鼠标滋养层干细胞从胚泡的推导
Summary
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。
Abstract
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>等。</em> 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>体外</em>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>等。</em> 2008年。
Protocol
Discussion
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（> 50％汇合），长大了。虽然原始内?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
Reagents
TS medium:
RPMI-1640, 20% FBS, 1 mM sodium pyruvate, 100 mM beta-mercaptoethanol and 100 mg/ml penicillin-streptomycin.
Mitomycin C:
Dissolve 2 mg mitomycin C (Sigma M-0503) in 2 ml PBS and add this 2 ml mixture into 200 ml TS medium (10 ng/ml). Make 20 aliquots (10 ml) and store at -20°C until use. Add one aliquot per 100 mm plate.
Mouse embryonic fibroblast-condition medium (MEF-CM):
Plate MEFs in 100 mm plates (1 x 106/plate). Next day, add 10 ml TS medium per dish and continue to culture for 48 hours. Collect the culture medium, filter them (0.45 mm) and store at -20°C in 35 ml aliquots. Thaw each aliquot when needed which can be stored at 4°C. The MEFs can be used to prepare for two more batches of CM before they become confluent.
PBS/BSA:
Dissolve BSA, fraction V (Sigma A3311, 0.1% (w/v)) in PBS (10 ml), filter through a 0.45 mm syringe filter and make 1 ml aliquots in 1.5 ml tubes. Store at -70°C and thaw one tube when needed to prepare FGF4.
FGF4 (1000x):
Resuspend lyophilized FGF4 (Sigma F8424, 25 mg) with 1 ml of the PBS/BSA solution. Mix well and make 10 aliquots (100 ml) into 1.5 ml tubes and store at -70°C. Thaw each tube when needed and store the remaining at 4°C, do not re-freeze. Dilute 1000 times in TS medium to obtain 1x FGF4 (25 ng/ml).
Heparin (1000x):
Resuspend heparin (Sigma H3149, 10,000 units) in PBS to a final concentration of 1 mg/ml (1000x). Make 100 ml aliquots into 1.5 ml tubes and store at -70°C. Thaw aliquots when needed and store the remaining at 4°C, do not re-freeze. Dilute 1000 time in TS medium to obtain 1x heparin (1 ng/ml).
Riferimenti
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