Sp7/Osterix is restricted to bone-forming vertebrates where it acts as a Dlx co-factor in osteoblast specification [ChIP-Seq 2]. Mus musculus

To identify Sp7’s molecular interactions in vivo, we used gene-targeting strategies to generate an Sp7-Biotin-3xFLAG knock-in mouse (Sp7-BioFL mouse). This approach appends a biotin (Bio) recognition motif and three copies of the FLAG (FL) epitope at the C-terminus of the Sp7 protein. To validate this knockin system, we compared ChIP-seq results in the MC3T3E1 osteoblast cell line, introducing Sp7 forms that were epitope-tagged at either the N- (N-terminal FLAG tag; MC3_FLAG-Sp7) or C-terminus (C-terminal Biotin-FLAG tag, as in the in vivo targeted allele; MC3_Sp7-BioFLAG). These data show an extensive overlap, suggesting that FLAG tagging at different positions gives comparable outcomes. Second, we examined ChIP on wild-type calvarial primary osteoblasts (POB) using an anti-Sp7 antibody (POB_Sp7Ab-ChIP). The majority of Sp7-Ab ChIP-seq peaks (95%) overlapped with the larger set identified by Sp7-BioFL ChIP-seq. Overall design: ChIP-seq experiments with primary osteoblasts and a osteoblast cell line were performed