我们感谢朱利奥·迪米宁博士对phaESC线的推导和雷莫·弗雷曼博士的流动细胞术。我们还感谢米歇尔·沙夫纳女士和托马斯·亨内克先生在胚胎移植方面提供的技术支持。这项工作得到了瑞士国家科学基金会的支持（赠款31003A_152814/1）。

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作者没有什么可透露的。

哺乳动物的克隆体细胞核转移（SCNT）已率先在20世纪90年代19，20，21。这些发展是继30年前在两栖动物22号进行的克隆研究之后发生的。相当的延迟反映了哺乳动物胚胎学和基因组印记的困难。哺乳动物SCNT的发展是应用HAESC替代精子的基础，本协议对此进行了详细的说明。
细胞周期同步是SCNT23成功的重要因素。本协议中也注射了 HAESC。将供体基因组引入接受者需要匹配细胞周期阶段，以避免染色体破裂或会破坏胚胎发育的增异。半克隆具有额外的复杂性，两个基因组和一个细胞质需要兼容。先前的一份报告显示，注射M相逮捕和生化haESCs比将G1相haESC注射到卵母细胞7中，能产生更好的半克隆胚胎发育率。本报告建议将M相作为半克隆的合适同步点。因此，噬菌体在元相中与脱甲基辛一起被分层逮捕，并被注射到MII卵母细胞的卵母细胞中，这些卵母细胞在肌瘤的元二期中自然被捕。重要的是，M相阻塞可以在小鼠ESC中高效实现，为供体和受体细胞周期提供出色的同步。
在间歇期间，核膜分解，并形成复制染色体的主轴形式。注射M相DKO-噬菌体后，姐妹色度被隔离成伪极地体和酶7（图3E）。因此，来自DKO-phaESC的单组染色体有助于半克隆胚胎。至关重要的是，DKO-phaESC染色体的姐妹色度在注射后可以正确隔离。完整的DKO-phaESC的等离子膜防止分离成伪极地体。我们确实观察到极少数情况下，DKO-噬菌体的血浆膜没有破裂，胚胎在注射后在卵石中表现出完整的DKO-噬菌体（图3D）。因此，必须小心通过管道去除DKO-噬菌体的血浆膜。在注射过程中，同样重要的是避免卵母细胞的髓质主轴中断，这可能导致染色体分离缺陷，并诱发厌食症。
在哺乳动物中，基因组印记限制了噬菌体作为精子替代物的应用。部分遗传性 HAESC 具有基因组印记的母体配置，而精子具有父系配置。因此，在注射野生型phaESC作为精子替代后，没有出现过全期幼崽的生成。为了克服这一限制，在分阶段的实验室中设计了IG和H19-DMR的删除。在母体Igf2-H19和Gtl2-Dlk1 loci上修改印迹表达，足以改变基因组印记的配置，使半克隆小鼠的生成频率超过5.1%，基于转移的2细胞胚胎。这些观察表明，针对两个印迹基因将噬菌体的基因组转换成一种功能性的父系结构，可以取代小鼠的精子。然而，这一战略需要对相文进行永久性的基因改造。作为替代策略，可以考虑雄激素haESC。雄激素从精子基因组中提取，并具有父系印记。有报道说，野生型和遗传性海斯特作为精子替代，以1.3%至1.9%的频率产生全期幼崽转移胚胎4，7，24。也通过注射和生化haESC与IG-和H19-DMR的删除频率20.2%的转移24的全期幼崽获得。使用改性雄激素 haESC 进行半克隆效率的提高可能是因为印记在文化中可能变得不稳定。打印缺陷通过关键 DMR 的遗传删除来纠正。
考虑到将基因改造直接引入卵母细胞或精子基因组的困难，HAESC是单独操纵父母基因组的宝贵工具。使用haESC作为精子的替代品，为小鼠生殖系的基因组编辑提供了显著的优势。最近的一项研究将CRISPR/Cas9的基因组编辑与haESC的应用相结合，用于对胚胎发育至关重要的印记区域的特征。这项研究分析了Rasgrf1-DMR与H19和IG-DMR在双子小鼠发育中的作用，以及7种不同的DMR在双父小鼠发育中的作用。用haESC代替精子的方法为基因筛选方法奠定了基础，用于识别DND1蛋白中原始生殖细胞发育中的关键氨基酸，以及识别骨骼发育中的基因24、25、26。基因组印记和基因筛选研究，以确定胚胎发育的关键因素，是将HAESC应用于游戏基因组的重要方法。

