Large scale sequencing of ESTs from human osteoclast cDNA library: “Electronic Northern Blot”

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• 作者：Drake ; F. ; Dodds ; R.A. ; James ; I. ; Connor ; J. ; Lee-Rykaczewski ; E. ; Hastings ; G. ; Rosen ; C. ; Gowen ; M.
• 刊名：Bone
• 出版年：1995
• 出版时间：December, 1995
• 年：1995
• 卷：17
• 期：6
• 页码：579
• 全文大小：150 K

文摘

Study of osteoclast gene expression has been hampered by the difficulty in obtaining sufficient numbers of cells and the lack of a suitable cell model system. To allow a better understanding of the pattern of gene expression of these cells, we have performed high throughput sequencing of randomly selected clones from a human osteoclast cDNA library. The cDNA library was prepared from human osteoclasts that were isolated from osteoclastoma-derived cell suspensions by anti-integrin (β₃) antibody attached to magnetic beads. Approximately 9300 clones were sequenced from this library and their expressed sequence tags (ESTs) were compared with known sequences by Blast analysis. Of the 9300 clones, 4357 did not match previously known sequences, while 2912 were known human proteins. The remainder showed homology to known human or non-human proteins. Based upon frequency in the library, the most abundant mRNA in the osteoclast was the matrix protein, osteopontin (11.5 % of ESTs). Cathepsin O was also expressed at high levels (4 % ), and was either not expressed or was expressed at very low levels in a number of other cDNA libraries. In situ hybridization and immunolocalization studies with probes and antibodies specific for osteopontin and cathepsin O confirmed their high level of expression in osteoclasts in cryostat sections of osteoclastoma and osteophyte. Cytokines IL-8 and IL-6 were present at 0.12 % and 0.05 % , respectively. Two ESTs for the calcitonin receptor were found, as well as 21 for the fibronectin receptor and 6 for the vitronectin receptor. The data indicate that large scale random sequencing of a cDNA library can provide a quantifiable analysis of gene expression in a cell type. Furthermore, as demonstrated by expression of cathepsin O, comparison of gene expression in cDNA libraries from different cell types allows analysis of relative expression of genes from different cells, essentially providing an “Electronic Northern blot”.