Kobori M, Ikeda Y, Nara H, Kato M, Kumegawa M, Nojima H, Kawashima H
Molecular Medicine Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd, Tsukuba, Ibakaki, Japan.
Genes Cells. 1998 Jul;3(7):459-75. doi: 10.1046/j.1365-2443.1998.00202.x.
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.
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.
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.
破骨细胞在骨吸收过程中发挥关键作用，而骨吸收会引发骨重塑。由于仅鉴定出有限数量的破骨细胞特异性基因，这些破骨细胞特异性生物学功能背后的分子机制仍不清楚。为了克服这一问题，我们通过制备高质量的消减cDNA文库，分离出大量破骨细胞特异性基因。
我们首先构建了一个能实现高效消减的质粒表达载体（pAP3neo）。然后，我们改进了cDNA文库制备及后续消减程序的标准方案。使用我们的方案，我们制备了一个复杂度高的兔破骨细胞cDNA文库。随后，我们通过生物素-抗生物素蛋白方法，用来源于兔脾脏的生物素标记mRNA对其进行消减，制备了一个复杂度高的破骨细胞特异性cDNA文库。所得文库包含高比例的全长cDNA插入片段。通过DNA斑点印迹分析，我们发现破骨细胞特异性cDNA克隆在这个消减cDNA文库中高度富集，即近70%的分析克隆主要在破骨细胞中检测到，而在脾脏中未检测到。多组织Northern分析也表明，这些克隆中的许多几乎只在破骨细胞中表达。对随机选择的克隆进行DNA测序表明，在1136个分析的cDNA物种中，有424个是新的。DNA测序还表明，我们的消减cDNA文库几乎是均一化的，这表明该文库可能包含几乎所有的破骨细胞特异性基因。
从这些数据中，我们得出结论，我们的消减方案以及这里开发的后续分离克隆分析程序，总体上对于全面分离和鉴定转录上调或下调的基因是有用的。
