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

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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.