Name: efficient genome editing of mice by crispr electroporation of zygotes
Uploaded: 2022-12-16T13:20:00
Description: Watch this Scientific Journal Video about Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes at JoVE.com

A.J.M.创造了导致CRISPR-EZ发展的原始概念并产生了这些数字。C.K.D.为当前手稿汇编并改编了内部和已发表的协议。A.J.M. 由 NIH （R00HD096108） 提供支持。

在整个协议中，所有动物护理和使用都遵守《动物福利法》政策，ILAR实验动物护理和使用指南，并遵循AVMA安乐死和宾夕法尼亚大学机构动物护理和使用委员会（IACUC）的指导方针和政策。宾夕法尼亚大学IACUC为该项目审查并批准了动物护理和使用协议。作为合规性和谨慎起见，请在尝试此协议之前寻求所有必要的授权。
1. sgRNA和可选的供体寡核苷酸设计
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这里介绍的是一种简单高效的小鼠基因组编辑技术。电穿孔可用于在1-2周内产生修饰的胚胎（图1），并且可以在6周内产生经过编辑的小鼠9。与同时期开发的基于电穿孔的RNPs7，10，11，12，13，14，15，17，<su...

自CRISPR编辑1，2，3出现以来，活小鼠的基因组编辑已经大大简化，并且变得容易获得且更实惠。最初的动物编辑尝试使用显微注射将CRISPR Cas9 mRNA / sgRNA递送到前核阶段胚胎中4，5，6。虽然显微注射非常有效，但完全掌握它所需的练习量可能不适合受训者和学生，并且还需要昂贵的设备，而资金有限的实验室无法负担。显微注射通常由转基因设施的专家技术人员进行，其时间表和服务价格对许多研究人员来说是限速的。一种更容易获得的方法是电穿孔，它已被证明对于将CRISPR Cas9 mRNA / sgRNA递送到前核阶段胚胎中非常有效7。CRISPR基因组编辑和递送策略的进一步改进表明，已经与sgRNA结合的预组装RNP可能是减少嵌合体的有效手段8。
开发和使用该方案背后的基本原理是绕过与显微注射相关的许多限制和障碍。顾名思义，一种简单、内部且经济高效的方法可以快速确定未经测试的 sgRNA 设计是否值得在显微进样实验中使用，这将是一个非常方便的首过质量控制步骤（图 1）。虽然这种方法不能取代显微注射用于更复杂的策略，例如为基于重组的结果引入长供体DNA序列，但它是不太复杂的策略的理想选择，如小的缺失或插入以及标记基因。这种方法适用于具有基本胚胎操作技能的研究人员，他们有简单的编辑需求，希望在植入前发育的时间范围内测试他们的假设，或者更喜欢在安排与显微注射专家的预约之前测试胚胎中的sgRNA。在这里，编辑试剂通过电穿孔（一系列电脉冲） 以 Cas9 / sgRNA RNP的形式瞬时递送到前核阶段胚胎中，以最大限度地提高效率，同时减少嵌合体8。使用胚胎基因分型方法，编辑结果可在大约 1 周内获得9，从而以显着降低的成本减少了对各种显微注射应用的需求。
该方法的有效性在原核胚胎阶段达到峰值，此时胚胎尚未融合母本和父本原核或进入S期（图2）。超排卵用于最大化受精卵的数量，但同时产生原核受精卵和未受精卵。健康的受精卵也可以在电穿孔前预先选择，以提高整体效率。由于其他电穿孔方案已经有效地编辑了受精卵，而无需包括类似的步骤7，10，11，12，13，14，15，因此该协议的可选步骤是透明带（ZP）的轻微侵蚀。ZP 是一种糖蛋白层，有助于精子结合、顶体反应和核前期胚胎周围的受精。根据我们的经验，我们发现对ZP的温和酸基侵蚀提供了可靠的Cas9 RNP电穿孔递送，对活性的影响很小。
我们已经观察到，在C57BL / 6J和C57BL /6N 9，16等研究中常用的小鼠品系中，通过电穿孔的RNP递送率高达100%。独立小组还开发了基于电穿孔的程序，其效率大于或匹配显微注射11，12，13，14，15，17，电穿孔方案在大鼠18，19，猪20，21，22和牛23中运行良好，因此我们建议读者比较协议以找到最适合其实验和设备需求的条件。这里描述的系统使用常见的材料和设备，只需要基本的胚胎操作技能。该技术对一系列编辑策略有效，使该方法被研究界广泛使用。
设计理想的小向导RNA（sgRNA）对于高效编辑至关重要。我们建议直接在小鼠胚胎中为每个靶位点筛选两到三种sgRNA策略，尤其是在需要生成小鼠系的情况下。一旦设计出来，建议使用体外转录（IVT）等无克隆方法来产生高质量的sgRNA3。将RNP和sgRNA与30-50个处理过的前核阶段胚胎混合，并暴露于一系列电脉冲中，首先暂时透化ZP和细胞膜，随后的脉冲保持孔隙打开并通过受精卵24对RNP进行电泳。优化后，我们发现在30 V下对大体胚胎（~50）的6个3 ms脉冲最适合编辑有效性和活力，提供高效的Cas9 / sgRNA RNP递送9，16，25。可以使用 CRISPR 编辑中常见的各种验证策略来确认单个小鼠桑葚中的编辑事件，例如限制性片段长度多态性 （RFLP）、T7 核酸内切酶消化和感兴趣区域的 Sanger 测序26。
目前的方法最适合简单的编辑方案（图3），例如插入/缺失（插入缺失），外显子大小的500-2000 bp缺失，以及点突变和小插入的传递，例如C或N端标签（例如，FLAG，HA或V5）9，16，27。复杂基因组编辑的潜力，如大量插入荧光标签或条件等位基因，仍然不确定，并且是即将进行的改进的当前重点。
该方法易于掌握，可用于在1周内 快速测试培养小鼠胚胎中的sgRNA（图1）。这项工作中提出的是一个六步方案，其中包括1）sgRNA设计;2）sgRNA合成;3）超排卵和交配;4）胚胎培养、采集和加工;5）RNP组装和电穿孔;6）胚胎培养和基因分型。提供有关所有所用材料的信息（材料表）。作为阳性对照，编辑 酪氨酸酶（Tyr）位点9，16 的试剂已包含在 补充表1中。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1-cm-gap electroporation cuvette</td>
			<td>Bio-Rad</td>
			<td>cat. no. 1652089</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>26-G, 1/2-inch needle</td>
			<td>BD</td>
