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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References
Zambrowicz, B.P. et al. Disruption of overlapping transcripts in the ROSA beta geo 26 gene trap strain leads to widespread expression of beta-galactosidase in mouse embryos and hematopoietic cells. Proc. Natl. Acad. Sci. USA 94, 3789–3794 (1997).
Birney, E. et al. Ensembl 2006. Nucleic Acids Res. 34, D556–D561 (2006).
Soriano, P. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70–71 (1999).
Friedrich, G. & Soriano, P. Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev. 5, 1513–1523 (1991).
Campbell, R.E. et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).
Siegel, R.W., Jain, R. & Bradbury, A. Using an in vivo phagemid system to identify non-compatible loxP sequences. FEBS Lett. 499, 147–153 (2001).
Luche, H., Weber, O., Nageswara Rao, T., Blum, C. & Fehling, H.J. Faithful activation of an extra-bright red fluorescent protein in “knock-in” Cre-reporter mice ideally suited for lineage tracing studies. Eur. J. Immunol. 37, 43–53 (2007).
Schnutgen, F. et al. A directional strategy for monitoring Cre-mediated recombination at the cellular level in the mouse. Nat. Biotechnol. 21, 562–565 (2003).
Mao, X., Fujiwara, Y., Chapdelaine, A., Yang, H. & Orkin, S.H. Activation of EGFP expression by Cre-mediated excision in a new ROSA26 reporter mouse strain. Blood 97, 324–326 (2001).
Bouhassira, E.E., Westerman, K. & Leboulch, P. Transcriptional behavior of LCR enhancer elements integrated at the same chromosomal locus by recombinase-mediated cassette exchange. Blood 90, 3332–3344 (1997).
Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. & Bongso, A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399–404 (2000).
Zwaka, T.P. & Thomson, J.A. Homologous recombination in human embryonic stem cells. Nat. Biotechnol. 21, 319–321 (2003).
Urbach, A., Schuldiner, M. & Benvenisty, N. Modeling for Lesch-Nyhan disease by gene targeting in human embryonic stem cells. Stem Cells 22, 635–641 (2004).
Costa, M. et al. A method for genetic modification of human embryonic stem cells using electroporation. Nat. Protoc. 2, 792–796 (2007).
Reubinoff, B.E. et al. Neural progenitors from human embryonic stem cells. Nat. Biotechnol. 19, 1134–1140 (2001).
Johansson, B.M. & Wiles, M.V. Evidence for involvement of activin A and bone morphogenetic protein 4 in mammalian mesoderm and hematopoietic development. Mol. Cell. Biol. 15, 141–151 (1995).
Kennedy, M., D'Souza, S.L., Lynch-Kattman, M., Schwantz, S. & Keller, G. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 109, 2679–2687 (2007).
Xu, R.H. et al. BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat. Biotechnol. 20, 1261–1264 (2002).
Costa, M. et al. The hESC line Envy expresses high levels of GFP in all differentiated progeny. Nat. Methods 2, 259–260 (2005).
Nolden, L. et al. Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase. Nat. Methods 3, 461–467 (2006).
Gerrard, L., Zhao, D., Clark, A.J. & Cui, W. Stably transfected human embryonic stem cell clones express OCT4-specific green fluorescent protein and maintain self-renewal and pluripotency. Stem Cells 23, 124–133 (2005).
Ma, Y., Ramezani, A., Lewis, R., Hawley, R.G. & Thomson, J.A. High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors. Stem Cells 21, 111–117 (2003).
Muotri, A.R., Nakashima, K., Toni, N., Sandler, V.M. & Gage, F.H. Development of functional human embryonic stem cell-derived neurons in mouse brain. Proc. Natl. Acad. Sci. USA 102, 18644–18648 (2005).
Lombardo, A. et al. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat. Biotechnol. 25, 1298–1306 (2007).
Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Kennedy, M. & Keller, G.M. Hematopoietic commitment of ES cells in culture. Methods Enzymol. 365, 39–59 (2003).
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.
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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.
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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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DOI: https://doi.org/10.1038/nbt1362
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