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.
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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
References
Bradley A, Evans M, Kaufman MH & Robertson E (1984) Formation of germ-line chimeras from embryo-derived teratocarcinoma cell line. Nature 309: 255–256.
Bradley A (1987) Production and analysis of chimaeric mice. In: Robertson EJ (ed.) Teratocarcinoma and embryonic stem cells: a practical approach. (pp. 113–151) IRL Press, Oxford.
Capecchi MR (1989) Altering the genome by homologous recombination. Science 244: 1288–1292.
Evans MJ & Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292: 154–156.
Gossler AG, Doetschman T, Korn R, Serfling E & Kelmer R (1986) Transgenesis by means of blastocyst-derived stem cell lines. Proc. Natl. Acad. Sci. USA 83: 9065–9069.
Gubbay J, Collignon J, Koopman P, Capel B, Economou A, Münsterberg A, Vivian N, Goodfellow P & Lovell-Badge R (1990) A gene mapping to the sex-determining region of the mouse Y-chromosome is a member of a novel family of embryonically expressed genes. Nature 346: 2145–2150.
Hogan B, Constantini F & Lacy E (1986) Manipulating the mouse embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Hooper M, Hardy K, Handyside A, Hunter S & Monk M (1987) HPRT deficient (Lesh-Nyhan) mouse embryos derived from germ line colonization by cultured cells. Nature 326: 292–295.
Kuehn MR, Bradley A, Robertson EJ & Evans MJ (1987) A potential animal model for Lesch-Nyhan syndrome through introduction of HPRT mutations into mice. Nature 326: 295–298.
Ledermann B & Bürki K (1991) Establishment of a germ-line component C57BL/6 embryonic stem cell line. Exp. Cell Res. 197: 254–258.
Lee K-F, Li E, Huber J, Landis SC, Sharpe AH, Chao MV & Jaenisch R. (1992) Targeted mutation of the gene encoding the low affinity NGF receptor p75 leads to deficits in the peripheral sensory nervous system. Cell 69: 737–749.
Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl. Acad. Sci. USA 78: 7634–7638.
McMahon AP & Bradley A (1990) The Wnt-1 (int-1) protooncogene is required for development of a large region of the mouse brain. Cell 62: 1073–1085.
Robertson EJ, Evans MJ & Kaufman MH (1983) X-chromosome instability in pluripotential stem cell lines derived from parthenogenetic embryos. J. Embryol. Exp. Morph. 74: 297–309.
Robertson EJ (1987) Embryo-derived stem cell lines. In: Robertson EJ (ed.) Teratocarcinoma and embryonic stem cells: a practical approach. (pp. 71–112) IRL Press, Oxford.
Tojo H, Kaisho Y, Nakata M, Matsuoka K, Kitagawa M, Abe T, Takami K, Yamamoto M, Shino A, Igarashi K, Aizawa S & Shiho O (1995) Targeted disruption of the neurotrophin-3 gene with lacZ induces loss of trkC-positive neurons in sensory ganglia but not in spinal cords. Brain Res. 669: 163–175.
Tokunaga T & Tsunoda Y (1992) Efficious production of viable germline chimera between embryonic stem (ES) cells and 8-cell stage embryos. Dev. Growth Differ. 34: 561–566.
Wood SA, Pascoe WS, Schmidt C, Kemler R, Evans MJ & Allen ND (1993) Simple and efficient production of embryonic stem cell-embryo chimeras by coculture. Proc. Natl. Acad. Sci. USA 90: 4582–4585.
Yagi T, Tokunaga T, Furuta Y, Nada S, Yoshida M, Tsukada T, Saga Y, Takeda N, Ikawa Y & Aizawa S (1993) A novel ES cell line, TT2, with high germline-differentiating potency. Anal. Biochem. 214: 70–78.
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Tojo, H., Nishida, M., Matsuoka, K. et al. Establishment of a novel embryonic stem cell line by a modified procedure. Cytotechnology 19, 161–165 (1995). https://doi.org/10.1007/BF00749771
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DOI: https://doi.org/10.1007/BF00749771