論文 - 詳細
| RRC ID | 50026 |
|---|---|
| 著者 | Kobori M, Ikeda Y, Nara H, Kato M, Kumegawa M, Nojima H, Kawashima H. |
| タイトル | Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library. |
| ジャーナル | Genes Cells |
| Abstract |
BACKGROUND:Osteoclasts play crucial roles in bone resorption, which triggers bone remodeling. Molecular mechanisms underlying these osteoclast-specific biological functions remain elusive because only a limited number of osteoclast-specific genes have been identified. To circumvent this, we isolated a large number of osteoclast-specific genes by preparing a subtracted cDNA library of high quality. RESULTS:We first constructed a plasmid expression vector (pAP3neo) that allowed an efficient subtraction. Then, we improved the standard protocols for preparation of the cDNA library and the subsequent subtraction procedure. Using our protocol, we prepared a rabbit osteoclast cDNA library of high complexity. Subsequently, we prepared an osteoclast-specific cDNA library of high complexity by subtracting it with biotin-labelled mRNA, derived from rabbit spleen through the biotin-avidin method. The resulting library included a high proportion of full-length cDNA inserts. Using DNA dot blot analysis, we found that the osteoclast-specific cDNA clones were highly enriched in this subtracted cDNA library, i.e. nearly 70% of the analysed clones were primarily detected in osteoclasts but not in spleen. Multiple-tissue Northern analysis also showed that many of these clones were expressed almost exclusively in osteoclasts. DNA sequencing of randomly selected clones showed that 424 cDNA species out of 1136 analysed were novel. DNA sequencing also showed that our subtracted cDNA library was almost equalized, suggesting that the library may contain almost all of the osteoclast-specific genes. CONCLUSION:From these data, we conclude that our subtraction protocols, and the subsequent procedure for the analysis of the isolated clones developed here, are useful for the comprehensive isolation and identification of transcriptionally up- or down-regulated genes in general. |
| 巻・号 | 3(7) |
| ページ | 459-75 |
| 公開日 | 1998-7-1 |
| DOI | 10.1046/j.1365-2443.1998.00202.x |
| PMID | 9753427 |
| MeSH | Animals Base Sequence Bone Morphogenetic Protein 2 Bone Morphogenetic Proteins / genetics Bone Resorption COS Cells Cloning, Molecular / methods* Cystatins / genetics Cystatins / pharmacology DNA, Complementary / genetics* Gene Expression Regulation, Developmental Gene Library* Genetic Vectors Humans Molecular Sequence Data Nucleic Acid Hybridization Organ Specificity Osteoclasts* / physiology RNA, Messenger / analysis Rabbits Sequence Analysis, DNA Transforming Growth Factor beta* |
| IF | 1.655 |
| 引用数 | 38 |
| リソース情報 | |
| 遺伝子材料 | pAP3neo cDNA Library Mouse 10T-half (RDB02903) |