Decades of research into gene targeting in mice have created a number of tools for knocking genes in and out of embryonic stem (ES) cells. But differences between mouse and human ES cells have hampered attempts to introduce site-specific gene modifications in human ES cells. By tinkering with the molecular recipes developed in mice, researchers are beginning to have some luck with homologous recombination in human ES cells1. But not all genomic sites are amenable to gene addition. Many sites are prone to silencing and introduced sequences can disrupt normal gene function. Now researchers have found a site that overcomes these problems.

The Rosa26 locus has become a preferred site for genetic modification in mice because it can be targeted with relative ease and shows broad expression across most cell types. Gordon Keller, of the University Health Network in Toronto, Canada, and his colleagues scoured the online genomic databases and identified a human homolog. As in mice, they found that human ROSA26 was expressed in all adult tissues tested and in three different human ES cell lines2.

The researchers then modified the widely used mouse Rosa26 vector and inserted a red fluorescent protein (RFP) gene into the locus in one of the human ES cell lines (HES 2). Their success rate was low — around 2% compared to close to 35% for the same locus in mice3 — but the modified ES cells showed typical morphology and RFP was expressed, even after differentiation. Furthermore, the construct allows the researchers to exchange the RFP marker for any other gene, which they demonstrated by swapping it for an antibiotic-resistance gene.

The vector used was specific to only one cell line and “there is still great room for improvement to get higher success rates,” says Keller, but other research groups can now use these tools to “create cell lines expressing any gene of interest.” The great advantage of the ROSA26 locus is that it resists silencing, and Keller is now trying to introduce more specific promoters to develop lineage-specific or inducible markers. The future of gene targeting in human ES cells, it seems, is coming up ROSA.

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