在哺乳动物中，游戏是唯一向下一代传递遗传信息的细胞。卵母细胞和精子的融合形成了一种双体卵母细胞，发育成成年动物。因此，游戏基因组的突变是由后代遗传的，并推动物种1的基因变异。引入生殖系突变已应用于生产转基因动物的多样化生物学研究，包括基因功能特征和疾病建模。卵母细胞和精子细胞都是绝分和高度专业化的细胞，已经停止增殖。因此，游戏玩家的直接修改在技术上是困难的，并且已经开发出专门的方法。基因改造可以通过将转基因ESC注射到胚波细胞中引入小鼠生殖系，在那里它们融入发育中的胚胎并殖民生殖系。此外，使用基因组编辑方法（包括CRISPR/Cas9系统）对受精卵进行基因改造已被广泛采用。
最近，有报道说，一个杰出的方法，它应用haESC作为游戏基因组3，4，5，6，7，8的替代品。HaESC是干细胞系，源自部分遗传学或雄激素单体胚波细胞的内细胞质量，拥有一组4、7、9、10染色体。已经证明，在细胞内质注射后，部分遗传学和雄激素haESC都可以促进半克隆小鼠的基因组。与其他方法不同，由于自身更新能力，HAESC的基因组可以在培养中直接改变。通过用haESC代替精子，将基因改造引入生殖系，是生物研究的重要方法。它规定了分别培养和操纵产妇或父系基因组的可能性，这些基因组分别来自部分遗传学或雄激素haESC。然后，HaESC 可以用作游戏基因组替代，这尤其有利于基因组印记、等位基因特异性表达和父母特定过程的研究。
在小鼠中，正常胚胎发育需要母体和母体基因组信息。因此，当注射野生型部分遗传体（phaESCs）以取代精子基因组5，8时，无法获得全期幼崽。为了克服发育障碍，需要将部分遗传性 HAESC 母体基因组的基因组印记更正为父系结构。这可以通过操纵不同甲基化区域 （DMR） 来实现。迄今为止，已研究有针对性地删除H19-DMR、Gtl2-Dlk1 IG-DMR和拉斯格夫1-DMR，以抑制第3、5、8、12期的母体表达基因。这些研究表明，删除H19-DMR和IG-DMR都足以将母体转换为可以替代精子染色体的母体印印结构。将两个DMR缺失物移植到卵母细胞中的噬细胞内质注射产生半克隆幼崽，其频率在5.1%至15.5%之间。
本议定书基于删除 H19-DMR 和 IG-DMR的 phaESC 的应用，我们称之为双敲除式噬菌体 （DKO-PHAESCs）。我们为改变噬菌体中的基因组印记以建立DKO-phaESC线、将DKO-phaESC注入卵母细胞以取代精子基因组、将半克隆胚胎培养成胚胎和获得半克隆小鼠提供详细说明。该协议是研究人员的参考，他们需要精确和直接地操纵父系基因组和半克隆胚胎和小鼠的生成。

