Summary
The manipulation of embryonic stem (ES) cells to introduce directional genetic changes into the genome of mice has become an important tool in biomedical research. Monitoring of cell morphology before and after DNA manipulation and special culture conditions are a prerequisite to preserve the pluripotent properties of ES cells and thus their ability to generate chimera and effective germline transmission (GLT). It has been reported that prolonged cell culturing may affect the diploid chromosomal composition of cells and therefore the percentage of chimerism and GLT. Herein, we report multicolor-fluorescence in situ hybridization (M-FISH) analysis of four different ES cell lines/clones. Although the morphology of all four ES cell lines/clones appeared normal and all four expressed the early markers Oct-3/4 and Nanog, two cell lines presented consistent numerical and structural chromosome aberrations. We demonstrate that M-FISH is a sensitive and accurate method for a comprehensive karyotype analysis of ES cells and may minimize time, costs, and disappointment due to inadequate ES cell sources.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Brown, D. G.; Willington, M. A.; Findlay, I.; Muggleton-Harris, A. L. Criteria that optimize the potential of murine embryonic stem cells for in vitro and in vivo developmental studies. In Vitro Cell Dev. Biol. 28A:773–778; 1992.
Cho, C.; Willis, W. D.; Goulding, E. H.; Jung-Ha, H.; Choi, Y. C.; Hecht, N. B.; Eddy, E. M. Haploinsufficiency of protamine-1 or-2 causes infertility in mice. Nat. Genet. 28:82–86; 2001.
Chomczynski, P.; Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem 162:156–159; 1987.
Dean, W.; Bowden, L.; Aitchison, A.; Klose, J.; Moore, T.; Meneses, J. J.; Reik, W.; Feil, R. Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes. Development 125:2273–2282; 1998.
Draper, J. S.; Smith, K.; Gokhale, P.; Moore, H. D.; Maltby, E.; Johnson, J.; Meisner, L.; Zwaka, T. P.; Thomson, J. A.; Andrews, P. W. Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat. Biotechnol. 22:53–54; 2004.
Evans, M. J.; Kaufman, M. H. Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156; 1981.
Jentsch, I.; Geigl, J.; Klein, C. A.; Speicher, M. R. Seven-fluorochrome mouse M-FISH for high-resolution analysis of interchromosomal rearrangements. Cytogenet. Genome Res. 103:84–88; 2003.
Liu, X.; Wu, H.; Loring, J.; Hormuzdi, S.; Disteche, C. M.; Bornstein, P.; Jaenisch, R. Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission. Dev. Dyn. 209:85–91; 1997.
Longo, L.; Bygrave, A.; Grosveld, F. G.; Pandolfi, P. P. The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism. Transgenic Res. 6:321–328; 1997.
Martin, G. R. 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; 1981.
Nagy, A.; Rossant, J.; Nagy, R.; Abramow-Newerly, W.; Roder, J. C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl. Acad. Sci. USA 90:8424–8428; 1993.
Nichols, J.; Evans, E. P.; Smith, A. G. Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity. Development 110:1341–1348; 1990.
Pease, S.; Braghetta, P.; Gearing, D.; Grail, D.; Williams, R. L. Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor (LIF). Dev. Biol. 141:344–352; 1990.
Rathjen, P. D.; Toth, S.; Willis, A.; Heath, J. K.; Smith A. G. Differentiation inhibiting activity is produced in matrix-associated and diffusible forms that are generated by alternate promoter usage. Cell 62:1105–1114; 1990a.
Rathjen, P. D.; Nichols, J.; Toth, S.; Edwards, D. R.; Heath, J. K.; Smith, A. G. Developmentally programmed induction of differentiation inhibiting activity and the control of stem cell populations. Genes Dev. 4:2308–2318; 1990b.
Robertson, E.; Bradley, A.; Kuehn, M.; Evans, M. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 323:445–448; 1986.
Smith, A. G.; Heath, J. K.; Donaldson, D. D.; Wong, G. G.; Moreau, J.; Stahl, M.; Rogers, D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336:688–690; 1988.
Suzuki, H.; Kamada, N.; Ueda, O.; Jishage, K.; Kurihara, Y.; Kurihara, H.; Terauchi, Y.; Azuma, S.; Kadowaki, T.; Kodama, T.; Yazaki, Y.; Toyoda, Y. Germ-line contribution of embryonic stem cells in chimeric mice: influence of karyotype and in vitro differentiation ability. Exp. Anim. 46:17–23; 1997.
Szabo, P.; Mann, J. R. Expression and methylation of imprinted genes during in vitro differentiation of mouse parthenogenetic and androgenetic embryonic stem cell lines. Development 120:1651–1660; 1994.
Ward, C. M.; Stern, P.; Willington, M. A.; Flenniken, A. M. Efficient germline transmission of mouse embryonic stem cells grown in synthetic serum in the absence of a fibroblast feeder layer. Lab. Invest. 82:1765–1767; 2002.
Williams, R. L.; Hilton, D. J.; Pease, S.; Willson, T. A.; Stewart, C. L.; Gearing, D. P.; Wagner, E. F.; Metcalf, D.; Nicola, N. A.; Gough, N. M. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336:684–687; 1988.
Ying, Q. L.; Nichols, J.; Chambers, I.; Smith, A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115:281–292; 2003.
Author information
Authors and Affiliations
Corresponding author
Additional information
Both authors contributed equally to this work.
Rights and permissions
About this article
Cite this article
Guo, J., Jauch, A., Holtgreve-Grez, H. et al. Multicolor karyotype analyses of mouse embryonic stem cells. In Vitro Cell.Dev.Biol.-Animal 41, 278–283 (2005). https://doi.org/10.1290/990771.1
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1290/990771.1