An efficient method for the derivation of mouse embryonic stem cells
An efficient method for the derivation of mouse embryonic stem cells
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DOI：
10.1634/stemcells.2005-0444
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发表时间：
2006-04-01
期刊：
影响因子：
5.2
通讯作者：
Arenas, Ernest
中科院分区：
文献类型：
--
作者：
Bryja, Vitezslav;Bonilla, Sonia;Arenas, Ernest
小鼠胚胎干细胞 (mESC) 对许多研究人员来说是一种独特的工具；然而，ESC 衍生过程通常效率非常低，并且需要高度专业化、培训和专业知识。为了规避这些限制，我们的目标是基于使用市售产品开发一种简单有效的协议。在这里，我们提出了一种优化方案，成功应用于从几种敲除小鼠品系（Wnt-1、Wnt-5a、Lrp6 和 Parkin）中获得 ESC，效率为 50%-75%。该方法基于使用小鼠胚胎成纤维细胞饲养层、敲除血清替代物 (SR) 以及对囊胚的最少处理。在此方案中，避免了所有离心步骤（以及胰蛋白酶抑制剂的使用），并在胰蛋白酶消化后用含有胎牛血清（FCS）的 ESC 培养基代替。我们定义了在协议的各个步骤中使用 SR 和 FCS 的潜在优点和缺点。我们还通过免疫组织化学、蛋白质印迹和干细胞聚焦微阵列来表征 ESC 的 ESC 标记物表达。总之，我们提供了一种简化和改进的方案来衍生 mESC，这对于旨在首次分离转基因 mESC 的实验室非常有用。
Mouse embryonic stem cells (mESCs) represent a unique tool for many researchers; however, the process of ESC derivation is often very inefficient and requires high specialization, training, and expertise. To circumvent these limitations, we aimed to develop a simple and efficient protocol based on the use of commercially available products. Here, we present an optimized protocol that we successfully applied to derive ESCs from several knockout mouse strains (Wnt-1, Wnt-5a, Lrp6, and parkin) with 50%-75% efficiency. The methodology is based on the use of mouse embryonic fibroblast feeders, knockout serum replacement (SR), and minimal handling of the blastocyst. In this protocol, all centrifugation steps (as well as the use of trypsin inhibitor) were avoided and replaced by an ESC medium containing fetal calf serum (FCS) after the trypsinizations. We define the potential advantages and disadvantages of using SR and FCS in individual steps of the protocol. We also characterize the ESCs for the expression of ESC markers by immunohistochemistry, Western blot, and a stem cell focused microarray. In summary, we provide a simplified and improved protocol to derive mESCs that can be useful for laboratories aiming to isolate transgenic mESCs for the first time.