			<td>cat. no. 305111</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>3&#8211;8-month-old male mice and 3- to 5-week-old female mice</td>
			<td>JAX</td>
			<td>cat. no. 000664</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>35-mm Tissue culture dish</td>
			<td>Greiner Bio-One,</td>
			<td>cat. no. 627-160</td>
			<td>Embryo Culture</td>
		</tr>
		<tr>
			<td>60-mm Tissue culture dish</td>
			<td>Greiner Bio-One,</td>
			<td>cat. no. 628-160</td>
			<td>Embryo Processing</td>
		</tr>
		<tr>
			<td>6x loading dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>cat. no. R0611</td>
			<td>sgRNA Synthesis and Genotyping</td>
		</tr>
		<tr>
			<td>Acidic Tyrode&#39;s (AT) solution, embryo culture grade</td>
			<td>Sigma-Aldrich,</td>
			<td>cat. no. T1788</td>
			<td>Embryo Processing</td>
		</tr>
		<tr>
			<td>BSA, embryo culture grade</td>
			<td>Sigma-Aldrich</td>
			<td>cat. no. A3311</td>
			<td>Embryo Processing and Culture</td>
		</tr>
		<tr>
			<td>Cas9 protein</td>
			<td>Alt-R S.p. Cas9 nuclease 3NLS</td>
			<td>cat. no. 1074181</td>
			<td>Electroporation</td>
		</tr>
		<tr>
			<td>DNase I, RNase-free</td>
			<td>New England BioLabs,</td>
			<td>cat. no. M0303</td>
			<td>sgRNA Synthesis</td>
		</tr>
		<tr>
			<td>DPBS(calcium and magnesium free)</td>
			<td>Gibco</td>
			<td>cat. no. 14190-144</td>
			<td>Embryo Processing</td>
		</tr>
		<tr>
			<td>EcoRI</td>
			<td>NEB</td>
			<td>cat. no. R3101S</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>EDTA, anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>cat. no. EDS-100G</td>
			<td>RNP Buffer</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Koptec</td>
			<td>cat. no. V1016</td>
			<td>sgRNA Synthesis</td>
		</tr>
		<tr>
			<td>Gelatin (powder) type B, laboratory grade</td>
			<td>Fisher,</td>
			<td>cat. no. G7-500</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>Glycerol,&#160;molecular-biology grade</td>
			<td>Fisher</td>
			<td>cat. no. BP229</td>
			<td>RNP Buffer</td>
		</tr>
		<tr>
			<td>Taq Polymerase</td>
			<td>Promega</td>
			<td>cat. no. M712</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>HEPES, cell culture grade</td>
			<td>Sigma-Aldrich</td>
			<td>cat. no. H4034</td>
			<td>RNP Buffer</td>
		</tr>
		<tr>
			<td>HinfI (10,000 U/mL)</td>
			<td>NEB</td>
			<td>cat. no. R0155S</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>HiScribe T7 High Yield RNA Synthesis Kit</td>
			<td>New England BioLabs,</td>
			<td>cat. no. E2040</td>
			<td>sgRNA Synthesis</td>
		</tr>
		<tr>
			<td>Human chorion gonadotropin, lyophilized&#160;(hCG)</td>
			<td>Millipore</td>
			<td>cat. no. 230734</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>Hyaluronidase/M2</td>
			<td>Millipore</td>
			<td>cat. no. MR-051-F</td>
			<td>Embryo Processing</td>
		</tr>
		<tr>
			<td>KSOMaa Evolve medium (potassium-supplemented simplex-optimized medium plus amino acids)</td>
