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Generation of mice from wild-type and targeted ES cells by nuclear cloning

Nature Genetics volume 24, pages 109–110 (2000)Cite this article

Mammalian cloning has recently been developed1,2,3,4,5, but a major obstacle has been the low frequency of viable clones, as most clones die during gestation or soon after birth. Parameters that affect cloning efficiency may include genetic background, passage number, cell-cycle stage of the donor cell6, loss of imprints, accumulated genetic damage of the donor cells or the ability of the oocyte to epigenetically reprogram the donor-cell nucleus. Cloning in mice5,7,8, the best mammalian model organism, has until recently been limited to freshly isolated or primary cultures of somatic cells, which limits study of the parameters that affect cloning efficiency. We have determined that established and targeted embryonic stem (ES) cells can generate cloned embryos and animals, thus making possible the study of parameters important for cloning.

To assess the most suitable genetic background for cloning, we compared cloning efficiencies using F1 (129SvJae×C57BL/6) ES cell lines and 129 (129SvJae) ES cell lines as nuclear donors. Nuclear transplantation resulted in blastocyst-stage clones at a similar frequency for all ES-cell donors (Table 1). After transfer into pseudopregnant females, 7 of 34 (21%) F1-cloned blastocysts developed to term and into healthy adults. In comparison, 8 of 76 (11%) inbred 129 donor ES cells developed to term, but all died within 24 hours of birth, demonstrating an effect of genetic background on survival. The viability of the F1-derived clones was also 5–70-fold greater than that reported for clones from other ES cell lines (R1, 4%, and E14, 0.3%; ref. 8). Thus, the F1 ES cells were more efficient as donors for nuclear transfer than the inbred 129 (J1 and E14) ES cells and the out-crossed 129 R1 ES cells. The ability of ES cells to generate viable cloned offspring might be correlated with their degree of polymorphism at 37 different SSLP markers (J1, 0/37; E14, 0/37; R1, 12/37; our F1 line, 28/37; ref. 9).

Table 1 Development of cloned embryos and mice from ES cells
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Figure 1: Targeted insertion at the ROSA26 locus in cloned mice.

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Acknowledgements

We thank H. Bujard for the rtTA2 construct. This work was supported by a grant from the NIH.

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  1. William M. Rideout and Teruhiko Wakayama: These authors contributed equally to this work.

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA

    William M. Rideout III, Anton Wutz, Kevin Eggan, Laurie Jackson-Grusby, Jessica Dausman & Rudolf Jaenisch

  2. Department of Anatomy and Reproductive Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii, USA

    Teruhiko Wakayama & Ryuzo Yanagimachi

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  1. William M. Rideout III
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Correspondence to Rudolf Jaenisch.

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Rideout, W., Wakayama, T., Wutz, A. et al. Generation of mice from wild-type and targeted ES cells by nuclear cloning. Nat Genet 24, 109–110 (2000). https://doi.org/10.1038/72753

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