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Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts

Nature volume 439, pages 212–215 (2006)Cite this article

Abstract

The derivation of embryonic stem (ES) cells by nuclear transfer holds great promise for research and therapy but involves the destruction of cloned human blastocysts. Proof of principle experiments have shown that ‘customized’ ES cells derived by nuclear transfer (NT-ESCs) can be used to correct immunodeficiency in mice1. Importantly, the feasibility of the approach has been demonstrated recently in humans2, bringing the clinical application of NT-ESCs within reach. Altered nuclear transfer (ANT) has been proposed as a variation of nuclear transfer because it would create abnormal nuclear transfer blastocysts that are inherently unable to implant into the uterus but would be capable of generating customized ES cells3. To assess the experimental validity of this concept we have used nuclear transfer to derive mouse blastocysts from donor fibroblasts that carried a short hairpin RNA construct targeting Cdx2 . Cloned blastocysts were morphologically abnormal, lacked functional trophoblast and failed to implant into the uterus. However, they efficiently generated pluripotent embryonic stem cells when explanted into culture.

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Figure 1: Derivation of NT-ESCs from Cdx2 -deficient blastocysts.
Figure 2: Cdx2 -deficient blastocysts and ES cell derivation.
Figure 3: Cdx2 -deficient cells maintain developmental potential but are unable to implant after nuclear transfer.

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Acknowledgements

We would like to thank K. Hochedlinger and L. Jackson-Grusby for discussion and critical reading of the manuscript, and in particular D. Fu for sections and stainings. We are also grateful to B. Hogan and P. Berg for critical comments on the manuscript. R.J. was supported by NIH/NCI grants. A.M. was supported by a Boehringer Ingelheim Fonds (BIF) PhD fellowship. Author Contributions R.J. and A.M. conceived and designed the experiments, A.M. performed the experiments, R.J. and A.M. wrote the paper.

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  1. Whitehead Institute and Department of Biology, Massachusetts Institute of Technology, Nine Cambridge Center, Massachusetts, 02142, Cambridge, USA

    Alexander Meissner & Rudolf Jaenisch

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  1. Alexander Meissner
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  2. Rudolf Jaenisch
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Correspondence to Rudolf Jaenisch.

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Supplementary information

Supplementary Figure 1

Selection of functional shRNAs. Knockdown efficiency of each shRNA (see Methods for design of shRNAs) was tested using a DsRed reporter construct. (PDF 2290 kb)

Supplementary Figure 2

Knockdown of endogenous Cdx2. The knockdown efficiency of the shRNA selected by the reporter assay (Supplementary Fig. 1) against endogenous Cdx2 was tested using ZHBTc4 ES cells. (PDF 734 kb)

Supplementary Figure Legends

Text to accompany the above Supplementary Figures. (DOC 23 kb)

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Meissner, A., Jaenisch, R. Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2-deficient blastocysts. Nature 439, 212–215 (2006). https://doi.org/10.1038/nature04257

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