<table>
	<tbody>
		<tr>
			<td>1.5 mLTube</td>
			<td>Eppendorf</td>
			<td>0030 125.150</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>15 ml Tube</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Thermo Scientific</td>
			<td>150628</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Thermo Scientific</td>
			<td>167008</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Merck</td>
			<td>M6250</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>BSA fraction</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>CHIR 99021</td>
			<td>Axon</td>
			<td>1386</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Demecolcine solution</td>
			<td>Merck</td>
			<td>D1925</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41965-039</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>DMEM / F-12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>DR4 mouse</td>
			<td>The Jackson Laboratory</td>
			<td>3208</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Merck</td>
			<td>E5134</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>biowest</td>
			<td>S1810-500</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Freezing medium</td>
			<td>amsbio</td>
			<td>11897</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Merck</td>
			<td>G1890</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Hepes solution</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Irradiated MEF</td>
			<td>Gibco</td>
			<td>A34966</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>LIF (Leukemia inhibitory factor)</td>
			<td>(Homemade)</td>
			<td>-</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Lipofection reagent</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>NDiff 227</td>
			<td>Takara</td>
			<td>Y40002</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>PD 0325901</td>
			<td>Axon</td>
			<td>1408</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>piggyBac plasmid carrying a CAG-EGFP transgene</td>
			<td>Addgene</td>
			<td>#40973</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>piggyBac transposase plasmid</td>
			<td>System Biosciences</td>
			<td>PB210PA-1</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>pX330 plasmid</td>
			<td>Addgene</td>
			<td>#42230</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>pX458 plasmid</td>
			<td>Addgene</td>
			<td>#48138</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>DNA polymerase</td>
			<td>Thermo Scientific</td>
			<td>F549S</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>dNTP Mix</td>
			<td>Thermo Scientific</td>
			<td>R0192</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Merck</td>
			<td>100317</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Merck</td>
			<td>109634</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Axon</td>
			<td>A3830</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Merck</td>
			<td>71729</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>106404</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>ThermoMixer</td>
			<td>Epeendorf</td>
			<td>5384000012</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Merck</td>
			<td>T1503</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>5 mL Tube</td>
			<td>Falcon</td>
			<td>352063</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>5 mL Tube with cell strainer cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>MoFlo Astrios EQ</td>
			<td>Beckman Coulter</td>
			<td>B25982</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Codan</td>
			<td>62.1640</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>30-gauge 1/2 inch needle</td>
			<td>Merck</td>
			<td>Z192362-100EA</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>B6D2F1 mouse</td>
			<td>The Jackson Laboratory</td>
			<td>100006</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>hCG</td>
			<td>MSD animal health</td>
			<td>49451</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010015</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>Avivasysbio</td>
			<td>OPPA1037 5000 IU</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>10-cm dish</td>
			<td>Thermo Scientific</td>
			<td>150350</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>4-well plate</td>
			<td>Thermo Scientific</td>
			<td>179830</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>6 cm dish</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Center-well dish</td>
			<td>Corning</td>
			<td>3260</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Digital rocker</td>
			<td>Thermo Scientific</td>
			<td>88880020</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>AppliChem</td>
			<td>A0878</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Merck</td>
			<td>H4272</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>KSOM</td>
			<td>Merck</td>
			<td>MR-020P-5F</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>M16 medium</td>
			<td>Merck</td>
			<td>M7292</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>Merck</td>
			<td>M7167</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Merck</td>
			<td>M8410</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>Merck</td>
			<td>P1049-1PAK</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Strontium chloride</td>
			<td>Merck</td>
			<td>13909</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>5ml syringe</td>
			<td>Codan</td>
			<td>62.5607</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Borosilicate glass cappilary</td>
			<td>Science product</td>
			<td>GB100T-8P</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>CellTram 4r Oil</td>
			<td>Eppendorf</td>
			<td>5196000030</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Fluorinert FC-770</td>
			<td>Merck</td>
			<td>F3556</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Holding pipette</td>
			<td>BioMedical Instruments</td>
			<td>-</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-U</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>MF-900</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Microinjection pipette</td>
			<td>BioMedical Instruments</td>
			<td>-</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Microlaoder tips</td>
			<td>Eppendorf</td>
			<td>5252 956.003</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Micromanipulaor arms</td>
			<td>Narishige</td>
			<td>NT-88-V3</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>PiezoXpert</td>
			<td>Eppendorf</td>
			<td>5194000016</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>PVP (Polyvinylpyrrolidine)</td>
			<td>Merck</td>
			<td>PVP360</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>SARSTEDT</td>
			<td>83.1826.001</td>
			<td>Microinjection</td>
		</tr>
	</tbody>
</table>

application of mouse parthenogenetic haploid embryonic stem cells as a substitute of sperm

