Fig. 2. Identification and targeted deletion of region XII (RXII). (A) Interspecies conservation within Evx2-Hoxd13 intergenic region. Sequence analyses revealed two stretches of significant conservation, referred to as region XII (RXII), which were found to be located within the insulating area (red bar). The position of these two sequences with respect to both Evx2 and Hoxd13 is schematised below for the mouse (m), chicken (ck) and zebrafish (zf). (B) Sequence alignment of region XII from mouse (m), chicken (ck) and zebrafish (zf) DNA. A high sequence similarity was observed between rodents and avian. The sequence conservation with the zebra fish DNA is less obvious, though significant whenever the respective positions of the two stretches are considered. (C) Strategy to delete region XII through targeted mutagenesis. A targeting vector was engineered lacking region XII and was recombined in ES cells to generate the Hoxd^RXII-neo mice. The selection cassette was further deleted after crossing these mice with a Cre deleter strain, to produce the Hoxd^RXII mice. In addition to the deletion of RXII, these mice also carried a loxP site at the exact integration site of the transgene shown under A. This loxP site was used for subsequent meiotic recombination approaches, as described in Fig. 4B,C.