Three inhibitors of FGF receptor, ERK, and GSK3 establishes germline-competent embryonic stem cells of C57BL/6N mouse strain with high efficiency and stability.
C57BL/6 mouse is the most standard strain in mouse genetics. The strain does, however, have several disadvantages; one being the difficulty in establishing embryonic stem (ES) cells. No reliable C57BL/6 ES cell line is widely available for creating mutant mice through gene targeting. It also greatly favors mouse genetics if one can routinely make multiple mutations by stably culturing germline-competent C57BL/6 ES cells or if one can routinely establish ES cells from C57BL/6-derived mutant mice to make multiple mutations. Recently, an ES culture method with three inhibitors (3i: SU5402 for FGFR, PD184352 for ERK, and CHIR99021 for GSK3) has been reported. Here we show that this 3i method is extremely instrumental in establishing and culturing germline-competent ES cells in the C57BL/6N strain.
Kiyonari
Hiroshi
H
Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Chuo-Ku, Kobe, Japan.
Kaneko
Mari
M
Abe
Shin-ichi
S
Aizawa
Shinichi
S
D000818:Animals; D001549:Benzamides / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors; D005260:Female; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D020869:Gene Expression Profiling; D005854:Germ Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D007150:Immunohistochemistry; D007621:Karyotyping; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D017468:Receptors, Fibroblast Growth Factor / Q000037:antagonists & inhibitors; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
C120227:2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; D001549:Benzamides; C473711:Chir 99021; D011725:Pyridines; D011743:Pyrimidines; D011758:Pyrroles; D017468:Receptors, Fibroblast Growth Factor; C105686:SU 5402; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3