An efficient method for the derivation of mouse embryonic stem cells.
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.
Bryja
Vítezlav
V
Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet S-171 77, Stockholm, Sweden.
Bonilla
Sonia
S
Cajánek
Lukás
L
Parish
Clare L
CL
Schwartz
Catherine M
CM
Luo
Yongquan
Y
Rao
Mahendra S
MS
Arenas
Ernest
E
D000818:Animals; D001755:Blastocyst / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods; D018920:Coculture Techniques; D003470:Culture Media; D005260:Female; D051379:Mice; D018345:Mice, Knockout; D011247:Pregnancy; D039901:Totipotent Stem Cells / Q000166:cytology* / Q000378:metabolism
D003470:Culture Media