Standfirst
By using recombinase-mediated exchange scientists have found a way to replace large regions in the mouse genome with their human counterpart.
If on hearing "humanized mouse" you think Stewart Little, your imagination has taken you a bit too far. Imagine instead a mouse with a human gene.
The goal of humanizing a mouse is to model human disease progression in the animal. This is done by replacing some of its genetic material with the human counterpart; the difficulty is to insert the human gene with all its regulatory elements in the right spot. Andrew Smith from Edinburgh University has now developed a strategy that allows the replacement of large genomic regions, as recently described in an article in Cell.
The Smith team focused on the 
globin locus; a mutation in the regulatory region of this locus causes thalassemia in humans, a fatal disease if both copies of the globin gene are affected. The corresponding regulatory sequence in mice has been identified; however, even in a homozygous animal the equivalent mutation only has a mild phenotype. Smith explains, "You can't actually model the human disease in a mouse, because there is something different about the regulation and that prompted us to do what we did."
Their genomic replacement strategy is based on the recombination between matching DNA sequences, lox sites (locus for crossing over), mediated by the enzyme Cre recombinase. To obtain a mouse that expresses human globin, the researchers first inserted heterospecific, that is, noninteracting, lox sites at either end of the globin gene in a mouse embryonic stem cell (ESC); this ensured that no recombination could take place that simply deleted the gene. They then placed the same lox sites in a bacterial artificial chromosome (BAC) on either side of the 120-kb human globin region, and electroporated the BAC together with a plasmid encoding Cre recombinase into the ESCs. Cre-mediated recombination took place between the matching lox sites at the proximal and the distal end of the genomic region, thus cleanly exchanging the human globin locus for that of the mouse.
Smith explains the advantage of this approach, "A large defined region of the BAC insert is precisely integrated as a single copy at a particular position in the genome;...within the BAC clone you can make a number of different mutations or you can, for example, delete transcription factor binding sites within a regulatory element and you can then reintegrate these back into the endogenous locus in a single step."
Although this exchange method is a powerful tool for the precise replacement of genetic regions of up to 300 kb, the present size limit of a BAC, technical innovations are needed to exchange much larger regions or multiple genes.
An example of a project that required the exchange of larger genomic regions was undertaken by scientists at the biotechnology company Regeneron. Their goal was to create a mouse with a humanized immune system, but instead of using recombination at carefully positioned lox sites, they engineered BACs in which human genes are flanked by mouse DNA. These mouse DNA arms insert the BAC at the correct genomic position through homologous recombination. With this approach the scientists succeeded in precisely exchanging 6 Mb of mouse DNA with the human counterpart, according to George Yancopoulos, chief scientific officer at Regeneron.
Different methods of making humanized mice will give researchers a choice when it comes to making their own version of Stewart Little's distant cousin.