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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</ol>

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>&amp;beta;-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 Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

科研

发育生物学

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

使用冷冻胚胎进行转基因小鼠高效生产

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

遗传学

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

CRISPR/Cas9介导的胚胎干细胞中的高效基因靶向，用于开发基因操纵的小鼠模型

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

<meta charset="utf8"></head><body>小鼠圆形精子细胞注射程序研究胚胎发育

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

<meta charset="utf8"></head><body>小鼠圆形精子细胞注射

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

小鼠圆形精子细胞注射

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

用于单细胞测序的原肠胚胚分离和基因分型

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

作者聚焦：以单细胞分辨率分析谱系特异性突变胚胎的管道

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

生殖细胞的移植和睾丸组织在小鼠异种移植

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

应用小鼠部分遗传类胚胎干细胞作为精子的替代品

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从小鼠胚胎植入前胚胎干细胞系的推导

使用冷冻胚胎进行转基因小鼠高效生产

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突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

突变全小鼠胚胎的单细胞 RNA 测序：从上胚层到原肠胚形成结束

生殖细胞的移植和睾丸组织在小鼠异种移植