Classical lissencephaly, a dominant human developmental disease characterized by a smooth cerebral surface and microscopic evidence of incomplete neuronal migration is often associated with small deletions or translocation at chromosome 17p13.3. Miller-Dieker syndrome (MDS) is associated with larger deletions and consists of classical lissencephaly and more complex phenotypes including facial abnormalities. To determine the contribution of various genes on 17p13.3 to MDS, we plan to make various microdeletions spanning the MDS critical region in mouse using the Cre-loxP system. We first obtained the gene order and orientation of three candidate genes, Lis1, L132 and 14-3-3epsilon in mouse. The orientation and distance of Lis1 and L132 were determined by fragmentation analysis of a YAC containing both genes. To determine the distance and orientation of 14-3-3epsilon with respect to Lis1 and L132, we introduced the super-rare cutter site VDE that is unique in the mouse genome into 14-3-3epsilon. Using the introduced a VDE site, the orientation of this gene was determined by PFGE and Southern blot analysis. Based on this information, we introduced VDE site into mouse ES cells with an adjacent loxP site in the same orientation within Lis1, L132, and 14-3-3epsilon. In addition, a second loxP site was introduced at the L132 locus in a Lis1 targeted ES clone. We identified ES clones in which two loxP sites were introduced into the same chromosome by digestion with VDE followed by detection of a DNA fragment of the expected size based on relative gene distances. These doubly-targeted ES clones were injected into mouse blastcysts to create chimeric mice, which will be mated to transgenic mice expressing the Cre recombinase to produce the microdeletions of the MDS region in the mouse.