在具有性繁殖的生物体中，生殖细胞是发育成新个体的托蒂能细胞的来源。在小鼠中，由精子卵母细胞受精会产生一种托蒂波特酶。最近，一些出版物报道说，单体胚胎干细胞（haESCs）可以替代游戏基因组，并有助于胚胎，发展成小鼠。在这里，我们提出了一个协议，应用部分遗传性haESCs作为精子的替代品，通过细胞内注入卵母细胞来构建胚胎。该协议包括准备作为精子替代的haESC，将海斯C染色体注射到卵母细胞中，以及培养半克隆胚胎的步骤。胚胎在胚胎移植后可以产生肥沃的半克隆小鼠。使用haESC作为精子的替代物，有助于基因组编辑的生殖系，胚胎发育的研究，基因组印记的研究。

本协议的目的是应用HAESC作为精子的替代品来获得半克隆胚胎和小鼠。为此，生成并用于将携带CAG-EGFP转基因的DKO-phaESC线注入MII卵母细胞。为了获得一个合适的phaESC线与父亲的印记配置，我们进行了基因工程使用Cas9核糖核酸酶。类倍体ESC线包含由于双倍体化10的人身机器人ESC的内在倾向而产生的双体细胞。一组双倍体染色体是成功替代精子基因组的先决条件。通过流动细胞学进行的DNA含量分析显示，在G0/G1-、S和G2/M相（图2A）中，单体细胞和双体细胞的分布情况。
为了建立DKO-phaESC线，野生型phaESC线被传染成 小猪Bac 转基因结构，用于稳定的转基因EGFP表达，并与CRISPR/Cas9质粒一起用于获取 H19和IG-DMR的删除。为了排除二倍体 ESC 和仅隔离表达 EGFP 的生物体 ESC，定义了特定的排序门（图 2A）。然后将单单倍体EGFP阳性细胞镀入96井板的单独井中，以获得亚克隆。MEF喂料机用于提高转染性噬菌体的电镀效率和存活率。在文化扩展之后，PCR 进行了第一轮基因化，以识别同时删除两个 DMR 的子克隆。在MEF馈线被从培养物中移除后，进行了第二次基因对比，以确认 H19和IG-DMR（图2B）中缺乏野生型等位基因。从总共135个亚克隆中，我们获得了5个单体DKO-phESC线，这些线携带删除的等位基因，并且没有 H19-DMR和IG-DMR的野生型等位基因。
在DKO-噬菌体中引入了一种CAG-EGFP转基因，通过显微镜下绿色荧光的可视化来研究它们对半克隆胚胎的贡献（图3A）。对于细胞内注射，DKO-phaESC接受脱甲基辛治疗，在M相中逮捕他们。因此，DKO-噬菌体的细胞周期与MII卵母细胞的细胞周期同步。流动细胞学分析显示，在接受脱甲基苯丙酮（图3B）治疗后，2个人群对应于G2/M相被捕的双体细胞（2n）和双体细胞（4n）。没有1n单体峰值表明细胞周期逮捕已基本完成。然后对M相单体ESC进行排序并注入卵母细胞。为此，将单个DKO-phaESC加载到微注射移液器中，并注射到MII卵母细胞的细胞质（图3C）中。DKO-phaESC 的等离子膜通过管道插入微注入移液器的尖端而破裂。
注射后，在构造的半克隆胚胎中很少检测到EGFP表达，因为DKO-phaESC的细胞质已经分散在卵母细胞的大细胞质（图3D）中。在极少数情况下，在卵母体内可以观察到强烈的EGFP表达的圆点。这一观察可能是由于无意间注射了完好无损的DKO-噬菌体。如果DKO-phaESC细胞膜不能破裂，很可能与进一步的胚胎发育不相容，应避免。注射一小时后，胚胎被氯化钛18治疗激活。启动6小时后，在显微镜下观察到多达3个极地天体（图3E）。这些极地天体对应于卵母细胞的第一和第二极体，以及来自DKO-phaESC7的伪极地体。此外，在微分干扰对比显微镜下观察到两个原核，这类似于精子正常受精后酶的亲核阶段。
为了展示发育能力，半克隆胚胎被培养到胚胎年代（图5A）。此外，在将半克隆的2细胞阶段胚胎转移到受体雌性（图5B）的卵泡后，还获得了一只完整的小鼠。不出所料，从半克隆胚胎中提取的老鼠是雌性，因为卵母细胞和卵母细胞衍生的噬细胞都没有携带Y染色体。半克隆小鼠是明显正常的，并产生健康的后代时，与野生型瑞士韦伯斯特雄配。到目前为止，半克隆小鼠已经存活了600多天，没有任何明显的健康问题。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61999/61999fig01v3.jpg"> 图1:DKO-噬菌体作为精子替代物的应用概述。（A） 议定书程序的时限。（B） 该计划显示了建立DKO-phaESC线路的步骤（步骤1-6）。（C） 该计划显示了通过将DKO-phaESC注射到MII卵母细胞（步骤7-14）中构建半克隆胚胎的步骤。缩写: DKO = 双敲击:phaESC = 部分遗传体位胚胎干细胞;FACS = 荧光激活细胞排序;MEF=小鼠胚胎成纤维细胞。 <a href="https://www.jove.com/files/ftp_upload/61999/61999fig01large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61999/61999fig02v2.jpg"> 图2:由CRISPR/Cas9调解删除H19和IG-DMR建立DKO-phaESC线。 （A） 流细胞学分析后，与小猪Bac质粒传输后，稳定EGFP表达和4CRISPR/Cas9质粒。非转染性噬菌体显示为控制。DNA图谱（上图）显示单体细胞和双体细胞的细胞周期分布。表示EGFP的G1/S相单体细胞由绿色门（下图，右图）指示。（B） 在MEF（第一基因型）上生长的phaESC子克隆的基因型，以及移除MEF（第二代基因型）后生长的基因。亚克隆的phaESC线1、2、3和4代表野生型细胞，具有H19-DMR删除的细胞，具有IG-DMR删除，并分别具有H19和IG-DMR删除的组合。DKO-phaESC 5–8线同时具有H19-DMR和IG-DMR删除，并且不含野生型等位基因。缩写: DKO = 双敲击:phaESC = 部分遗传体位胚胎干细胞;DMR=微分甲基化区域;MEF=小鼠胚胎成纤维细胞:EGFP = 增强型绿色荧光蛋白;WT=野生类型。<a href="https://www.jove.com/files/ftp_upload/61999/61999fig02large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61999/61999fig03v3.jpg"> 图3:将被密密麻麻地逮捕的DKO-噬菌体注射到MII卵母细胞中。（A） 携带CAG-EGFP转基因和删除H19-和IG-DMR的DKO-phaESC文化的形态学: 比例杆 = 50μm. （B） DKO-噬菌体在被捕后与脱甲基苯丙氨酸8小时的代表流动细胞学分析。没有脱甲基辛治疗的DKO-噬菌体显示为控制。（C） 微注射移液器中DKO-噬菌体的形态学。显示在（左）和（右）通过管道破裂等离子膜之前（左）和之后的一个完整DKO-phaESC:比例杆 = 20μm. （D） 在将 DKO-噬菌体注射到 MII 卵母细胞后，在 1 小时内构建胚胎。显示了EGFP荧光和明亮场的合并图像。黑色箭头表示注射完好的DKO-噬菌体后强烈EGFP表达的圆点，应避免:比例杆 = 50 μm. （E） 显示使用氯化钛启动后 6 h 的半克隆胚胎的微分干扰对比图像。白色箭头表示有3个极地体的胚胎，包括注射的DKO-phaESC中的一个伪极地体:比例栏=50μm。缩写: DKO = 双敲击:phaESC = 部分遗传体位胚胎干细胞;EGFP = 增强型绿色荧光蛋白。<a href="https://www.jove.com/files/ftp_upload/61999/61999fig03large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61999/61999fig04.jpg"> 图4:将DKO-噬菌体注射到卵母细胞的细胞内质的设置方案。（A） 在注射室中显示注射移液器、手持式移液器和卵母细胞的排列情况。α，微投射移液器的弯曲角度。（B） 微操纵盘中用于内质注射的滴的布局。缩写: DKO = 双敲击:噬菌体=部分遗传体质胚胎干细胞。 <a href="https://www.jove.com/files/ftp_upload/61999/61999fig04large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61999/61999fig05.jpg"> 图5:半克隆胚胎的发育。（A） 体外半克隆胚胎的预植发育。EGFP表达最初在细胞内注射后的第2天在四细胞胚胎中观察到。在第 4 天，可在爆炸细胞中观察到强烈的 EGFP 表达:比例杆 = 100μm. （B） 2细胞胚胎移植给受体雌性后获得的半克隆小鼠。74 岁的 dpp ，半克隆小鼠（箭头）在与野生型瑞士韦伯斯特雄配后，自然分娩产下她的第一只幼崽（星号）。图中显示的半克隆胚胎和半克隆小鼠与艾泽等人所报道的胚胎相同。缩写:EGFP=增强型绿色荧光蛋白;。dpp，产后天数。 <a href="https://www.jove.com/files/ftp_upload/61999/61999fig05large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="4">明胶解决方案</td>
		</tr><tr><td>元件</td>
			<td>库存集中度</td>
			<td>体积/重量</td>
			<td>最终浓度</td>
		</tr><tr><td>水</td>
			<td>-</td>
			<td>500毫升</td>
			<td>-</td>
		</tr><tr><td>明胶</td>
			<td>-</td>
			<td>1 克</td>
			<td>0.2%</td>
		</tr><tr><td colspan="4"></td>
		</tr><tr><td colspan="4">类固醇胚胎干细胞（海斯）介质</td>
		</tr><tr><td>元件</td>
			<td>库存集中度</td>
			<td>体积/重量</td>
