Name: trans-inner cell mass injection of embryonic stem cells leads to higher chimerism rates
Uploaded: 2018-05-29T13:00:00
Description: Watch this Scientific Journal Video about Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates at JoVE.com

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) ?...

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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>b-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&#60;c-2J&#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 ...

Research

JoVE Journal

Biology

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

胚胎干细胞衍生的内皮细胞治疗肢体缺血

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

肝细胞样细胞从人类胚胎干细胞群的鲁棒代

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

使用LysoTracker检测及鉴别胚胎干细胞在胚胎细胞程序性死亡

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Induction and Analysis of Epithelial to Mesenchymal Transition

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...