Generation Of A Knock-In Murine Model For Osteogenesis Imperfecta Reproducing The Biochemical, Molecular, Histological Characteristic Of Human Patients.

Antonella Forlino and Joan C. Marini
Heritable Disorders Branch, NICHD, NIH, USA

Purpose: To develop a murine model for Osteogenesis Imperfecta reproducing a characteristic glycine substitution in Type I collagen, present in single copy in the genome under the control of the endogenous promoter and with dominant transmission, we generated the first knock-in OI mouse (BrtlIV) using the cre/lox recombination system. Our model reproduces both the alpha1 (I) gly349cys substitution already described in a patient with the moderately severe type IV OI and the resultant clinical outcome.

Methods: By site-directed mutagenesis on a subcloned fragment of murine col1a1, a G->T transversion was introduced in exon 23 at nt 1456 to generate the causative molecular defect. Also a C->T transition was created at nt 1461 to generate a GUC ribozyme cleavage site in the mutated allele. A transcription/translation stop cassette flanked by two directly repeating lox sequences was inserted in intron 22. By homologous recombination in ES cells and standard procedures, 2 male chimeras were obtained. They were mated with transgenic mice expressing cre recombinase to eliminate the conditional stop cassette. The offspring were mosaic for the two forms of mutant allele, with and without the stop cassette. By mating the mosaic mice with wild-type CD1 females, non-lethal OI mice with full expression for the gly349cys substitution were generated.

Results: Expression of the mutant transcript was demonstrated by Hind III digestion of RT-PCR products amplified from the region coding for exons 21-25 of RNA extracted from embryos and cultured ication differed in the two cell types.

X-Ray and skeletal staining of BrtlIV mice confirmed the classical OI phenotype: general undermineralization of the skeleton, especially of the skull, deformity of the chest and smaller vertebral bodies were evident. Long bone fractures were detected only in the more severe mice.

Histological analysis of different tissues from Brtl IV mice and control littermates revealed the presence of immature and disorganized bone tissue in both calvarium and vertebral bodies. The calvarium was thinner than controls. Long bones were osteoporotic and trabecular structure was disorganized. Dentinogenesis imperfecta was also detected: necrotic pulp cavity, osteoid proliferation and fibrous tissue, rather than the well organized osteoid present in control mice, were evident in Brtl IV maxilla.

Phenotypic variability of the gly349cys mice was detected from matings of BrtlIV with females of either identical or different background. Matings with C57BL/6 and 129Sv produced exclusively lethal mice with death from respiratory insufficiency within hours of birth. Matings with CD1, as well as intercrosses of the F1, generated mice with variable outcome. About half of the mutant mice have a non-lethal outcome, with smaller size than wt littermates, flattened calvarium, flared chest and bowed limbs. They survive beyond breeding age and are reproductively successful. The remaining Brtl mice have a lethal outcome from respiratory failure within day 1 of life.

Conclusion: We generated and characterized the first knock-in murine model for OI. Brtl mice reproduce the genetic transmission, molecular defect, histology and phenotype of human OI. Because these mice have physiological and tissue-specific expression of mutant collagen, they are an ideal model for studies of OI pathophysiology and for testing of therapeutic agents such as bisphosphonates. In particular, because of the presence of the GUC ribozyme cleavage site linked to the mutation, they are an excellent model to develop hammerhead ribozyme for gene therapy of OI. Furthermore the phenotypic variability in the OI outcome of the knock-in mice reproduces a common characteristic for human dominant negative disorders and offers a valid model to better understand its molecular basis.

Reference: Proceedings of the 7th International Conference on Osteogenesis Imperfecta. Montreal, Canada, 1999.