Abstract
In this chapter, methods are described that permit the enucleation of mouse embryonic stem (ES) cells and the transfer of donor nuclei to these cells before or after enucleation has taken place. The small size and high nucleus-to-cytoplasm volume ratio of ES cells poses a challenge to their enucleation. The first step describes the production of lines of larger, polyploid ES cells, which are more suited to enucleation than diploid ES cells. In a second step, a simple centrifugal enucleation technique is described that allows efficient bulk production of ES cell cytoplasts and karyoplasts. Finally, techniques for nuclear transfer to ES cells are described, involving either transfer of karyoplasts to cytoplasts or the formation of heterokaryons between donor and recipient cells followed by the selective elimination of the polyploid nucleus. These methods have potential applications in the generation of autologous, diploid pluripotent cells from donor somatic cells. Also, they provide a novel dynamic model for studying nucleocytoplasmic interactions in ES cells.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Matveeva, N. M., Shilov, A. G., Kaftanovskaya, E. M., et al. (1998) In vitro and in vivo study of pluripotency in intraspecific hybrid cells obtained by fusion of murine embryonic stem cells with splenocytes. Mol. Reprod. Dev. 50, 128–138.
Tada M., Takahama Y., Abe K., Nakatsuji N., and Tada T. (2001) Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr. Biol. 11, 1553–1558.
Ying, Q., Nichols, J., Evans, E. P., and Smith, A. G. (2002) Changing potency by spontaneous fusion. Nature 416, 545–548.
Terada, N., Hamazaki, T., Oka, M., et al. (2002) Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 416, 542–545.
Pells, S., Di Domenico, A. I., Gallagher, E. J., and McWhir, J. (2002) Multipotentiality of neuronal cells after spontaneous fusion with embryonic stem cells and nuclear reprogramming in vitro. Cloning Stem Cells 4, 331–338.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Adra C. N, Boer P. H., and McBurney M.W. (1987) Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter. Gene 60, 65–74.
Morgenstern J. P. and Land H. (1990) Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18, 3587–3596.
Pfarr D. S., Rieser L. A., Woychik R. P., Rottman F. M., Rosenberg M., and Reff M. E. (1986) Differential effects of polyadenylation regions on gene expression in mammalian cells. DNA 5, 115–122.
Hobbs, S., Jitrapakdee, S., and Wallace. J. C. (1998) Development of a bicistronic vector driven by the human polypeptide chain elongation factor 1alpha promoter for creation of stable mammalian cell lines that express very high levels of recombinant proteins. Biochem. Biophys. Res. Commun. 252, 368–372.
Doestschman, T. C., Eistetter, H., Katz, M., Schmidt, W. and Kemler R. (1985) The in-vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J. Embryol. Exp. Morphol. 87, 27–45.
Rathjen J., Lake, J., Bettess, M. D., Washington, J. M., Chapman, G., and Rathjen P. (1999) Formation of a primitive ectoderm-like cell population, EPL cells, from cells in response to biologically derived factors. J. Cell Sci. 112, 601–612.
Dinnyes A., De Sousa P., King T., and Wilmut I. (2002) Somatic cell nuclear transfer: recent progress and challenges. Cloning Stem Cells 4, 81–90.
Ringertz, N. R., Krondahl, U., and Coleman, J. R. (1978) Reconstitution of cells by fusion of cell fragments. I. Myogenic expression after fusion of minicells from rat myoblasts (L6) with mouse fibroblasts (A9) cytoplasm. Exp. Cell Res. 113, 233–246.
Degaetano, D. and Schindler, M. (1987) Enucleation of normal and transformed cells. J. Cell. Physiol. 130, 301–309.
Celis, J. E. and Celis, A. (1994) Preparation of karyoplasts and cytoplasts from HeLa cell monolayers, in Cell Biology: A Laboratory Handbook, Academic Press, Inc., New York, pp. 619–621.
Chiu, C. and Blau, H. M. (1984) Reprogramming cell differentiation in the absence of DNA synthesis. Cell 37, 879–887.
Tatham, B. G., Dowsing, A. T. and Trounson AO (1995) Enucleation by centrifugation of in vitro-matured bovine oocytes for use in nuclear transfer. Biol. Reprod. 53, 1088–1094.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Pralong, D., Verma, P.J. (2006). Techniques for Nuclear Transfer to Mouse Embryonic Stem Cells. In: Verma, P.J., Trounson, A.O. (eds) Nuclear Transfer Protocols. Methods in Molecular Biology™, vol 348. Humana Press. https://doi.org/10.1007/978-1-59745-154-3_19
Download citation
DOI: https://doi.org/10.1007/978-1-59745-154-3_19
Publisher Name: Humana Press
Print ISBN: 978-1-58829-280-3
Online ISBN: 978-1-59745-154-3
eBook Packages: Springer Protocols