Chromosomal engineering to produce a model of human 5q-syndrome

Initial chromosomal engineering has been undertaken on the syntenic region of mouse chromosome 11. Chromosomal deletions were generated using Cre-loxP technology.  A primary loxP site was inserted at a determined anchor point, the sparc gene, present within the syntenic region identified on chromosome 11. Deletions were then created in ES cells following the insertion of a second loxP site into the same copy of chromosome 11, either using a random integration strategy or a defined targeting methodology, and the expression of Cre recombinase. A defined deletion of 150 kb was successfully generated by targeting a second loxP site to the glral gene which represented one end of the delineated critical region identified in humans. In addition, a series of nested deletions were generated successfully, varying in size from a few kb to over 5 Mb, from the sparc gene anchor point by the random integration of a second loxP site. Three mouse strains have been generated that are heterozygous for a deletion, although none have demonstrated a significant disruption to haematopoiesis. To complement the deletion strategy, a gene targeting approach was used to disrupt a candidate tumour suppressor gene present in the syntenic region identified on chromosome 11. The gene selected was fat2, a member of the cadherin gene family and homologue of the Drosophila fat tumour suppressor gene. Fat2-deficient mice were generated and analysis thus far has demonstrated the fat2 gene is not critical for normal haematopoiesis in 6 month old animals. Mice generated in this study have provided novel reagents with which to investigate the mechanisms of 5q- syndrome. Their use in conjunction with other reagents developed in the laboratory should prove useful in providing insight into the cellular and molecular controls that contribute to this disease.