Identification of mutations in the mouse gene encoding spp24 and analysis of protein interactions

Secreted phosphoprotein 24 (spp24) was first isolated from bovine cortical bone. The mature human protein consists of 182 amino acids and is encoded by a gene designated SPP2. The function of spp24 is unknown, however, evidence suggests that it may be a growth hormone responsive protein, involved with calcification and the cytoskeleton, as well as cancer and osteoarthritis. The aim of this project was to identify more information regarding the function of spp24. Four different but complementary approaches were used; screening DNA samples of ENU-­mutagenised mice to identify mice with mutations in spp24 and screening an ENU-mutagenised ES-cell library, using the yeast two-hybrid system to identify proteins that interact with spp24 and assessing the effect of spp24 mutations on these interactions, generating a mouse with non-functional spp24 (a knockout mouse), and expressing and purifying spp24. Using these approaches five mutations that alter the amino acid sequence of spp24 were identified. Fourteen additional proteins that potentially interact with spp24 were identified but the effect of mutations in spp24 on the interaction strength could not be accurately determined. A spp24 targeting vector to generate a knockout mouse was assembled and transfected but no homologous recombinants could be identified, so it was not possible to generate a knockout mouse. Spp24 expression was attempted using a baculovirus insect cell system, an in vitro system and an E. coli system but no spp24 expression was detected with these systems. Future work will involve generating and characterising mice with mutated spp24 and further analysis of the interaction of spp24 and the proteins identified.