			<td>Zenith Biotech</td>
			<td>cat. no. ZEKS-050</td>
			<td>Embryo Culture</td>
		</tr>
		<tr>
			<td>LE agarose, analytical grade</td>
			<td>BioExpress</td>
			<td>cat. no. E-3120-500</td>
			<td>sgRNA Synthesis and Genotyping</td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>Zenith Biotech</td>
			<td>cat. no. ZFM2-050</td>
			<td>Embryo Processing</td>
		</tr>
		<tr>
			<td>Magnesium chloride, anhydrous (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td>cat. no. M8266</td>
			<td>RNP and Lysis Buffer</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Millipore</td>
			<td>cat. no. ES-005C</td>
			<td>Embryo Culture</td>
		</tr>
		<tr>
			<td>Nonidet P-40,substitute (NP-40)</td>
			<td>Sigma-Aldrich</td>
			<td>cat. no. 74385</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>Nuclease-free water, molecular-biology grade</td>
			<td>Ambion</td>
			<td>cat. no. AM9937</td>
			<td>sgRNA Synthesis and Genotyping</td>
		</tr>
		<tr>
			<td>Oligos for sgRNA synthesis, donor oligo and PCR primers for genotyping</td>
			<td>Integrated DNA Technologies</td>
			<td>custom orders</td>
			<td>sgRNA Design</td>
		</tr>
		<tr>
			<td>Reduced serum medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>cat. no. 31985062</td>
			<td>Embryo Culture</td>
		</tr>
		<tr>
			<td>High-fidelity DNA polymerase</td>
			<td>New England BioLabs,</td>
			<td>cat. no. M0530</td>
			<td>sgRNA Synthesis</td>
		</tr>
		<tr>
			<td>Potassium chloridemolecular-biology grade (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td>cat. no. P9333</td>
			<td>RNP and Lysis Buffer</td>
		</tr>
		<tr>
			<td>Pregnant mare serum gonadotropin lyophilizd ((PMSG)</td>
			<td>ProspecBio</td>
			<td>cat. no. HOR-272</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>Proteinase K, molecular-biology grade</td>
			<td>Fisher</td>
			<td>cat. no. BP1700-100</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>RNase-free 1.5-mL microcentrifuge tube</td>
			<td>VWR</td>
			<td>cat. no. 20170-333</td>
			<td>sgRNA Synthesis and Genotyping</td>
		</tr>
		<tr>
			<td>RNase-free eight-well PCR strip tubes</td>
			<td>VWR</td>
			<td>cat. no. 82006-606</td>
			<td>sgRNA Synthesis and Genotyping</td>
		</tr>
		<tr>
			<td>Magnetic purification beads</td>
			<td>GE Healthcare</td>
			<td>cat. no. 65152105050250</td>
			<td>sgRNA Synthesis</td>
		</tr>
		<tr>
			<td>Tris (2-carboxyethyl) phosphine hydrochloride (TCEP)</td>
			<td>Sigma-Aldrich</td>
			<td>cat. no. C4706</td>
			<td>RNP Buffer</td>
		</tr>
		<tr>
			<td>Tris-HCl solution, pH 8.5 molecular-biology grade</td>
			<td>Teknova</td>
			<td>cat. no. T1085</td>
			<td>Lysis Buffer</td>
		</tr>
		<tr>
			<td>Tween 20 molecular-biology grade</td>
			<td>Sigma-Aldrich</td>
			<td>cat. no. P7949-500</td>
			<td>Lysis Buffer</td>
		</tr>
	</tbody>
</table>

