Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library.

Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library. Research Abstract Details 

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Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library. Abstract Text:

M Kobori,Y Ikeda,H Nara,M Kato,M Kumegawa,H Nojima,H Kawashima,

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.

Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library. Publishing Authors By Initials

M Kobori,Y Ikeda,H Nara,M Kato,M Kumegawa,H Nojima,H Kawashima,

For similar peptides: intercellular signaling peptides and proteins: cytokines: transforming growth factor beta research abstracts see: peptides: intercellular signaling peptides and proteins: cytokines: transforming growth factor beta research

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Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Genes to cells : devoted to molecular & cellular m

VOLUME: 3

Page Numbers: 459-75

Journal Abbreviation: Genes Cells

ISSN: 1356-9597

DAY: 19

MONTH: Jul

YEAR: 1998

Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library. Information

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LANGUAGE: eng

NlmUniqueID: 9607379

Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library. Keywords Mesh Terms:

KEYWORDS: Transforming Growth Factor beta

MESH TERMS: analysis

Chemical & Substance for Abstract: Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library. Information

Substance Name: bone morphogenetic protein 2

Registry Number: 0

Grant and Affiliation Information for Large scale isolation of osteoclast-specific genes by an improved method involving the preparation of a subtracted cDNA library.

AFFILIATION: Molecular Medicine Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd, Tsukuba, Ibakaki, Japan.

Country: ENGLAND

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MEDLINETA: Genes Cells

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