Bone phenotype of P2X4 receptor knockout mice: implication of a P2X7 receptor mutation?
Maria Ellegaard1, Tanja Hegner1, Ming Ding2,3
1Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark.
Abstract:
Transgenic and knockout animal models are widely used to investigate the role of receptors, signaling pathways, and other peptides and proteins. Varying results are often published on the same model from different groups, and much effort has been put into understanding the underlying causes of these sometimes conflicting results. Recently, it has been shown that a P2X4R knockout model carries a so-called passenger mutation in the P2X7R gene, potentially affecting the interpretation of results from studies using this animal model. We therefore report this case to raise awareness about the potential pitfalls using genetically modified animal models, especially within P2 receptor research. Although purinergic signaling has been recognized as an important contributor to the regulation of bone remodeling, the process that maintains the bone quality during life, little is known about the role of the P2X4 receptor (P2X4R) in regulation of bone remodeling in health and disease. To address this, we analyzed the bone phenotype of P2rx4tm1Rass (C57BL/6J) knockout mice and corresponding wildtype using microCT and biomechanical testing. Overall, we found that the P2X4R knockout mice displayed improved bone microstructure and stronger bones in an age- and gender-dependent manner. While cortical BMD, trabecular BMD, and bone volume were higher in the 6-month-old females and 3-month-old males, this was not the case for the 3-month-old females and the 6-month-old males. Bone strength was only affected in the females. Moreover, we found that P2X4R KO mice carried the P2X7 receptor 451P wildtype allele, whereas the wildtype mice carried the 451L mutant allele. In conclusion, this study suggests that P2X4R could play a role in bone remodeling, but more importantly, it underlines the potential pitfalls when using knockout models and highlights the importance of interpreting results with great caution. Further studies are needed to verify any specific effects of P2X4R on bone metabolism.
Insights
Genetically modified mouse models may have unintended mutations, impacting research. This study found P2X4R knockout mice showed altered bone structure, but a co-occurring P2X7R mutation complicates results.
Area of Science:
- Bone biology and purinergic signaling research.
- Genetically modified animal models and their limitations.
- Investigating receptor function in skeletal health.
Background:
- Purinergic signaling influences bone remodeling, but the role of P2X4 receptor (P2X4R) remains unclear.
- Genetically modified models are crucial but can harbor confounding mutations.
- A P2X4R knockout model was found to possess a passenger mutation in the P2X7R gene.
Purpose of the Study:
- To investigate the role of P2X4R in bone remodeling using knockout mice.
- To raise awareness about potential pitfalls in using genetically modified animal models.
- To analyze the bone phenotype of P2X4R knockout mice and wildtype controls.
Main Methods:
- Microcomputed tomography (microCT) for bone microstructure analysis.
- Biomechanical testing to assess bone strength.
- Genotyping to identify mutations in P2X7R gene.
Main Results:
- P2X4R knockout mice exhibited improved bone microstructure and strength in an age- and gender-dependent manner.
- Cortical and trabecular bone mineral density and bone volume varied by age and sex.
- A significant finding was the presence of a P2X7R wildtype allele in knockout mice and a mutant allele in wildtype mice.
Conclusions:
- P2X4R may play a role in bone remodeling.
- This study highlights critical pitfalls associated with knockout models, emphasizing cautious interpretation of results.
- Further research is necessary to confirm P2X4R's specific effects on bone metabolism.