efficient genome editing of mice by crispr electroporation of zygotes

凭借卓越的效率、准确性和易用性，CRISPR/Cas9系统显著改善了细胞培养和实验动物实验中的基因组编辑。在生成动物模型时，受精卵电穿孔可提供更高的效率、简单性、成本和通量，作为显微注射金标准方法的替代方案。电穿孔也更温和，具有更高的活力，并且可靠地将Cas9/单向导RNA（sgRNA）核糖核蛋白（RNP）递送到常见实验室小鼠品系（例如C57BL / 6J和C57BL / 6N）的受精卵中，接近100%的递送效率。该技术可实现插入/缺失（插入缺失）突变、点突变、整个基因或外显子的缺失，以及 100-200 bp 范围内的小插入以插入 LoxP 或短标签，如 FLAG、HA 或 V5。在不断改进的同时，我们在这里展示了CRISPR-EZ的当前状态，其中包括通过 体外 转录，胚胎处理，RNP组装，电穿孔和植入前胚胎的基因分型产生sgRNA。具有最少操作胚胎经验的研究生水平的研究人员可以使用该协议在不到 1 周的时间内获得经过基因编辑的胚胎。在这里，我们提供了一种简单，低成本，高效，高容量的方法，可用于小鼠胚胎。

该方法可产生超过 100 μg 的 sgRNA（浓度为 >6，000 ng/L 时为 20 μL），可实现高效的 Cas9/sgRNA RNP 组装。这里描述的常规超排卵方法通常为每个插入的雌性产生10-20个活胚胎。由于与胚胎操作相关的处理错误和典型损失，预计80%的胚胎在电穿孔后受精，存活且状况良好。为了帮助研究人员执行成功的实验，我们提供了一个将小鼠Tyr位点作为阳性对照的示例策略（图6），包括寡核苷酸设计（图6A，...

Watch this Scientific Journal Video about Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes at JoVE.com

Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes

在这里，我们描述了一种用于高效生成转基因小鼠的简单技术，称为CRISPR RNP受精卵电穿孔（CRISPR-EZ）。该方法通过电穿孔将编辑试剂以接近100%的效率输送到胚胎中。该方案对哺乳动物胚胎中的点突变、小基因组插入和缺失有效。

通过受精卵的CRISPR电穿孔对小鼠进行高效基因组编辑

With exceptional efficiency, accuracy, and ease, the CRISPR/Cas9 system has significantly improved genome editing in cell culture and lab animal ...

