A LONG-RANGE REGULATORY ELEMENT OF HOXC8 IDENTIFIED BY USING THE PCLASPER VECTOR

Hox genes are located in highly conserved clusters. The significance o f this organization is unclear, but one possibility is that regulatory regions for individual genes are dispersed throughout the cluster and shared with other Hox genes. This hypothesis is supported by studies on several Hox genes in which even large genomic regions immediately s urrounding the gene fail to direct the complete expression pattern in transgenic mice. In particular, previous studies have identified proxi mal regulatory regions that are primarily responsible for early phases of mouse Hoxc8 expression. To locate additional regulatory regions go verning expression during the later periods of development, a yeast ho mologous recombination-based strategy utilizing the pClasper vector wa s employed. Using homologous recombination into pClasper, we cloned a 27-kb region around the Hoxc8 gene from a yeast artificial chromosome. A reporter gene was introduced into the coding region of the isolated gene by homologous recombination in yeast. This large fragment recapi tulates critical aspects of Hoxc8 expression in transgenic mice. We sh ow that the regulatory elements that maintain the anterior boundaries of expression in the neural tube and paraxial mesoderm are located bet ween 11 and 19 kb downstream of the gene.