Background: Generating controlled spatial and temporal knockouts in mice is a lengthy and expensive process. In bone, it is also often hindered by issues of embryonic lethality and confounding phenotypes in other tissues. Recombinant adeno-associated viral vectors (rAAV) offer an alternative method for gene editing applicable to neonatal and adult mice. Our group has previously produced a bone-specific rAAV (AAV8-Sp7-Cre) that demonstrates high efficiency and specificity for osseus tissues. It was hypothesized that this AAV-Cre vector could be employed to create targeted gene disruption in postnatal animals. This study aimed to knockout the murine Sclerostin (Sost) gene, previously shown to be a key negative regulator of bone mass.
Methods: 8-week-old Sostflox/flox mice were systemically injected with either AAV8-Sp7-Cre at 5x1011 vg/mouse or saline. Bone density was measured by longitudinal DXA for 6 weeks. Fore and hind limbs and vertebrae were harvested for genetic analysis, microCT, biomechanical testing, standard bone histology and dynamic histomorphometry.
Results: Sostflox/flox:AAV-Cre mice showed enhancement of numerous bone parameters including trabecular and cortical thickness, trabecular bone volume fraction, and cortical bone volume. This was associated with a +25% increase in mineral apposition rate in the cortical bone. Analysis of AAV8-Sp7-Cre mediated recombination in an Ai9 fluorescent reporter mouse model confirmed efficient and bone-targeted vector activity.
Discussion: Consistent with prior reports of Sost null mice, Sostflox/flox:AAV-Cre demonstrate a high bone mass phenotype and increased bone anabolism. This technology represents a new and streamlined approach to generate conditional gene knockout mice restricted to postnatal bone, where floxed mouse strains are available. This technology is not only economical and versatile, but it also overcomes challenges with studying genes where developmental disruption is embryonically lethal.