CHARACT. OF A NOVEL RESORP.-REL. GENE IN THE OSTEOCLAST

人们对影响生物多样性的分子知之甚少 破骨细胞前体或随后的部位中心特异性招募 体内的形成和激活事件。鉴定表达的基因 在破骨细胞发育的早期，我们使用了分化 利用发育期小鼠跖骨RNA的显示技术 作为起始材料的雏形。从跖骨中提取总RNA 胚胎第14天(初级软骨化)和第17天的骨骼，当Trap+ 单个核前体细胞迁移到周围骨膜 导致软骨的融合和侵袭。RT-PCR法扩增mRNAs 用简并锚定的3‘端和随机的5’端引物(10mer) 存在放射性标记的三磷酸腺苷。差异表达的产物是 在6%聚酰胺凝胶上的重复反应中鉴定，切除，和 用相同的一套引物进行聚合酶链式反应。重新扩增的产物是 纯化的琼脂糖凝胶，连接到TA克隆载体(在Vitgen中)和 采用双脱氧链终止法进行测序。序列同源性是 用DNASIS检索GenBank/EMBL进行分析。到目前为止，有几个 这些产物与已知基因高度同源；这些基因包括 4个含有小鼠F1/FO ATPase亚基91%的产品，1个含90%， 与半胱氨酸Stefin家族成员的同源性分别为72%和73% 与人类降钙素密切相关的蛋白酶抑制剂 受体。许多被分析的产品与 这些数据库和代表潜在的新基因产品。这些数据 演示差异显示在以下方面的潜在用处 鉴定参与破骨细胞成熟的基因产物和参与 破骨细胞发育的调控。

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.