Bryja Vítezlav, Bonilla Sonia, Cajánek Lukás, Parish Clare L, Schwartz Catherine M, Luo Yongquan, Rao Mahendra S, Arenas Ernest
Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet S-171 77, Stockholm, Sweden.
Stem Cells. 2006 Apr;24(4):844-9. doi: 10.1634/stemcells.2005-0444. Epub 2005 Dec 9.
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.
小鼠胚胎干细胞（mESCs）对许多研究人员来说是一种独特的工具；然而，胚胎干细胞的获取过程往往效率很低，需要高度专业化、培训和专业知识。为了克服这些限制，我们旨在基于使用市售产品开发一种简单高效的方案。在此，我们展示了一种优化方案，该方案已成功应用于从几种基因敲除小鼠品系（Wnt-1、Wnt-5a、Lrp6和parkin）中获取胚胎干细胞，效率达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）以及对囊胚的最少操作。在本方案中，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）的胚胎干细胞培养基替代。我们定义了在方案的各个步骤中使用SR和FCS的潜在优缺点。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对胚胎干细胞进行胚胎干细胞标志物表达的特征分析。总之，我们提供了一种简化且改进的方案来获取小鼠胚胎干细胞，这对于首次旨在分离转基因小鼠胚胎干细胞的实验室可能有用。