			<td>最终浓度</td>
		</tr><tr><td>恩迪夫 227</td>
			<td>-</td>
			<td>10升</td>
			<td>-</td>
		</tr><tr><td>奇尔 99021</td>
			<td>10米</td>
			<td>3μL</td>
			<td>3 微米</td>
		</tr><tr><td>PD 0325901</td>
			<td>10米</td>
			<td>1μL</td>
			<td>1 微米</td>
		</tr><tr><td>利夫</td>
			<td>1 x 106 IU/mL</td>
			<td>10 微升</td>
			<td>1，000 国际联盟/ML</td>
		</tr><tr><td>青霉素-链霉素</td>
			<td>100倍</td>
			<td>100μL</td>
			<td>1x</td>
		</tr><tr><td colspan="4"></td>
		</tr><tr><td colspan="4">海斯C维护缓冲区</td>
		</tr><tr><td>元件</td>
			<td>库存集中度</td>
			<td>体积/重量</td>
			<td>最终浓度</td>
		</tr><tr><td>海斯中等</td>
			<td>-</td>
			<td>1升</td>
			<td>-</td>
		</tr><tr><td>赫佩斯解决方案</td>
			<td>2 .</td>
			<td>20 微升</td>
			<td>20立方米</td>
		</tr><tr><td colspan="4"></td>
		</tr><tr><td colspan="4">洗涤缓冲区</td>
		</tr><tr><td>元件</td>
			<td>库存集中度</td>
			<td>体积/重量</td>
			<td>最终浓度</td>
		</tr><tr><td>德国马克/F-12</td>
			<td>-</td>
			<td>100毫升</td>
			<td>-</td>
		</tr><tr><td>BSA 分数</td>
			<td>7.5%</td>
			<td>7.1升</td>
			<td>0.5%</td>
		</tr><tr><td colspan="4"></td>
		</tr><tr><td colspan="4">梅夫介质</td>
		</tr><tr><td>元件</td>
			<td>库存集中度</td>
			<td>体积/重量</td>
			<td>最终浓度</td>
		</tr><tr><td>德梅姆</td>
			<td>-</td>
			<td>500毫升</td>
			<td>-</td>
		</tr><tr><td>FBS</td>
			<td>-</td>
			<td>56升</td>
			<td>10%</td>
		</tr><tr><td>β-默卡普托乙醇</td>
			<td>14.3 毫升/升</td>
			<td>3.9 微升</td>
			<td>100 微米</td>
		</tr><tr><td>青霉素-链霉素</td>
			<td>100倍</td>
			<td>5.6升</td>
			<td>1x</td>
		</tr><tr><td colspan="4"></td>
		</tr><tr><td colspan="4">裂解缓冲区</td>
		</tr><tr><td>元件</td>
			<td>库存集中度</td>
			<td>体积/重量</td>
			<td>最终浓度</td>
		</tr><tr><td>水</td>
			<td>-</td>
			<td>8.25毫升</td>
			<td>-</td>
		</tr><tr><td>特里斯-赫克 （pH 8.5）</td>
			<td>1 M</td>
			<td>1升</td>
			<td>100立方米</td>
		</tr><tr><td>埃塔</td>
			<td>0.5 米</td>
			<td>100μL</td>
			<td>5米</td>
		</tr><tr><td>SDS 解决方案</td>
			<td>10%</td>
			<td>200μL</td>
			<td>0.2%</td>
		</tr><tr><td>纳克</td>
			<td>5M</td>
			<td>400μL</td>
			<td>200立方米</td>
		</tr><tr><td>蛋白酶 K</td>
			<td>20毫克/升</td>
			<td>50 微升</td>
			<td>100微克/升</td>
		</tr><tr><td colspan="4"></td>
		</tr><tr><td colspan="4">激活介质</td>
