"Making mice with mutations is really the best way of trying to define gene function, whether it's a mouse gene or a human gene," says genetic engineer Andrew Smith of the University of Edinburgh in Scotland and lead author of a report in Cell that describes the technique. "By doing it the way we did it, you can be absolutely precise."
Researchers already knew how to trade a single mouse gene for its human counterpart to create a so-called transgenic mouse. But the activity of a gene is also regulated by bits of DNA outside of the gene. The problem the field faced was that the equivalent gene could be regulated a different way in the mouse than it had been in humans.
For example, the blood disorder α-thalassemia had resisted attempts to model it by deleting part of the mouse gene for α hemoglobin, a form of the oxygen-carrying blood protein. Although the same deletion causes the disease in people, regulatory DNA outside of the mouse gene compensated for the loss, resulting in a weak version of the disease.
To insert the human regulatory machinery for α hemoglobin, Smith and his colleagues adapted a system called Cre/lox for exchanging two gene segments. In their modified technique, the researchers inserted two different short pieces of the bacterial lox DNA into the genome of a mouse embryonic cell so that they flanked the mouse gene and its regulatory elements.
They also injected into the mouse cells an equivalent human DNA fragment containing matching lox sequences in the same positions. They then used an enzyme called Cre recombinase, which connected matching lox sequences to each other, to switch out the mouse DNA for the human fragment.
Sure enough, although the genetic exchange happened rarely, when the resulting transgenic mice were again altered to mimic α-thalassemia they had the same changes in hemoglobin gene expression and red blood cell shape as people with the human disorder, the group reports.
In addition, Smith says, there were no random insertions of human DNA into other locations, or insertions of multiple copies or fragments pointing the wrong direction—problems which typically occur when researchers insert big pieces of DNA into the mouse genome by other means.