Identification and targeting of the ROSA26 locus in human embryonic stem cells

Abstract

The derivation of human embryonic stem (hES) cells has opened new avenues for studies on human development and provided a potential source of cells for replacement therapy. To reveal the full potential of hES cells, it would be advantageous to be able to genetically alter them as is routinely done with mouse ES cells through homologous recombination. The mouse Rosa26 locus is particularly useful for genetic modification as it can be targeted with high efficiency and is expressed in most cell types tested. Here we report the identification of the human homolog of the mouse Rosa26 locus. We demonstrate targeting of a red-fluorescent protein (tdRFP) cDNA to this locus through homologous recombination and expression of this targeted reporter in multiple hES cell–derived lineages. Through recombinase-mediated cassette exchange, we show replacement of the tdRFP cDNA with other cDNAs, providing a cell line in which transgenes can be readily introduced into a broadly expressed locus.

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Figure 1: Identification, expression and targeting of the hROSA26 locus.
Figure 2: Morphology and differentiation of HES2.R26 cells.
Figure 3: Multilineage differentiation of HES2.R26-targeted cells in vitro.
Figure 4: Mesodermal differentiation of HES2.R26_puro cells.

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Acknowledgements

We thank members of the Keller lab for their contribution and helpful discussions, Sunita D'Souza and Macarena Lynch-Kattman for an initial karyotypic analysis, John Fallon for evaluation of the histology slides and Kirsten Bomblies for assistance with the EST analysis. We would also like to thank the members of the Linden and Jessberger labs for assistance with Southern blots, the Mount Sinai flow cytometry shared research facility for cell sorting, the real time PCR core for the qPCR analysis and the histology core for assistance with the slide preparation. tdRFP was a kind gift of R. Tsien. This work was supported by National Institutes of Health grant HL80627 and P20GM075019 to G.K. S.I. was supported by a grant from the Deutsche Akademie der Naturforscher, Leopoldina, Halle. H.L. and H.J.F. are supported by Sonderforschungsbereich (SFB) 497-Projekt A7.

Author information

S.I. performed the experiments in this study, H.L. and S.I. designed the targeting vector, H.L. and H.J.F. performed the electronic screen of hROSA26 transcripts, P.G. contributed to the RMCE experiments, M.K. established the hES cell cultures and mesoderm differentiation. S.I., H.L., H.J.F. and G.K. contributed to the planning and design of the project. S.I., H.L., H.J.F. and G.K. wrote the manuscript.

Correspondence to Gordon Keller.

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Supplementary Figures 1–6, Supplementary Methods (PDF 2292 kb)

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Irion, S., Luche, H., Gadue, P. et al. Identification and targeting of the ROSA26 locus in human embryonic stem cells. Nat Biotechnol 25, 1477–1482 (2007). https://doi.org/10.1038/nbt1362

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