A novel method for somatic cell nuclear transfer to mouse embryonic stem cells

Danièle Pralong, Krzysztof Mrozik, Filomena Occhiodoro, Nishanthi Wijesundara, Huseyin Sumer, Antonius L. Van Boxtel, Alan Trounson, Paul J. Verma

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39 Citations (Scopus)

Abstract

Nuclear reprogramming by somatic cell nuclear transfer (SCNT) provides a practical approach for generating autologous pluripotent cells from adult somatic cells. It has been shown that murine somatic cells can also be reprogrammed to a pluripotent-like state by fusion with embryonic stem (ES) cells. Typically, the first step in SCNT involves enucleation of the recipient cell. However, recent evidence suggests that enucleated diploid ES cells may lack reprogramming capabilities. Here we have developed methods whereby larger tetraploid ES cells are first generated by fusion of two mouse ES cell lines transfected with plasmids carrying different antibiotic-resistance cassettes, followed by double antibiotic selection. Tetraploid ES cells grown on tissue culture disks or wells can be efficiently enucleated (up to 99%) using a combination of cytochalasin B treatment and centrifugation, with cytoplasts generated from these cells larger than those obtained from normal diploid ES cells. Also, we show that the enucleation rate is dependent on centrifugation time and cell ploidy. Further, we demonstrate that normal diploid ES cells can be fused to tetraploid ES cells to form heterokaryons, and that selective differential centrifugation conditions can be applied where the tetraploid nucleus is removed while the diploid donor nucleus is retained. This technology opens new avenues for generating autologous, diploid pluripotent cells, and provides a dynamic model for studying nuclear reprogramming in ES cells.

Original languageEnglish
Pages (from-to)265-271
Number of pages7
JournalCloning and Stem Cells
Volume7
Issue number4
DOIs
Publication statusPublished - 1 Dec 2005

ASJC Scopus subject areas

  • Biotechnology
  • Developmental Biology

Cite this

Pralong, D., Mrozik, K., Occhiodoro, F., Wijesundara, N., Sumer, H., Van Boxtel, A. L., ... Verma, P. J. (2005). A novel method for somatic cell nuclear transfer to mouse embryonic stem cells. Cloning and Stem Cells, 7(4), 265-271. https://doi.org/10.1089/clo.2005.7.265