这项研究得到了 (部分) 的支持, 由美国国立卫生研究院 NIH 的内部研究计划。特别感谢 Shyamal Peddada 的统计分析。我们还要感谢汉弗瑞, 加里鸟, 和雪荒尾照片, 以审查这份手稿, 和佛朗哥 DeMayo 继续支持和建议。

所有操作都是作为 NIEHS 击出鼠标核心设施的正常操作的一部分进行的。所有的老鼠都保持在12:12 小时的光: 黑暗的循环, 并喂养 NIH-31 和水的饮食ad 随意.所有的实验都是按照国家环境健康科学研究所的动物护理和使用委员会进行的。
1. 动物制剂
<ol><li>培育捐赠小鼠, B6 (cg)-Tyr < c-2J >/J 雌性小鼠在 8-10 周自然 B6 (Cg)-Tyr < c-2J >/j 雄性小鼠在8和26周的年龄。第二天早上 (E0.5) 目视检查插头。</li>
	<li>养殖受体小鼠, 瑞士韦氏雌性小鼠在6和9周的年龄是自然繁殖输精管切除术瑞士韦氏雄性小鼠。第二天早上 (E0.5) 目视检查插头。</li>
</ol>2. ES 细胞制剂
<ol><li>对于 129-derieved AB2.2 ES 细胞, DMEM 的培养细胞补充15% 的血清, 1% 谷氨酰胺, 1% 佩恩/链球菌, 0.1% β巯基乙醇。使用推荐的协议, 每 es 细胞供应商的说明中培养所有其他 es 细胞。</li>
</ol>3. 胚胎采集
<ol><li>
动物解剖
	<ol>
<li>准备60毫米的菜肴, 大约1每5小鼠加上3额外, 与10毫升爆炸收集媒体 (立方米) (10% DMEM) 和一个4个好菜与 0.5 mL 每井。平衡在37°c, 5% CO2。</li>
		<li>弄死 B6 (Cg)-Tyr < c-2J >/J 母鼠 E3.5 使用 CO2窒息, 流速为10%。</li>
		<li>euthanizing 后, 将小鼠置于仰卧位, 用70% 乙醇彻底湿润腹部。</li>
		<li>通过皮肤的第一切口, 在腹部的中点, 使开口尽可能宽。接下来通过腹壁打开, 再次使切口尽可能宽。 注意: 在两个步骤中做到这一点有助于减少或消除毛皮污染。</li>
		<li>从卵巢开始, 用钝钳抱住卵巢的脂肪垫, 小心地移除卵巢、输卵管和子宫, 格外小心, 使脂肪组织保持最小。将子宫作为单个单位或单独的角移除, 以下过程不发生任何变化。</li>
		<li>取出后, 将子宫放置在含有爆炸收集介质的60毫米盘中。</li>
	</ol>
</li>
	<li>
胚胎切除
	<ol>
<li>准备一个10毫升的注射器, 装满了立方米, 并配合25克针。</li>
		<li>一次子宫, 将其从所持皿中取出, 将其涂抹在组织上以去除多余的血液, 并在低放大率解剖显微镜下放置在60毫米的盘子中。</li>
		<li>当仔细地和轻轻地举在远端末端 (在输卵管附近) 与解剖钳的子宫, 插入针在子宫的中心在平行一架飞机尽可能。插入针只是在钳的尖端。</li>
		<li>对镊子施加一些压力, 将子宫放到针上, 然后通过子宫角排出大约1毫升的立方米。 注意: 在这一步必须小心, 因为太多的压力, 注射器会导致一个无法控制的媒体流, 经常离开盘子。</li>
		<li>冲洗后的子宫, 多达5整个子宫每60毫米菜, 把菜放回孵化器, 直到收集。</li>
	</ol>
</li>
	<li>
胚胎采集
	<ol>
<li>使用手拉玻璃吸管和嘴吸管装置, 收集在解剖范围内的囊胚, 设置在25X 和35X 之间。(图 1)。 注: 口吸管装置由 30-40 厘米的挠性油管组成, 在5毫米的 id 下插入1、000µL 吸管尖端, 首先指向一端, 并在其上贴上玻璃吸管。添加注射器过滤器到另一端, 附有 1 cc 注射器桶, 没有柱塞, 作为一个口片。</li>
		<li>从孵化器中取出菜肴, 使用网格模式, 检查盘子中的每一部分是否有胚胎。胚胎被单独拾起, 并放置在第二道菜的一滴。</li>
		<li>从所有的菜肴中收集后, 再在一滴新鲜的立方米中再洗一次, 然后放在4井的盘子里等待注射。</li>
	</ol>
</li>
</ol>4. 胚胎注射
<ol><li>根据确认的注射针分别取 ES 细胞。<ol>
<li>通过加入 5-7 毫升的注射培养基 (DMEM, 10% 血清和20毫米 HEPES), 将注射盘准备成一个玻璃底部注射皿。注射是在室温下进行的。</li>
		<li>使用空气或油 microinjector, 将真空应用于注射针, 以吸引 ES 细胞。注意尽量在细胞间保持尽可能少的空间。计算 ES 细胞在这里, 或在注射步骤。</li>
		<li>选择与普通细胞相比, 大小适中的 ES 细胞, 表面光滑, 无明显的缺陷, 如空泡。 注意: ES 细胞的形态质量有很大差异, 所使用的标准将需要与之进行调整。</li>
	</ol>
</li>
	<li>注射大约 15 ES 细胞的胚胎 (图 1)。<ol>
<li>将持有吸管尖端连接到第二个空气 microinjector, 并将胚胎定位在支架的开口附近。应用真空将胚胎固定在支架上。</li>
		<li>通过将保持吸管向上或向下移动来调整 z 轴, 这样胚胎才会接触到滑块, 然后细聚焦在透明的上。接下来, 调整注射针的 Z 轴, 使尖端附近的 ES 细胞处于焦点。</li>
		<li>现在调整胚胎, 通过轻轻推从顶部或底部注射针, 旋转它把 ICM 在理想的位置。</li>
		<li>使用一个坚定但控制的推力插入注射针通过透明的, 或者 ICM 或滋养层。对注射针的 microinjector 施加轻微的压力, 以帮助防止囊胚崩溃。</li>
		<li>一旦针穿透囊胚, 施加 microinjector 的压力, 将 ES 细胞转移到胚胎的囊胚。短暂停顿, 因为针的斜角开始离开胚胎, 使胚胎中的压力恢复正常, 同时仍保留细胞。 注: 参考组 es 细胞正常注射, 遵循传统 es 细胞注射协议9, 注意不要接触 ICM。ES 细胞也被注入通过 icm, 直接推针通过 icm (TICM) (图 2, 补充电影)。</li>
	</ol>
</li>
</ol>5. eEmbryos 的转让
<ol><li>在 E2.5, 使用非手术胚胎转移装置, 根据制造程序, 将胚胎移植到瑞士韦伯斯特小鼠体内。这种非手术程序不需要麻醉或特殊术后护理。在胚胎移植后, 通过分娩和随后的断奶, 单独喂养小鼠。</li>
</ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+knockout+mouse+project.">The knockout mouse project.</a> Nat Genet. 36 (9), 921-924 (2004).</li><li>Coleman, J. L., Brennan, K., Ngo, T., Balaji, P., Graham, R. M., Smith, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Knockout+and+Reporter+Mouse+Line+Generation+and+Breeding+Colony+Establishment+Using+EUCOMM+Conditional-Ready+Embryonic+Stem+Cells:+A+Case+Study.">Rapid Knockout and Reporter Mouse Line Generation and Breeding Colony Establishment Using EUCOMM Conditional-Ready Embryonic Stem Cells: A Case Study.</a> Front Endocrinol (Lausanne). 30, 105 (2015).</li><li>Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., Roder, J. 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作者没有相互竞争的利益来披露。