这种方法可以检查胚胎中的引导RNA，测试早期发育问题或创建编辑小鼠。CRISPR Cas9的任何进展都可以用这种方法使用。这种方法的主要优点是易于学习，并且使用可用于接触小鼠的实验室的试剂。将来，该技术将有助于纠正伴侣动物或农业重要动物后代的疾病或突变。此方法最适合生成鼠标模型。在我们的实验室中，我们一直在使用这种方法的变体来研究围绕受精和早期植入前发育的事件，特别是胚胎效力中的基因调控。最具挑战性的部分是使用玻璃针将胚胎从一个盘子移动到另一个盘子。演示这个程序的将是Chantal Diallo，我实验室的研究专家。要开始胚胎收集和处理，请将安乐死的雌性小鼠放在其背上，并在腹部喷洒70%乙醇以手术打开。要打开腹腔并移除输卵管，请用手术剪刀切割皮肤层并找到卵巢。接下来，通过手术切除位于卵巢近端的两个输卵管，并将它们放入单个 50 微升的 M2 加 BSA 培养基液滴中。在体视显微镜下，使用解剖钳切开输卵管的壶腹以释放含有卵母细胞的卵丘-卵母细胞复合物或COC，然后为避免携带碎片，转移并将每个卵丘-卵母细胞合并为单个50微升的M2液滴加BSA，手柄移液器设置为20至30微升。接下来，为了从受精卵中去除卵丘细胞，将带有少量额外培养基的COC转移到100微升M2加透明质酸酶的液滴中，并通过移液轻轻混合，直到胚胎明显与更小的积云细胞分离。要稀释透明质酸酶并清除松散的积云细胞，请使用口服移液管将受精卵移动到新鲜的 50 微升 M2 加 BSA 液滴中。接下来，为了侵蚀胚胎的透明带并减少试剂递送的额外物理障碍，将胚胎转移到预热的 100 微升液滴中 酸性 Tyrode 或 AT 溶液，在 60 毫米板上。在体视显微镜下连续观察受精卵，直到30%的透明带被侵蚀，然后稀释AT，将胚胎通过四个50微升的M2加BSA液滴。接下来，通过在体视显微镜下计数来确定已处理的适当数量的胚胎。通过将胚胎通过两滴50微升还原血清培养基来稀释BSA。接下来，将25至30个胚胎移液到10微升的还原血清培养基液滴中，然后使用手持式移液管加入10微升核糖核蛋白或RNP复合物。从RNP复合物中稀释粘性甘油，并通过移液10次或直到混合物不再显示粘度来均质化样品。接下来，将 20 微升胚胎和 RNP 混合物移液到 0.1 厘米的电穿孔比色皿中，同时避免气泡，然后将编辑试剂递送到受精卵中，使用 30 伏、4 至 6 个脉冲、3 毫秒脉冲长度和 100 个脉冲间隔的方波方案电穿孔胚胎。接下来，将 50 微升 M2 加 BSA 加入比色皿顶部，从比色皿中冲洗胚胎，然后将该混合物轻轻移液到盘子上。对于胚胎培养，将20至30个受精卵转移到预平衡培养板中的KSOM加BSA液滴中，并将胚胎移动到液滴中。将板在 37 摄氏度下在 5% 二氧化碳和 95% 湿度中孵育过夜。第二天，仅将双细胞胚胎转移到KSOM加BSA混合物的新鲜液滴中，并将板返回培养箱。留下或丢弃死亡或未受精的胚胎。在基因分型之前，用两滴DPBS洗涤桑葚囊胚胚胎，以稀释可能干扰PCR反应的培养基成分。使用口移液管，将单个胚胎转移到8孔PCR条的一个孔中，并加入10微升裂解缓冲液。接下来，为了裂解胚胎，将热循环仪编程为55摄氏度4小时，然后在95摄氏度下孵育10分钟使蛋白酶K失活。在这项研究中，展示了一种靶向小鼠酪氨酸酶位点作为阳性对照的示例策略，包括寡核苷酸设计和非同源末端连接和同源定向修复的基因分型策略。当递送单个单向导或sgRNA时，C57黑色6J和C57黑6N小鼠品系中RNP的递送率高达100%，插入缺失形成率为50%至100%，基于寡核苷酸的小替换率为17%至63%当工程基因组缺失时，编辑效果从3%到100%不等胚胎应保持在尽可能接近理想条件，以便越快协议完成了，越好。此外，尽量减少接触透明质酸酶和酸性泰罗德。该方法描述了生成编辑过的动物，测试植入或妊娠活力，或多次执行实验以在种植前开发期间收集测量值或图像以及各种指标。

Research

JoVE Journal

Developmental Biology

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

在小鸡视网膜有效的基因转移的原代细胞培养研究：<em&gt;前OVO</em&gt;电穿孔法

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

科研

发育生物学

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

小鼠胚胎植耳蜗的文化和基因转移通过电穿孔

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

Genome-wide Snapshot of Chromatin Regulators and States in Xenopus Embryos by ChIP-Seq

染色质监管机构和各国的全基因组快照<em&gt;爪</em&gt;通过胚胎的ChIP-SEQ

The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and ...

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

基因转移到鸡听觉器官通过<em&gt;在卵</em&gt;微电

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

显微注射转基因的基因组，并在编辑Threespine刺鱼

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

CRISPR 介导的小脑颗粒细胞在宫内电穿孔基因转移中的功能分析损失

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

基于基因组编辑的果蝇组织特异二进制转录系统的高效生成策略

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

使用mRNA电穿孔快速有效地表达Avian胚胎中的多种蛋白质

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

卵子微弹射和有效交配的基因组编辑在火布拉特 热比亚家

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to ...