNA电泳的1％TAE凝胶评估RNA的质量。运行200-400 ng的DNA模板和相应的sgRNA并行。 RNA带（≈100bp）应略大于DNA条带（ 见图3A ）。 </li></ol></li></ol></li></ol>捐助者模板<ol><li>订购商业合成的单链寡核苷酸（ssOligos，参见材料表 ），用于点突变或用于整合至50 bp的小序列;同源臂通常为60-90 bp长。 </li><li>对于较大的插入，使用供体质粒作为模板。使用经典的克隆方法或从头孢菌素合成生成合适的质粒商业来源。 注意:建议每个同源臂至少800 bp。主干的大小对同源直接修复（HDR）的效率没有影响。 </li></ol> 4.注射混合<ol><li>使用不含核酸酶的显微注射缓冲液进行基因敲除实验，将Cas9 mRNA稀释至50ng /μL，sgRNA至12.5ng /μL（总共为25ng /μL）。添加浓度为200 ng /μL的供体模板（ssOligo或质粒），用于基于同源性直接修复的基因组编辑实验。 注意:可以使用在线工具（如重悬浮计算器18）轻松确定稀释量。 </li><li>在显微注射会话期间将每个等分试样保持在冰上，然后丢弃（不要重新冷冻注射组合）。 </li></ol> 5.阴道输精管切除术注意:两种类型的输精管切除术通常在小鼠中进行:腹部和阴囊。后者是较少侵入性的，之前已经描述过15 。 <ol><li>高压灭菌所有不锈钢手术器械。 </li><li>使用称重确定小鼠的体重，并用注射麻醉药（氯胺酮100mg / kg，赛拉嗪10mg / kg）腹膜内（ip）注射麻醉麻醉男性。 </li><li>监测脚趾反射的损失，一旦鼠标被镇静放置在加热垫的顶部。 </li><li>用局部消毒剂如氯己定和70％乙醇的交替擦拭来消毒睾丸周围的皮肤。 </li><li>将睾丸推入阴囊，并用手术刀进行皮肤切口。 </li><li>可视化睾丸，睾丸附睾和输精管。 </li><li>用镊子抓住输精管，握住镊子并在镊子的两侧灼烧，以移除3 mm的输精管。 </li><li>对第二睾丸执行相同的程序。 </li><li>用伤口夹闭合皮肤切口并监视鼠标紧密直到完全恢复。 </li></ol> 6.超排卵（卵母细胞供体）和时间交配（假怀孕女性） 注意:在其他地方已经描述了产生合适数量的受精卵母细胞和插入寄生雌虫进行再植入的技术。 <ol><li>在第1天下午12点左右，注射10只雌性ip与5IU的怀孕母马血清促性腺激素（PMSG;100μL）。 </li><li>在46-48小时后（第3天上午11点左右），用5IU的人绒毛膜促性腺激素（hCG;100μL）注射雌性ip。 </li><li>在一夜之间立即与单身男性交配的女性。 </li></ol>原核（随机整合）和细胞质（基因靶向）注射<ol><li>如先前所述，通过颈部脱位剔除超排卵小鼠，暴露腹部，并进入卵巢和输卵管。解剖o导管并在立体构架仪15下收获卵丘 - 卵母细胞复合物（COCs）。将它们放入一滴补充有氨基酸（KSOMaa）的单纯疱疹优化培养基，按照传统方法15 。 </li><li>通过使用吸气嘴口将近似的1μL透明质酸酶（10mg / mL）添加到KSOMAA培养基的滴中并将培养皿放置在37℃/ 5％CO 2培养箱中30秒至1分钟来净化卵母细胞。 </li><li>使用吸气嘴口用4个新鲜的KSOMAA培养基洗涤卵母细胞，并将其转移到用矿物油（约2mL）覆盖的最后一滴KSOMaa培养基中。将盘放在37°C / 5％CO 2培养箱中，直至卵母细胞准备注射。 </li><li> 原核注射（随机整合）。 <ol><li>在立体显微镜下可视化鸡蛋，并将大约50个鸡蛋转移到注射室p的M2介质用矿物油覆盖）使用吸气嘴。 </li><li>将注射室转移到倒置显微镜，并注射受精的卵母细胞（两个可见原核）与几个注射液混合物，靶向原核（任何原核可以靶向）。通过观察原核的肿胀来观察成功的注射。 </li></ol></li><li> 细胞质注射（基因靶向）。 <ol><li>使用口器将大约50个卵转移到含有5μg/ mL细胞色素B的KSOMaa的一滴，并在37℃/ 5％CO 2培养箱中孵育5分钟。 </li><li>使用嘴巴将鸡蛋转移到注射室。 </li><li>使用自动化显微注射器的补偿压力尽可能在极低压（50-100hPa））将注射混合物的少量皮下注射入细胞质。 </li><li>注射后，将卵母细胞转移到一滴KSO中哇哇哇哇哇哇哇哇哇哇咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔咔</li></ol></li></ol> 8.重新植入<ol><li>高压灭菌所有不锈钢手术器械。 </li><li>使用称重确定小鼠的体重，并通过注射麻醉剂（氯胺酮100mg / kg，赛拉嗪10mg / kg）腹膜内（ip）注射雌性麻醉。 </li><li>监测脚趾反射的损失，一旦鼠标被镇静放置在加热垫的顶部。 </li><li>将大部分皮毛围绕在雌鼠的背部中线上。 </li><li>用局部消毒剂如氯己定和70％乙醇的交替擦拭来消毒暴露的皮肤。 </li><li>按照传统方法15暴露生殖道。做一个1厘米长的皮肤切口平行于背部中线，切割肌肉，并抓住脂肪垫wi镊子，然后轻轻拉出卵巢，直到其附着的输卵管和子宫清晰可见。 </li><li>用容器夹固定脂肪垫。在立体显微镜下可视化输卵管，并使用一把微型剪刀在壶腹上游几毫米的输卵管壁上进行切口。 </li><li>在立体显微镜下，将25个微注射的卵装入连接到口部的玻璃毛细管（毛细管的内径应为120μm宽）。将玻璃毛细管引入输卵管并排出直到壶腹内可见气泡。 </li><li>轻轻取出玻璃毛细管并将生殖道放回腹部。用3-0不可吸收的外科缝合线缝合切口，然后用伤口夹闭合。密切监视鼠标直到完全恢复。 </li></ol>基因分型策略/排序注意:隔离基因组根据相关动物伦理规定，从2毫米尾部或耳朵活组织检查DNA。 <ol><li> 快速基因组DNA提取（随机整合）。 <ol><li>在100μL碱性裂解试剂（25mM氢氧化钠和0.2mM EDTA，pH = 12）中将组织样品（〜2mm）在95℃下裂解1小时。 </li><li>加入100μL中和剂（40 mM Tris-HCl，pH = 5）。 </li><li>在12,000g和4℃下离心5分钟。 </li></ol></li><li> 高质量的基因组DNA提取（基因靶向）。 <ol><li>加入500μL尾缓冲液（50mM Tris，pH = 8; 100mM EDTA，pH = 8; 100mM NaCl; 1％SDS和0.5mg / mL蛋白酶K，新鲜加入）。 </li><li>在55℃下孵育过夜。 </li><li>通过反转混合5分钟（不要旋涡）。 </li><li>加入250μL饱和NaCl（6 M），倒置搅拌5分钟（不要旋涡）。 </li><li>在12,000 xg和4℃下旋转5-10分钟，将上清液倒入新管中。 </l我&gt; <li>加入500μL异丙醇，倒置搅拌5 min（不要旋涡）。 </li><li>在12,000 xg和室温下旋转10分钟。 </li><li>倾倒上清液（颗粒不可见，粘在管上）。 </li><li>用1 mL 70％乙醇洗涤。 </li><li>在12,000 xg和室温下旋转5-10分钟。去除上清液，小心，因为白色颗粒不粘，可以丢失。 </li><li>空气干燥〜1 h。 </li><li>加入400μLTE缓冲液（10mM Tris和1mM EDTA，pH = 8）。 </li><li>将DNA在55-60℃溶解2小时。 </li></ol></li><li> 底漆设计。 <ol><li>使用计算工具设计引物至少20 bp长。 注意:对于随机整合，引物应与转基因杂交，产生200-800bp的不同片段。为了基因靶向，设计引物使其与基因组DNA杂交，产生不同的f在切割地点周围数百bp的碎片。对于大的插入，引物可以位于转基因上，但是随后使用引物步行或类似技术来进行靶向整合的确认。 </li></ol></li><li> PCR基因分型。 <ol><li>对于每个基因组修饰，设计一个新的PCR方案并经验检验。考虑产生的片段的长度和每对引物的解链温度（Tm）。 </li></ol></li><li> 测序。 <ol><li>对于基因定位，将PCR产物发送给Sanger测序服务提供商。 </li></ol></li></ol>

