Okihana H, Yamada K
Fujimoto Pharmaceutical Corporation, Osaka, Japan.
J Bone Miner Res. 1999 Feb;14(2):304-10. doi: 10.1359/jbmr.1999.14.2.304.
Cartilage is an inconvenient tissue for the isolation of mRNA, and this has hampered studies of its component mRNAs conducted to date. Here, we describe the preparation of a good quality cDNA library from mouse growth cartilage (mGC). A total of 1.7 microg of poly(A)+ RNA was obtained from about 1200 pieces of the mGC zone of 60 young mice (BALB/c, 4 weeks old). Using this poly(A)+ RNA, we constructed a cDNA library using the pAP3neo vector by the linker-primer method. The complexity of the cDNA library was 2.6 x 106 colony-forming units (cfu), which signified that almost all of the mRNA components in the mGC were present in this cDNA library. From this library, 1401 clones were randomly selected and their insert sizes were examined. Of these clones, 166 (12%) had no inserts, 466 (33%) had inserts ranging in size from 0-0.9 kbp, 480 (34%) had inserts of 1. 0-1.9 kbp, 162 (12%) had inserts of 2.0-2.9 kbp, and 127 (9%) had sizes greater than 3.0 kbp. The average insert size was 1.45 kbp. The number of cfu and the insert size data qualified this library as of reasonably good quality. Clones with an insert size greater than 1 kbp (769 clones) were sequenced from their 5' ends. Among the 769 clones examined, 608 gave sequence data. Among these, 196 (32%) were unknown, 2 were only poly A, and 410 (67%) coded for known proteins. Of these, 55 clones coded for type II (pro)collagen, 54 for osteonectin, and 22 for other cartilage collagens (type IX, type X, and type XI). The rest included cartilage extracellular matrix genes, general cellular genes, and others. To judge further the quality of the library, 45 species coding for type II collagen chain were aligned based on their 5' end sequences. Three species (7%) contained almost the full-length insert, and the shortest one was 1. 5 kbp in length (full-length 5.6 kbp). These data show that this cDNA library is of reasonably good quality, making it likely that the large number of unknown inserts (32%) will provide a suitable pool for the identification and functional determination of new GC genes.
软骨是一种不利于分离mRNA的组织，这一直阻碍着迄今为止对其组成mRNA的研究。在此，我们描述了从小鼠生长软骨（mGC）制备高质量cDNA文库的方法。从60只4周龄的幼鼠（BALB/c）的约1200块mGC区域中获得了总计1.7μg的聚腺苷酸加尾（poly(A)+）RNA。利用这些poly(A)+ RNA，我们通过接头引物法使用pAP3neo载体构建了一个cDNA文库。该cDNA文库的复杂度为2.6×10⁶集落形成单位（cfu），这表明mGC中几乎所有的mRNA成分都存在于这个cDNA文库中。从该文库中随机挑选了1401个克隆，并检测了它们的插入片段大小。在这些克隆中，166个（12%）没有插入片段，466个（33%）的插入片段大小在0 - 0.9kbp之间，480个（34%）的插入片段大小为1.0 - 1.9kbp，162个（12%）的插入片段大小为2.0 - 2.9kbp，127个（9%）的插入片段大小大于3.0kbp。平均插入片段大小为1.45kbp。cfu数量和插入片段大小数据表明该文库质量相当不错。对插入片段大小大于1kbp的克隆（769个克隆）从其5'端进行测序。在检测的769个克隆中，608个给出了序列数据。其中，196个（32%）是未知的，2个仅为聚腺苷酸，410个（67%）编码已知蛋白质。其中，55个克隆编码II型（原）胶原蛋白，54个编码骨连接蛋白，22个编码其他软骨胶原蛋白（IX型、X型和XI型）。其余的包括软骨细胞外基质基因、一般细胞基因等。为了进一步判断文库的质量，基于它们的5'端序列对45种编码II型胶原链的序列进行了比对。三种序列（7%）包含几乎全长的插入片段，最短的一个长度为1.5kbp（全长5.6kbp）。这些数据表明这个cDNA文库质量相当不错，这使得大量未知插入片段（32%）有可能为新的GC基因的鉴定和功能测定提供一个合适的资源库。
