Molecular Genetic Analysis of the Role of the HoxD Complex in Skeletal Development

Abstract

There is an ever-growing list of human and mouse genes with implications in skeletal development. Among them, Hox genes represent a particular class for at least two reasons: first, in absence of Hox gene products, skeletal development becomes altered in many respects, including variation in spatial anatomical pattern and temporal tissue-maturation sequence. Second, Hox genes show clustered genomic organization, which in itself is expected to exert profound, as yet poorly understood, influences in executing their function through influencing gene expression control. Therefore, analyzing the role of Hox genes in skeletal development must involve large-scale manipulations at the level of gene complexes. In this chapter, we give a brief overview of our experimental strategies that involve site-specific recombination at the HoxD gene cluster. These are experimental manipulations of the mouse genome applying the loxP/Cre recombination system (Fig. 1). The most important aspect of this system for targeted mutagenesis is that it allows sequential modifications of the genome at predefined positions as a result of the specificity of recognition and polarity of recombination induced by the Cre enzyme between loxP sites (1). This 35-nucleotide long recombination target site, the loxP site, does not normally occur in the mammalian genome but can be introduced to selected genomic positions by homologous recombination thanks to embryonic stem (ES) cell technology. When two loxP sites and the Cre recombinase are present in a cell, the recombination reaction can occur either in vitro or in vivo in individuals derived from such modified ES cells. A large number of alleles can be generated in this way, facilitating the genetic analysis of complex loci, like the Hox gene clusters (Fig. 2).