Our goal was the generation of a murine model for Osteogenesis Imperfecta(OI), suitable for development of a gene therapeutic approach using hammerhead ribozymes. Site directed mutagenesis was perform on a subcloned collagen fragment to insert in exon 23 a G→A, changing α1(I) gly349→cys and a C→T, creating a ribozyme cleavage site. The glycine substitution reproduced the causative mutation detected in one of our moderately severe patients. Since the expected phenotype could compromise F1 survival, a transcription/translation stop cassette under the control of the cre/lox system was inserted in intron 22. We expected an F1 with null expression of the mutant allele, as associated with mild OI. Mating of the F1 generation with wt mice expressing cre under the control of the EIIa promoter was predicted to yield an F2 with expression of OI in a phenotypically severe non-lethal form. By standard procedures, three chimeras were generated and used to produce two murine models with a skeletal phenotype characteristic of human OI.

A knock-out mouse, from a chimera x wt mating, was lethal due to the alternative splicing generated by the presence of the lox-stop-lox cassette itself. One alternatively spliced form was in-frame. Radiography, histology and skeletal staining confirmed a phenotype with multiple rib fractures and poor skeletal mineralization. No mutant protein was secreted; the phenotype may result from protein suicide effects.

The chimeras were also mated with mice expressing cre recombinase, to eliminate the stop cassette and allow expression of the collagen mutation. The majority of the pups were mosaics of the two forms of mutant allele in each tissue examined. Their lethal OI phenotype was confirmed by radiography, histology, skeletal staining, and biochemical and molecular analysis. We obtained two surviving mice from more than 100 examined pups. Their mating with wt mice will permit us to obtain mice with full expression of the mutant allele carrying the causative glycine mutation. The cre/lox system yielded conditional expression of the mutant allele. This opens the possibility of using cre/lox to generate animal models for other dominant disorders with severe/lethal forms.