		</tr><tr><td>元件</td>
			<td>库存集中度</td>
			<td>体积/重量</td>
			<td>最终浓度</td>
		</tr><tr><td>克索姆</td>
			<td>-</td>
			<td>1升</td>
			<td>-</td>
		</tr><tr><td>氯化钛</td>
			<td>0 升</td>
			<td>5 微升</td>
			<td>5米</td>
		</tr><tr><td>埃格塔</td>
			<td>0.5 米， pH 8.0</td>
			<td>4μL</td>
			<td>2米</td>
		</tr><tr><td colspan="4"></td>
		</tr><tr><td colspan="4">PVP 解决方案</td>
		</tr><tr><td>元件</td>
			<td>库存集中度</td>
			<td>体积/重量</td>
			<td>最终浓度</td>
		</tr><tr><td>M2 介质</td>
			<td>-</td>
			<td>5升</td>
			<td>-</td>
		</tr><tr><td>PVP</td>
			<td>-</td>
			<td>0.6 克</td>
			<td>12%</td>
		</tr><tr><td colspan="4"></td>
		</tr><tr><td colspan="4">PMSG 解决方案</td>
		</tr><tr><td>元件</td>
			<td>库存集中度</td>
			<td>体积/重量</td>
			<td>最终浓度</td>
		</tr><tr><td>PBS</td>
			<td>-</td>
			<td>450μL</td>
			<td>-</td>
		</tr><tr><td>PMSG</td>
			<td>500 国际联盟/mL</td>
			<td>50 微升</td>
			<td>50 国际大学/百万升</td>
		</tr><tr><td colspan="4"></td>
		</tr><tr><td colspan="4">hCG 解决方案</td>
		</tr><tr><td>元件</td>
			<td>库存集中度</td>
			<td>体积/重量</td>
			<td>最终浓度</td>
		</tr><tr><td>PBS</td>
			<td>-</td>
			<td>450μL</td>
			<td>-</td>
		</tr><tr><td>hCG</td>
			<td>500 国际联盟/mL</td>
			<td>50 微升</td>
			<td>50 国际大学/百万升</td>
		</tr><tr><td colspan="4"></td>
		</tr><tr><td colspan="4">海卢罗尼达塞介质</td>
		</tr><tr><td>元件</td>
			<td>库存集中度</td>
			<td>体积/重量</td>
			<td>最终浓度</td>
		</tr><tr><td>M2 介质</td>
			<td>-</td>
			<td>380 微升</td>
			<td>-</td>
		</tr><tr><td>透明质酸酶</td>
			<td>10毫克/升</td>
			<td>20 微升</td>
			<td>0.5毫克/升</td>
		</tr><tr><td colspan="4"></td>
		</tr><tr><td colspan="4">缩写:LIF = 白血病抑制因子;MEF=小鼠胚胎成纤维细胞:</td>
		</tr><tr><td colspan="4">FBS=胎儿牛血清;DMEM = 杜尔贝科的改装鹰介质;BSA=牛血清白蛋白;</td>
		</tr><tr><td colspan="4">EDTA = 乙酰胺乙酰酸;SDS = 钠二硫酸盐;EGTA = 乙烯-比斯（氧化乙烯三氯苯甲酸）四乙酸;</td>
		</tr><tr><td colspan="4">PBS = 磷酸盐缓冲盐水;PVP=聚乙烯丙酮;hCG=人类胆汁性腺激素:</td>
		</tr><tr><td colspan="4">PMSG=怀孕的母马血清性腺激素。</td>
		</tr></tbody></table>表1:中等、缓冲和解决方案的配方。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>名字</td>
			<td colspan="2">序列 （5' 到 3'）</td>
			<td>应用</td>
		</tr>
<tr>
<td>H19-DMR-P1</td>
			<td colspan="2">格特格特·阿格特·阿塔·塔格·格格</td>
			<td>基因型平</td>
		</tr>
