Figure 1. Methods of humanised mouse synthesis.

There are multiple ways of introducing the human genomic region of interest into the mouse germline. (a) Traditionally this has been via an additive process, where a YAC or BAC vector is introduced via pronuclear injection or cell fusion, resulting in random incorporation into mouse genome, while the endogenous mouse locus is unmodified. (b, c) An alternative is the specific targeting and replacement of genomic loci, either using (b) homologous recombination or (c) the SSR-based technologies RMCE and RMGR. Homologous recombination with a genomic fusion (mouse-human) BAC vector results in the endogenous mouse locus being replaced by equivalent human sequence, using the large regions of homology provided by the BAC vector for increased targeting efficiency (b). RMCE and RMGR require prior modification of the mouse genome, to introduce heterotypic SSR sites to flank the region of interest. A BAC vector containing equivalent human genomic region flanked by same SSR sites then acts as a donor for the swap of genetic material mediated by expression of recombinase (c). (d) A non-integrative approach to creating a humanised mouse is by the introduction of a HAC into ES cells via MMCT. The HAC vector is synthesised via a top-down or bottom-up approach, where the genomic region of interest is introduced by homologous recombination or SSR. The HAC is mitotically stable and maintained as an extra-chromosomal element, leaving the mouse genome unmodified.