Establishment of a novel embryonic stem cell line by a modified procedure |
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Authors: | Hideaki Tojo Mayumi Nishida Kunio Matsuoka Koichi Igarashi and Osamu Shiho |
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Institution: | (1) Discovery Research Laboratories II, Takeda Chemical Industries Ltd., Wadai 10, 300-42 Tsukuba, Ibaraki, Japan;(2) Research on Research, Takeda Chemical Industries Ltd., Wadai 10, 300-42 Tsukuba, Ibaraki, Japan |
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Abstract: | To generate mutant mice, embryonic stem (ES) cells are used as a vehicle for introducing mutations. The establishment of ES cells is diffucult because it requires specific skills and it is time-consuming. We established a novel ES cell line derived from hybrid mice between C57BL/6 and DBA/2 using a modified method. To collect a large number of preimplantational embryos, we collected embryos at the 8-cell stage and cultured them to blastocysts, whereas the usual procedure of preparing the delayed blastocysts demands technical skills. To eliminate unnecessary female cells at an initial stage of inner cell mass culture, male clones were selected by polymerase chain reaction to detect the mouseSry gene. The established ES cell line efficiently contributed to the germ-line when injected into 8-cell embryos of ICR mice. This potency was maintained after manipulation throughout gene targeting.Abbreviations DMEM
Dulbecco's modified Eagle's medium
- FBS
fetal bovine serum
- FIAU
1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil
- LIF
leukemia inhibitory factor
- NEAA
non-essential amino acids |
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Keywords: | 8-cell stage embryos embryonic stem cells gene targeting germ-line transmission sex-determination |
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