长期以来, 人们一直认为 ICM 的任何干扰都可能导致死亡率, 事实上, 目前的胚泡微注射协议仍然警告此12,13。我们在这里显示的是, 通过 ICM 的显微注射不损害胚胎, 并增加合体的产量。
这种效果的确切机制尚未确定。然而, ICM 的机械干扰, 以及伴随的 hypoblast, 应将 ES 细胞的发病率增加到新形成的 ICM 后, 显微注射。最近在 iPSCs 领域的...

近25年来, 基因靶向小鼠的产生一直是一个缓慢的过程, 经常受到胚胎干细胞微注射阶段的瓶颈的阻碍, 生殖传送合体的产生。最近的进展, 包括 CRISPR/Cas91、2针对 ES 细胞和高吞吐量 es 细胞生成联盟 (如 KOMP), 改善了基因改良 ES 细胞的生成/可用性。然而, 生殖合体的产生仍然是从这些 ES 细胞中创建基因修饰小鼠的瓶颈3,4。高吞吐量 es 细胞生成项目受到 es 细胞质量高变异性的困扰, 这对于成功创建生殖合体至关重要。此外, 一些常用 ES 细胞系以高体检著称, 生殖主管合体5难以生产。
已经开发了许多方法来创建具有更高的嵌合体的老鼠, 或完全 ES 细胞衍生的老鼠6,7,8,9,10。虽然这些系统都有自己的优点, 但也有其缺点。生成的二、四倍体杂交合体, 同时生成 100% ES 细胞派生的小鼠, 是低效的, 通常限于 outbred线5, 6.新的注入桑椹胚的方法可以产生高百分比合体, 接近 100%, 但通常需要大量额外设备 (激光器或 piezos) 和训练 10, 11.在这些技术已经到位的实验室里, 可能没有必要采用这种新的方法。
本研究的目的是寻找一种利用常用技术和现成设备来提高嵌合体生成率的方法, 增加突变等位基因生殖传播的几率, 建立后续的小鼠群。

