Abstract
Reprogramming of somatic cells in the enucleated egg made Dolly, the sheep, the first successfully cloned mammal in 1996. However, the mechanism of sheep somatic cell reprogramming has not yet been addressed. Moreover, sheep embryonic stem (ES) cells are still not available, which limits the generation of precise gene-modified sheep. In this study, we report that sheep somatic cells can be directly reprogrammed to induced pluripotent stem (iPS) cells using defined factors (Oct4, Sox2, c-Myc, Klf4, Nanog, Lin28, SV40 large T and hTERT). Our observations indicated that somatic cells from sheep are more difficult to reprogram than somatic cells from other species, in which iPS cells have been reported. We demonstrated that sheep iPS cells express ES cell markers, including alkaline phosphatase, Oct4, Nanog, Sox2, Rex1, stage-specific embryonic antigen-1, TRA-1-60, TRA-1-81 and E-cadherin. Sheep iPS cells exhibited normal karyotypes and were able to differentiate into all three germ layers both in vitro and in teratomas. Our study may help to reveal the mechanism of somatic cell reprogramming in sheep and provide a platform to explore the culture conditions for sheep ES cells. Moreover, sheep iPS cells may be directly used to generate precise gene-modified sheep.
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Acknowledgements
This research was partially supported by grants from the National Key Basic Research and Development Program of China (2007CB947902, 2007CB948003, 2009CB941101, 2009CB940903), the National Natural Science Foundation of China (30971682, 30901019, 31025016), the Ministry of Agriculture (2009ZX08010-016B), the National High Technology Research and Development Program (863 program) of China (2010AA100504) and the Chinese Academy of Sciences Knowledge Creation Program (KSCX2-YW-R-223).
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
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Supplementary information, Figure S1
Immunostaining of OCT4, SOX2 and NANOG in sheep iPS cells. (PDF 57 kb)
Supplementary information, Table S1
Primers for RT-PCR (PDF 17 kb)
Supplementary information, Table S2
PCR primers for Bisulfite genomic sequencing (PDF 28 kb)
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Bao, L., He, L., Chen, J. et al. Reprogramming of ovine adult fibroblasts to pluripotency via drug-inducible expression of defined factors. Cell Res 21, 600–608 (2011). https://doi.org/10.1038/cr.2011.6
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DOI: https://doi.org/10.1038/cr.2011.6
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