Title

Development and phenotypic characterization of transgenic mice with bone-directed overexpression of insulin-like growth factor-I

Date of Completion

January 2002

Keywords

Biology, Molecular

Degree

Ph.D.

Abstract

Insulin-like growth factor (IGF-I) is an anabolic growth factor for bone formation. To explore the local effect of IGF-I on bone remodeling, transgenic mice were developed having bone-directed overexpression of FLAG epitope tagged murine IGF-I driven by a 3.6 kb fragment of the col1a1 gene (pOBCol3.6-IGF). ^ Transgene expression was first demonstrated in vitro. Five CD-1 pOBCol3.6IGF lines were generated. Transgenic mice appeared healthy, bred normally and had growth rates similar to wild-type littermates. Analysis of transgene mRNA expression indicated that the transgene was targeted to bone tissue, with some extent of expression in tendon and skin. Transgene mRNA expression was strong in young animals and then gradually decreased. The level of transgene mRNA expression varied among the different founder lines. ^ The level of transgene expression was correlated with the bone phenotype in three independent founder lines. Percent collagen synthesis (PCS) in calvarial cultures was significantly greater in both the high and intermediate expressing lines than in wild-type. However, PCS of the low expressing line was not different than wild-type. Static histomorphometry showed that 8-week old transgenic calvariae from the high and intermediate expressing lines were significantly wider and had greater marrow area and osteoclast number/bone area than wild-type calvariae; however, calvariae from the low expressing line and wild-type mice were similar. Calvariae of the intermediate expressing line maintained the phenotype at 7 months. Eight-week old transgenic femurs had increased cortical width. Trabecular bone was increased and decreased in the high and intermediate expressing lines, respectively. There was no significant change in the low expressing line. ^ To assess bone resorption in vitro, calvariae from 2–4 day-old neonatal mice were labeled in utero with [45Ca] and cultured ex vivo. In 5 day cultures, the release of [45Ca] was significantly higher in transgenic calvariae from the intermediate expressing line. To assess ostoeclastogenesis, bone marrow cells were cultured in the presence of RANKL and M-CSF. Osteoclast formation was 2-fold greater in bone marrow cultures from transgenic mice compared to wild-type mice. ^ In summary, pOBCol3.6-IGF mice showed transgene dose-dependent bone phenotype that likely reflects simulation of bone resorption and formation by osteoblast-directed IGF-I expression. ^