ECTS2016 Poster Presentations Cell biology: Osteocytes, mechanobiology (3 abstracts)
Transgene expression by Dmp1 promoter fragments occurs in various organs
Hiroaki Saito 1 , Hanna Taipaleenmäki 1 , Ahmed Al-Jazzar 2 , Andreas Gasser 1 , Behzad Javaheri 2 , Cheryl Scudamore 2 , Teresita Bellido 3 , Andrew A Pitsillides 2 & Eric Hesse 1,
1Heisenberg-Group for Molecular Skeletal Biology, Department of Trauma, Hand & Reconstructive Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 2Comparative Biomedical Sciences, The Royal Veterinary College, London, UK; 3Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Analysis of osteocyte function often uses promoter elements of osteocyte-specific genes i.e. SOST or Dentin-matrix-protein 1 (Dmp1) to overexpress genes or the Cre-recombinase for conditional deletion studies. However, evidence suggests that these promoters may not be osteocyte-specific, which would be critical for subsequent data interpretation. To investigate the selectivity of supposedly osteocyte-specific in vivo models, we crossed the 8kb-Dmp1-Cre mice (i) with Ai9 tomato reporter mice (Dmp1-Cre+;Ai9T/wt), and (ii) with mice, in which the expression of the diphtheria toxin receptor (DTR) is controlled in a Cre-inducible manner (Dmp1-Cre+;iDTRT/wt) to ablate Dmp1-positive cells. Furthermore, we ablated Dmp1-positive cells in mice harboring human DTR (hDTR) regulated by a 10kb-Dmp1 promoter fragment (Dmp1-hDTR). Immunohistochemical staining of tibiae harvested from 8-week old Dmp1-Cre+;Ai9T/wt mice revealed a strong tomato expression in all osteocytes and osteoblasts covering endocortical and trabecular surfaces. Furthermore, we detected tomato expression in muscle, brain, testis, and in vessels in the heart, spleen, lung, and intestine. Consistently, histological analyses of bones five days after diphtheria toxin (DT) administration in Dmp1-Cre+;iDTRT/wt and Dmp1-hDTR mice revealed an efficient ablation of not only osteocytes but also osteoblasts and lining cells. In addition, DT injection resulted in liver vacuolation, acute kidney necrosis, splenic atrophy, and disturbance of bone marrow composition in Dmp1-hDTR mice, which is consistent with expression of hDTR mRNA in several tissues including muscle, liver, kidney and spleen. Taken together, our results indicate that in 8kb-Dmp1-Cre mice as well as in Dmp1-hDTR mice, expression of the respective transgenes during mouse development and growth is not restricted to osteocytes, but also takes place in other osteolineage cells and in several organs. Our findings therefore suggest that despite the great usefulness of these in vivo systems, the expression pattern of the gene of interest should be determined carefully and the results need to be interpreted accordingly.
Volume 5
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Bone Abstracts
ISSN 2052-1219 (online)
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