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Zinc fingers splice genes in human pluripotent stem cells

Technique will make genetic engineering easier

Researchers at the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, and Sangamo Biosciences in Richmond, California, have created protein tools capable of introducing new genes into human pluripotent stem cells. The technique uses enzymes called zinc-finger nucleases, which can be engineered to recognize and cut DNA sequences of around a couple dozen base pairs.

Although the genomes of mouse embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are relatively easy to manipulate, genomes of their human counterparts resist tinkering. Available techniques are so laborious, say the researchers, that only a dozen or so genes have been swapped into human ES cells (hESCs). In contrast, say the researchers, zinc fingers should be easier to use, perhaps allowing modification of hundreds of genes.

Led by Rudolf Jaenisch, the researchers created sets of zinc fingers capable of modifying three different spots in the genome. They inserted a reporter gene into the Oct4 locus, which is expressed in pluripotent cells, the AAVS1 locus, which encodes a ubiquitously expressed gene, and PITX3, which is expressed in certain differentiated cells but not in iPS or ES cells. They were able to target five different loci on the three different genes and also insert drug-inducible cassettes into them. This extends earlier work published by Sangamo in which zinc fingers were demonstrated to function on a different locus.

“It's very important. I love it,” says Thomas Zwaka of the Baylor College of Medicine in Houston, Texas, who has worked on homologous recombination in hESCs and was not involved in this work. “I'm only aware of 20 papers in the last 10 years on hESC targeting.” The technique won't solve everything, however. Introducing high concentrations of DNA-cutting enzymes into cells will require researchers to be vigilant about searching out unwanted DNA damage. Although the researchers convincingly showed that such off-target effects were certainly manageable in their hands, that might not be the case for other nucleases or cell lines. (The Jaenisch team demonstrated the ability of the zinc fingers with one hESC line and one iPS cell line.) Another limitation, Zwaka says, is that researchers will likely want to modify sites in the genome other than those described in the paper.

That shouldn't be a problem, says Fyodor Urnov of Sangamo, who was an author on the paper. The zinc fingers in the published paper will be provided under “standard MTAs [material transfer agreements” to academic labs. Plenty of zinc fingers designed using Sangamo's methods and targeting other genes are distributed by Sigma-Aldrich, based in St. Louis, Missouri.

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References

  1. 1

    Hockemeyer, D. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nature Biotechnol. advance online publication, 10.1038/nbt.1562 (13 August 2009).

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Baker, M. Zinc fingers splice genes in human pluripotent stem cells. Nat Rep Stem Cells (2009). https://doi.org/10.1038/stemcells.2009.115

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