Summer Students/2000 Meghan Kibbey's project. "The Design and Production of Antisera to Recombinant Human Dentin Sialophosphoprotein (DSPP)." Megan M Kibbey, Songtao Shi, Larry W.Fisher. National Institute of Dental and Craniofacial Research, Craniofacial and Skeletal Diseases Branch. Dentin Sialoprotein (DSP) and Dentin Phosphoprotein (DPP) are the principal noncollagenous proteins found in dentin extracellular matrix. However, it was recently found that they are a protease-cleavage product of the translation of a single gene, Dentin Sialophosphoprotein (DSPP). The DSPP gene is located on human chromosome 4q21, within the locus of dentinogenesis imperfecta type II and dentin dysplasia type II, and may play a role in their pathogenesis'. Although message RNA for DSPP has been shown to be expressed in odontoblasts and preameloblasts, its function is unknown. To help elucidate questions of function, we have produced portions of the human DSP and mouse DSP proteins,and had antiserums made against these products in rabbits. We have used these antisera in Western blot analysis and in immunolocalization. We cloned the human DSP and mouse DSP genes by polymerase chain reaction (PCR), ligated the fragments into a pET15b vector, transformed DE3 bacterial cells, released the overexpressed proteins through sonication, and purified the Hisx6 fusion proteins on a Ni2+-charged column. Western blot analysis indicated that the antiserum (LF-151) made against the human DSP domain cross-reacted with human and cow, but not mouse dentin. The same antiserum on paraffin-embedded sections revealed similar specificity, staining human odontoblasts and dentin, but not mouse. The antiserum against the mouse DSP domain (LF-153) was specific for mouse in both the Western blot analysis and in immunolocalization. In conclusion, we have produced polyclonal antisera against the DSP portion of human and mouse DSPP that work will in both Western blot analysis and immunolocalization. The failure of the two antisera to cross-react with both human and mouse will make them particularly useful in studies involving the implantation of human dental cells in immune-suppressed mice.