Little is known about the molecules which influence the site-center specific recruitment of osteoclast precursors or subsequent formation and activation events in vivo. To identify the genes expressed early in the development of osteoclasts, we have employed differential display technique using RNA from developmentally-staged mouse metatarsal rudiments as starting material. Total RNA was isolated from metatarsal bones at embryonic day 14 (primary chondrogenic) and day 17, when TRAP+ mononuclear precursors have migrated to the surrounding periosteum prior to fusion and invasion of the cartilage. mRNA was amplified by RT-PCR using degenerate anchored 3' primers and random 5' primers ( 10mer) in the presence of radiolabelled ATP. Differentially expressed products were identified in duplicate reactions on 6% polyacylamide gels, excised, and reamplified by PCR using the same primer set. Reamplified products were agarose gel purified, ligated into a TA cloning vector (In Vitrogen) and sequenced using dideoxy chain termination method. Sequence homology was analyzed by searching GenBank/EMBL using DNASIS. To date, several of these products have high degrees of homology to known genes; these include four products with >91% to the murine F1/FO ATPase subunit, one with 90%, 72%, and 73% homology to members of the stefin family of cysteine proteinase inhibitors, and closely related to the human calcitonin receptor. Many of the products analyzed do not closely match sequences in the databases and represent potentially novel gene products. These data demonstrate the potential usefulness of differential display for identifying gene products involved in osteoclast maturation and in the regulation of osteoclast development.
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