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EMBRYONIC STEM CELLS

An Efficient Method for the Derivation of Mouse Embryonic Stem Cells

Vítzlav Bryja^a, Sonia Bonilla^a, Luká ajánek^a, Clare L. Parish^a, Catherine M. Schwartz^a,b, Yongquan Luo^b, Mahendra S. Rao^b,c, Ernest Arenas^a

^a Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;
^b Gerontology Research Center, Stem Cell Biology Unit, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland, USA;
^c Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA

Key Words. Mouse embryonic stem cells • Derivation from blastocyst • Efficient protocol • Serum-free media

Correspondence: Ernest Arenas, M.D., Ph.D., Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet S-171 77, Stockholm, Sweden. Telephone: +46-8-52487663; Fax: +46-8-34 1960; e-mail: ernest.arenas{at}ki.se

Received September 13, 2005; accepted for publication December 5, 2005.
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.