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作者通过新南威尔士大学Mark Wainwright分析中心为小鼠提供学术转基因服务。

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 ...

generation-genetically-modified-mice-through-microinjection

Research

JoVE Journal

Developmental Biology

20.4K 次观看.  University of New South Wales. 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。 

视频: Generation of Genetically Modified Mice through the Microinjection of Oocytes

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

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

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

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and 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

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 embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

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 ...

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

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 functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

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

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

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

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 blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB 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

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to promote biodiversity preservation. In felids, most species are threatened with extinction, and domestic breeds are used as a model to increase the efficiency of protocols for germplasm banking. Among oocyte cryopreservation techniques, vitrification is more and more popular in human and veterinary assisted reproduction. Cryotop vitrification, which was at first developed for human oocytes and embryos, has demonstrated to be well-suited for cat oocytes. This method offers several advantages, such as the feasibility in field conditions and the speed of the procedure, which can be helpful when several samples need to be processed. However, the efficiency is strongly dependent on the operator&#8217;s skills, and intra- and inter-laboratory standardization are needed, as well as personnel training. This protocol describes minimum volume vitrification of immature feline oocytes on a commercial support in a step by step field-friendly protocol, from oocyte collection to warming. Following the protocol, preservation of oocyte integrity and viability at warming (as high as 90%) can be expected, although there is still room for improvement in post-warming maturation and embryonic development outcomes.

This manuscript describes a protocol for the minimum volume vitrification of immature cat oocytes with laboratory-made media on commercial supports. It covers every step from oocyte isolation from ex vivo gonads to vitrification and warming.

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

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 oocytes and surrounding follicular cells. It is vital to separate both follicular cells and oocytes for various research purposes such as for primary culture of follicular cells, analysis of gene expression, oocyte maturation and in vitro fertilization, etc. The conventional method uses forceps to separate both compartments, which is laborious, time consuming and has high damage to the oocyte. Here, we have established a simple method to separate both compartments using a pulled glass capillary. Under a stereomicroscope, oocytes and follicular cells can be easily separated by pipetting in a pulled fine glass capillary (the diameter depends on the follicle diameter). Compared with the conventional method, this new method has high efficiency in separating both oocytes and follicular cells and has low damage to the oocytes. More importantly, this method can be applied to early-stage follicles including at the pre-vitellogenesis stage. Thus, this simple method can be used to separate follicular cells and oocytes of zebrafish.

Here, we present a simple method for separating follicular cells and oocytes in zebrafish ovarian follicles, which will facilitate investigations of ovarian development in 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

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. Cardiac differentiation from human induced pluripotent stem cells (iPSCs) is modulated by defined signaling pathways that are essential for embryonic heart development. Numerous cardiac differentiation methods on 2-D and 3-D platforms have been developed with various efficiencies and cardiomyocyte yield. This has puzzled investigators outside the field as the variety of these methods can be difficult to follow. Here we present a comprehensive protocol that elaborates robust generation and expansion of patient-specific cardiomyocytes from peripheral blood mononuclear cells (PBMCs). We first describe a high-efficiency iPSC reprogramming protocol from a patient&#39;s blood sample using non-integration Sendai virus vectors. We then detail a small molecule-mediated monolayer differentiation method that can robustly produce beating cardiomyocytes from most human iPSC lines. In addition, a scalable cardiomyocyte expansion protocol is introduced using a small molecule (CHIR99021) that could rapidly expand patient-derived cardiomyocytes for industrial- and clinical-grade applications. At the end, detailed protocols for molecular identification and electrophysiological characterization of these iPSC-CMs are depicted. We expect this protocol to be pragmatic for beginners with limited knowledge on cardiovascular development and stem cell biology.

Here, we present a protocol to robustly generate and expand 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. ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

受精卵显微注射用于在小鼠中产生基因盒敲入和絮状等位基因

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...