Use of the Cre/lox recombination system to develop a non-lethal knock-in murine model for osteogenesis imperfecta with an alpha 1(I) G349C substitution - Variability in phenotype in BrtlIV mice

We utilized the Cre/lox recombination system to develop the first knock-in murine model for osteogenesis imperfecta (OI). The moderately severe OI phe notype was obtained from an alpha 1(I) Gly(349) --> CyS substitution in typ e I collagen, reproducing the mutation in a type IV OI child. We introduced four single nucleotide (nt) changes into murine collal exon 23: the diseas e causing G --> T transversion (nt 1546), an adjacent G --> T change (nt 15 51) to generate a GUC ribozyme cleavage site, and two transversions (nt 156 7 C --> A and nt 1569 C --> G;) to cause a Leu --> Met substitution, We als o introduced a 3.2-kilobase pair transcription/translation stop cassette in intron 22, flanked by directly repeating lox recombination sites. After ho mologous recombination in ES cells, two male chimeras were obtained, Chimer as were mated with transgenic females expressing Cre recombinase to remove the stop cassette from a portion of the progeny's cells. To generate mice w ith full expression of the Gly(349) --> CYS mutation, these offspring were then mated with wild-type females. Skeletal staining and bone histology of the F2 revealed a classical OI phenotype with deformity, fragility, osteopo rosis and disorganized trabecular structure. We designate these mice Brt1IV (Brittle IV). Brt1IV mice have phenotypic variability ranging from perinat al lethality to long term survival with reproductive success. The phenotypi c variability is not associated with differences in expression levels of th e mutant allele in total RNA derived from tissue extracts. Expression of th e mutant protein is also equivalent in different phenotypes. Thus, these mi ce are an excellent model for delineation of the modifying factors postulat ed to affect human OI phenotypes. In addition, we generated knock-in mice c arrying an "intronic" inclusion by mating chimeras with wild-type females. Alternative splicing involving the stop cassette results in retention of no n-collagenous sequences. These mice reproduce the lethal phenotype of simil ar human mutations and are designated BrtIII.