Name: derivation of mouse trophoblast stem cells from blastocysts
Uploaded: 2010-06-08T17:03:00
Description: Watch this Scientific Journal Video about Derivation of Mouse Trophoblast Stem Cells from Blastocysts at JoVE.com

作者感谢珍妮特Rossant原协议。这项研究是由国家健康授予CA106308到WH研究所的支持。

在这个协议中，我们描述了小鼠囊胚的编制和TS细胞株的建立。一般的鼠标操作前囊胚的集合，包括设立自然交配和超排卵诱导，基本上是按照标准的协议，由纳吉等人说明。2003年（pp146 - 150）。 Ts细胞的推导和维护<ol><li>设置之间的利益老鼠繁殖的十字架。 </li><li>准备作为饲养层细胞的小鼠胚胎成纤维细胞（MEFs）。前两天收集囊胚，MEFs（2 × 10 6细胞...

<ol>
	<li>Chiu, S. Y., Asai, N., Costantini, F. &. a. m. p. ;. a. m. p., Hsu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-specific+protease+2+is+essential+for+modulating+p53-Mdm2+in+development+of+trophoblast+stem+cell+niches+and+lineages.">SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.</a> PLoS Biol. 6, E310-E310 (2008).</li><li>Kunath, T., Arnaud, D., Uy, D. G., Okamoto, I., Chureau, C., Yamanaka, Y., Heard, E., Gardner, R. L., Avner, P., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinted+X-inactivation+in+extra-embryonic+endoderm+cell+lines+from+mouse+blastocysts.">Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.</a> Development. 132, 1649-1661 (2005).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: The Laboratory Manual. , (2003).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

在这个视频中，我们展示的过程来收集E3.5从子宫和实验程序的囊胚建立TS细胞株。我们还描述了条件，保持Ts细胞的干性和诱导分化的细胞，其分化。两个关键的步骤，以取得纯TS细胞株的产物分解的时间（步骤9）和加工的第一段（第11步）。据报道，原始内胚层来源的细胞可以在文化产生广泛的囊胚后在第11步（Kunath等人，2005年）在第9步或繁茂的TS殖民地（&gt; 50％汇合），长大了。虽然原始内?...

试剂 TS介质： RPMI - 1640年，20％胎牛血清，1毫米丙酮酸钠，100毫米β-巯基乙醇和100毫克/毫升的青霉素，链霉素。 丝裂霉素C： 2毫克丝裂霉素C（Sigma公司的M - 0503）2毫升PBS溶解，并加入到200毫升的TS中（10毫微克/毫升）2毫升的混合物。直到使用20等分（10毫升）并储存在-20 ° C。新增100毫米板的每一个等份。 小鼠胚胎成纤维细胞条件培养基（MEF - CM）： 板MEFs（1 × 10 6 /板）在100毫米的钢板。第二天，添加10毫升，每道菜的TS介质，并持续48小时的文化。收集培养液，过滤（0.45毫米），并储存在-20 ° C在35毫升分装。解冻每个等分需要时可在4 ° C储存MEFs可用于准备之前，他们成为融合两个批次的CM。 PBS / BSA： 通过0.45毫米的注射器过滤溶解BSA的PBS（10毫升），过滤器，分数在五（西格玛A3311，0.1％（W / V））和1毫升分装在1.5毫升管。贮存于-70℃，解冻一管时，需要准备FGF4。 FGF4（1000倍）： 悬浮冻干FGF4（西格玛F8424，25毫克），1毫升的PBS / BSA溶液。充分混匀，10等份（100毫升）1.5毫升管和存储，在-70 ° C。在需要时解冻每管和储存其余4 ° C时，不要再冻结。在TS液稀释1000倍获得1X FGF4（25毫微克/毫升）。 肝素（1000倍）： 悬浮肝素在PBS（Sigma公司H3149，10000台）至终浓度为1毫克/毫升（1000倍）。设为100毫升分装到1.5毫升管和储存在-70 ° C。解冻等份，当需要存储的其余4 ° C时，不重新冻结。在TS培养基稀释1000时间获得1X肝素（1毫微克/毫升）。

derivation of mouse trophoblast stem cells from blastocysts

滋养层的规范，是最早的哺乳动物发育分化事件之一。从滋养层介导植入派生和滋养细胞谱系产生胎儿胎盘的一部分。因此，本宗族的发展是必不可少的胚胎存活率。最初是由田中滋养干细胞（TS）从小鼠囊胚细胞的推导<em&gt;等。</em&gt; 1998年。 Ts细胞的能力，以保持适当的刺激后的滋养特定的属性和阶段和细胞类型特异性标志物的表达提供了一个有价值的模型系统研究滋养细胞谱系的发展，即扼要早期的胎盘事件。此外，滋养细胞，少数细胞发生的自然基因组扩增的类型之一。虽然底层滋养多倍体的分子途径已经开始显现出来，滋养基因组扩增的生理作用和优势在很大程度上仍然难以捉摸。多倍体的文化滋养细胞的干细胞二倍体的发展，使得这<em&gt;体外</em&gt;系统阐明基因组复制和不稳定的监管机制，在健康和疾病的优良工具。在这里，我们描述在邱修改以前发表的报告为基础的协议<em&gt;等。</em&gt; 2008年。

Watch this Scientific Journal Video about Derivation of Mouse Trophoblast Stem Cells from Blastocysts at JoVE.com

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

在这段视频中，我们展示了小鼠囊胚的隔离和滋养干细胞从胚泡的推导。我们还介绍了干细胞财产的维护，以及在文化的分化诱导条件。

鼠标滋养层干细胞从胚泡的推导

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Research

JoVE Journal

Biology

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 ...

科研

生物学

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 ...

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 ...

Isolation of Retinal Stem Cells from the Mouse Eye

从鼠眼视网膜干细胞的分离

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

分离小鼠原代滋养细胞及滋养细胞侵袭实验

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

高效的推导人类心脏的前体和从多能干的小分子诱导人类胚胎干细胞的心肌细胞

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

在文化序列富集的精原干细胞和祖细胞（SSCS）推导长期成年小鼠SSC线

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

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 ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...