<tr>
<td>H19-DMR-P2</td>
			<td colspan="2">阿加T格格特猫TCT抄送</td>
			<td>基因型平</td>
		</tr>
<tr>
<td>H19-DMR-P3</td>
			<td colspan="2">泰克塔克阿格特克特克特克特克特克特格特</td>
			<td>基因型平</td>
		</tr>
<tr>
<td>IG-DMR-P1</td>
			<td colspan="2">特格特·格卡·加卡·阿格·卡格·阿格·阿格</td>
			<td>基因型平</td>
		</tr>
<tr>
<td>IG-DMR-P2</td>
			<td colspan="2">卡卡A一个CCT C卡</td>
			<td>基因型平</td>
		</tr>
<tr>
<td>IG-DMR-P3</td>
			<td colspan="2">阿塔· Cga 塔克 · 格格 · 阿卡 · 卡亚 Cg</td>
			<td>基因型平</td>
		</tr>
<tr>
<td>H19-DMR-格纳1-F</td>
			<td colspan="2">中航CCA TGA法案卡格阿格阿加CTG</td>
			<td>格纳</td>
		</tr>
<tr>
<td>H19-DMR-格纳1-R</td>
			<td colspan="2">A CCA GTC技术通信技术贸易委员会</td>
			<td>格纳</td>
		</tr>
<tr>
<td>H19-DMR-格纳2-F</td>
			<td colspan="2">中航卡格GTG阿加AA行动GCT加格</td>
			<td>格纳</td>
		</tr>
<tr>
<td>H19-DMR-格纳2-R</td>
			<td colspan="2">A CCT卡格卡格特克特克特克</td>
			<td>格纳</td>
		</tr>
<tr>
<td>IG-DMR-格纳1-F</td>
			<td colspan="2">中航 CCG TAC 阿加 GCT 中航</td>
			<td>格纳</td>
		</tr>
<tr>
<td>IG-DMR-格纳1-R</td>
			<td colspan="2">A CGT广电联ATG加格贸易委员会TGT交流</td>
			<td>格纳</td>
		</tr>
<tr>
<td>IG-DMR-格纳2-F</td>
			<td colspan="2">中航CCT GCT标签AGG塔克塔克广义</td>
			<td>格纳</td>
		</tr>
<tr>
<td>IG-DMR-格纳2-R</td>
			<td colspan="2">A卡格·克格特·阿格特·阿格·阿格·卡格</td>
			<td>格纳</td>
		</tr>
</tbody></table>表2:寡核苷酸清单。

Watch this Scientific Journal Video about Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm at JoVE.com

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

本文旨在证明使用部分遗传性单体胚胎干细胞作为精子的替代品，用于构建半克隆胚胎。

应用小鼠部分遗传类胚胎干细胞作为精子的替代品

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

application-mouse-parthenogenetic-haploid-embryonic-stem-cells-as

Research

JoVE Journal

Developmental Biology

4.5K 次观看.  ETH Zurich. 本文旨在证明使用部分遗传性单体胚胎干细胞作为精子的替代品，用于构建半克隆胚胎。

视频: Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

从胚胎干细胞衍生心脏祖细胞

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

科研

发育生物学

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

心脏分化的表观遗传调控胚胎干细胞和组织

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

小鼠胚胎Hemogenic内皮细胞的分离

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

在无进料和异种定义的条件下,从人类多能干细胞中血性内皮物分化

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...