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http://www.nature.com/naturebiotechnology
Web End = Identication and targeting of the ROSA26 locus in human embryonic stem cells
http://www.nature.com/naturebiotechnology
Stefan Irion1, Herv Luche2, Paul Gadue3, Hans Joerg Fehling2, Marion Kennedy1 & Gordon Keller1
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 modication as it can be targeted with high efciency and is expressed in most cell types tested. Here we report the identication of the human homolog of the mouse Rosa26 locus. We demonstrate targeting of a red-uorescent protein (tdRFP) cDNA to this locus through homologous recombination and expression of this targeted reporter in multiple hES cellderived 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.
Using the sequence of mouse Rosa26 transcripts1 as a template to search the Ensembl database2, we located a region with several highly homologous sequences on human chromosome 3. The highest degree of sequence similarity (485%) was found in a stretch of nonrepetitive DNA corresponding to the 5 portion of exon 1 plus B1.0 kb of the putative promoter region in the mouse Rosa26 locus (Supplementary Fig. 1a online). An electronic screen of the Ensembl gene expression database for this genomic region revealed a large number of uncharacterized expressed sequence tags (ESTs) and transcripts. These transcripts are derived from a broad spectrum of adult cell types and three embryonic stem cell lines. They map to specic positions within the region of ROSA26 synteny, most likely demarcating exons (Supplementary Fig. 1b). Together with the conserved location of a anking gene (Supplementary Fig. 1b), these ndings provide convincing evidence that the region on human chromosome 3 identied here represents the equivalent of the mouse Rosa26 locus and should thus be referred to as human ROSA26 (hROSA26).
The mouse Rosa26 locus has become a preferred site for the integration of transgenes and various reporter...