<table><tbody><tr><td>ES Cell injection needle</td><td>Humagen</td><td>MSC-20-0</td><td></td></tr><tr><td>NSET device</td><td>Paratechs</td><td>60010</td><td></td></tr><tr><td>DMEM</td><td>Gibco (life technologies)</td><td>11965-092</td><td></td></tr><tr><td>FBS</td><td>Gibco (life technologies)</td><td>10439-024</td><td>lots should be individually tested for ES Cell compatibility.</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H0887</td><td></td></tr><tr><td>&amp;#946;-mercaptoethanol</td><td>Sigma</td><td>M7522</td><td></td></tr><tr><td>Micro manipulator</td><td>Leica</td><td>Micro manipulator</td><td></td></tr><tr><td>micro injector</td><td>Eppendorf AG</td><td>Cell Tram Air 5176</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>DM IRB</td><td></td></tr><tr><td>4 well dish</td><td>Thermo Scientific</td><td>176740</td><td></td></tr><tr><td>60 mm dish</td><td>Sarstedt</td><td>83.3901</td><td></td></tr><tr><td>B6(Cg)-Tyr&amp;#60;c-2J&amp;#62;/J</td><td>Jackson Labs, Bar Harbor, Maine, USA</td><td>000058</td><td></td></tr><tr><td>Swiss Webster mice</td><td>Taconic, USA</td><td>Tac:SW</td><td></td></tr><tr><td>Injection Dish</td><td>MatTek Corp.</td><td>P50G-0-30-F</td><td></td></tr></tbody></table>

trans-inner cell mass injection of embryonic stem cells leads to higher chimerism rates

为了通过 ES 细胞方法提高转基因小鼠的效率, 我们提出了一种对目前囊胚注射协议的适应。在这里, 我们报告, 胚胎的简单旋转, 并通过跨内细胞质量 (TICM) 的注射增加了嵌合体小鼠的百分比从31% 到 50%, 没有额外的设备或进一步的专门训练。在9月内注射了26种不同的近交无性系克隆和35株总无性系。传统注射技术与 TICM 的妊娠率、胚胎恢复率均无显著性差异。因此, 如果没有任何重大改变的注射过程和简单的定位胚泡和注射通过 ICM, 释放 ES 细胞进入囊胚腔可以有可能提高嵌合生产的数量和随后生殖传输。

以细胞系为随机效应, 采用混合效应线性模型分析软件中的数据 (如 SAS (9.2 版))。每种分析都控制着移植胚胎的数量和细胞系。第二种分析还控制了存活的幼崽数量。
在这项研究中, 每种 ES 细胞系和克隆均采用传统的和 TICM 的技术, 在每个胚胎后交替使用。注射后, 胚胎被隔离在盘子上, 并分组的转移, 一般在12和15胚胎每接受女?...

Watch this Scientific Journal Video about Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates at JoVE.com

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

在这里, 我们提出了一个协议, 以增加嵌合体生产不使用新设备。一个简单的方向改变的胚胎注射可以增加胚胎的数量, 并有可能缩短时间的生殖传播。

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

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10.2K 次观看.  NIEHS/NIH. 在这里, 我们提出了一个协议, 以增加嵌合体生产不使用新设备。一个简单的方向改变的胚胎注射可以增加胚胎的数量, 并有可能缩短时间的生殖传播。

视频: Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

与预先定义的T细胞受体特异性Transnuclear小鼠对弓形虫获得通过体细胞核移植

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

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免疫与感染

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

方法细胞或DNA的精确本地化转移到着床后早期小鼠胚胎

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

发育生物学

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.

We describe a dramatically improved method for mouse cloning using trichostatin A, vitamin C, and deionized bovine serum albumin. We show a simplified, reproducible protocol that supports efficient development of cloned embryos. Hence, this method could become a standardized procedure for mouse cloning.

Trichostatin a 和维生素 C 的组合治疗提高体细胞细胞核移植克隆小鼠的效率

Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

生物学

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Intravenous and Intra-amniotic In Utero Transplantation in the Murine Model

We describe a protocol for performing an in utero transplantation (IUT) through intravenous and intra-amniotic routes of injection in the murine model. This protocol can be used to introduce cells, viral vectors, and other substances into the unique immune-tolerant fetal environment.

小鼠宫内移植中静脉和羊膜内植入的研究

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.

Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. This video shows step-by-step procedures for how to manipulate medaka embryos, including dechorionation, mounting in agarose for imaging and cell transplantation for the production of chimeras. These procedures are essential to use medaka and zebrafish in a laboratory to take full advantage of their complementary features for the genetic dissection of vertebrate genome functions.

鳉胚胎和细胞移植嵌合体生成Dechorionation

Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

人类干细胞异种移植到鸡胚

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

单细胞转染鸡胚

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

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 ...