Generation and Characterization of Osterix-mCherry Reporter Mice

Objectives: Osterix is a zinc finger transcription factor which functions as a master regulator of osteoblast differentiation. We report here on the generation of  Osterix-mCherry reporter mice.

Methods: We subcloned a 40 kb region encompassing the Osterix gene from BAC clone RP24-362M3. An mCherry fluorescent reporter was then inserted into this subcloned genomic DNA region. Pronuclear injection was carried out with this construct and two founder lines were generated.

Results: Preliminary characterization of reporter gene expression at early postnatal ages indicates Osterix expression is largely restricted to cells of the osteoblast lineage. Robust mCherry expression can be detected in cells of the osteoblast lineage in the skull, long bones, and axial skeleton.  Reporter expression can also be detected in the kidney. At adult ages, Osterix expression is not exclusive to the osteoblast lineage and is moderately expressed in hypertrophic chondrocytes and weakly expressed in cells present within the bone marrow.  Osterix expressing cells in the bone marrow appear close to, but are not on the bone surface.  These cells appear similar to reticular cells with regards to their cell morphology. 

To validate the expression pattern of Osterix-mCherry reporter expression, we carried out immunostaining studies for  endogenous Osterix protein on tissue sections. Also, qRT-PCR analysis for Osterix on FACS isolated cells from day 5 stromal cultures revealed that the mCherry positive cell fraction was approximately 35 fold higher in endogenous Osterix gene expression relative to the negative cell fraction. 

Conclusions: These studies substantiate that our reporter accurately represents endogenous Osterix expression in the cortical bone and cells of the bone marrow. The identity and significance of these mCherry positive cell types is currently under investigation. Future studies will further validate and characterize the  mCherry + reporter expression relative to endogenous Osterix gene expression.