Tool to delete DNA

A Canadian team has developed a tool to rapidly delete regions of chromosome in mammalian cells, with particular import for embryonic and adult stem cells.

Guy Sauvageau and team from the University of Montreal used the Cre protein, a site-specific DNA enzyme that can catalyse the recombination of DNA between specific sites known as loxP.

By putting the loxP sites in separate retroviral vectors and sequentially infecting stem cells, the researchers ensured a random distribution of loxP sites in the genome.

Addition of the enzyme Cre led to deletion of chromosomal regions, ranging in size from 6kb to 23Mb.

"Chromosomal deletions, as a genetic tool for functional genomics, remain underexploited for vertebrate stem cells mostly because presently available methods are too labour-intensive," the researchers write in a paper published online in the February issue of Nature Methods.

"To address this, we developed and validated a set of complementary retroviruses that creates a wide range of nested chromosomal deletions.

"When applied to mouse embryonic stem cells (ESCs), this retrovirus-based method yielded deletions ranging from 6 kb to 23 Mb (average 2.9 Mb), with an efficiency of 64 per cent for drug-selected clones. Notably, several of the engineered ESC clones, mostly those with large deletions, showed major alteration in cell fate.

"In comparison to other methods that have also exploited retroviruses for chromosomal engineering, this modified strategy is more efficient and versatile because it bypasses the need for homologous recombination, and thus can be exploited for rapid and extensive functional screens in embryonic and adult stem cells."

The new technique makes it possible to screen many different genomic regions and identify those essential for specific functions in adult and embryonic stem cells.

The paper, A tool to delete DNA appears online in Nature Methods. See www.nature.com/nmeth