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Updated: Jul 28, 2026
A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders
Published on: June 8, 2014
Targeted postnatal knockout of Sclerostin using a bone-targeted adeno-associated viral vector increases bone
Alexandra K O'Donohue1, Ya Xiao2, Lucinda R Lee1
1Bioengineering & Molecular Medicine Laboratory, The Children's Hospital at Westmead and the Westmead Institute for Medical Research, Westmead, NSW, Australia; The Children's Hospital at Westmead Clinical School, The University of Sydney, Camperdown, NSW, Australia.
Purpose:
The creation of murine gene knockout models to study bone gene functions often requires the resource intensive crossbreeding of Cre transgenic and gene-floxed strains. The developmental versus postnatal roles of genes can be difficult to discern in such models. For example, embryonic deletion of the Sclerostin (Sost) gene establishes a high-bone mass phenotype in neonatal mice that may impact on future bone growth. To generate a postnatal skeletal knockout of Sost in adult mice, this study used a single injection of a bone-targeted recombinant adeno-associated virus (rAAV) vector.
Methods:
8-week-old Sostflox/flox mice were injected with saline (control) or a single injection containing 5 × 1011 vg AAV8-Sp7-Cre vector. Ai9 fluorescent Cre reporter mice were dosed in parallel to confirm targeting efficiency. After 6 weeks, detailed bone analysis was performed via microCT, biomechanical testing, and bone histology on vertebral and long bone specimens.
Results:
The AAV8-Sp7-Cre vector induced widespread persistent recombination in the bone compartment. Regional microCT analyses revealed significant increases in bone with vector treatment. In the L3 vertebrae, Sostflox/flox:AAV-Cre showed a 22 % increase in bone volume and 21 % in trabecular bone fraction compared to controls; this translated to a 17 % increase in compressive strength. In the tibiae, Sostflox/flox:AAV-Cre led to small but statistically significant increases in cortical bone volume and thickness. These were consistent with a 25 % increase in mineral apposition rate, but this did not translate into increased four-point bending strength. Ploton silver nitrate stain on histological sections revealed an unexpected increase in canalicular density associated with Sost ablation.
Conclusion:
This report demonstrates a proof-of-concept that the AAV8-Sp7-Cre vector can efficiently produce postnatal skeletal knockout mice using gene-floxed strains. This technology has the potential for broad utility in the bone field with existing conditional lines. These data also confirm an important postnatal role for Sost in regulating bone homeostasis, consistent with prior studies using neutralizing Sclerostin antibodies, and highlights a novel role of Sost in canalicular remodeling.
Insights
This study developed a new method using recombinant adeno-associated virus (rAAV) to create postnatal Sclerostin (Sost) gene knockout mice. This approach efficiently generates skeletal knockout models for studying bone gene function.
Area of Science:
- Molecular Biology
- Genetics
- Orthopedics
Background:
- Creating gene knockout models for bone research is resource-intensive.
- Distinguishing developmental from postnatal gene roles is challenging with traditional crossbreeding methods.
- Embryonic deletion of Sclerostin (Sost) leads to high bone mass, complicating the study of its postnatal functions.
Purpose of the Study:
- To establish a postnatal skeletal knockout of the Sclerostin (Sost) gene in adult mice.
- To utilize a bone-targeted recombinant adeno-associated virus (rAAV) vector for gene deletion.
- To investigate the postnatal role of Sost in bone homeostasis.
Main Methods:
- Adult Sostflox/flox mice received a single injection of AAV8-Sp7-Cre vector or saline control.
- Bone analysis included microCT, biomechanical testing, and histology after 6 weeks.
- Cre reporter mice confirmed vector targeting efficiency in the bone compartment.
Main Results:
- AAV8-Sp7-Cre induced widespread and persistent recombination in bone.
- Vector treatment significantly increased bone volume and trabecular bone fraction in vertebrae, enhancing compressive strength.
- Tibial analysis showed increased cortical bone volume and mineral apposition rate, with an unexpected increase in canalicular density.
Conclusions:
- AAV8-Sp7-Cre efficiently generates postnatal skeletal knockout mice from gene-floxed strains.
- This technology offers broad utility for studying gene function in existing conditional mouse lines.
- Sclerostin (Sost) plays a significant postnatal role in bone homeostasis and canalicular remodeling.
