Gateway非放射标记法构建小鼠破骨细胞前体细胞cDNA文库

背景：作者前期研究发现一个在破骨细胞形成中起关键作用的基因，其在细胞之间相互识别及膜融合中发挥关键作用。 目的：采用Gateway的非放射标记技术构建小鼠破骨细胞早期形成细胞的cDNA基因文库，并检测文库质量。 方法：采集C57BL/6小鼠长骨骨髓，收集贴壁的骨髓单核细胞，加入巨噬细胞集落刺激因子和RANKL诱导形成破骨细胞，在巨噬细胞开始融合至形成破骨细胞的不同阶段开始收集细胞，提取总RNA，用CloneMiner TM cDNA文库构建试剂盒，采用非放射标记方法构建小鼠破骨细胞全长cDNA文库。 结果与结论：构建的小鼠骨髓巨噬细胞cDNA文库滴度为2.15×107 CFU/ mL，库容量为10.5×107 CFU，重组率为100%，重组子插入cDNA平均片段大小约为1.7 kb，范围为0.1~5.8 kb。表明非放射标记法构建同样可以构建小鼠骨髓巨噬细胞全长cDNA质粒文库，避免了放射性物质的接触，且文库质量良好。

Construction of an osteoclast precursor cell cDNA library using Gateway non-radiolabelingtechnology in mice

BACKGROUND:A crucial gene in the osteoclast formation is found in the authors’ previous research, which plays a key role in the mutual recognition between cells and membrane fusion. OBJECTIVE:To construct an osteoclast precursor cell cDNA library using Gateway non-radiolabeling technology in mice and to test the quality of the cDNA library. METHODS:Bone marrow of the long bone in C57BL/6 mice was collected for the adherent bone marrow mononuclear cells. Macrophage colony-stimulating factor and RANKL was added to induce osteoclast formation. Cells were collected for total RNA extraction at different stages from the beginning of macrophage fusion to the formation of osteoclasts. A full-length cDNA library of mouse osteoclast precursor cells was constructed using CloneMinerTM cDNA construction kit by Gateway non-radiolabeling technology. RESULTS AND CONCLUSION:The titer of the constructed cDNA library of mouse bone marrow macrophages was 2.15×107 CFU/mL; the storage capacity was 10.5×107 CFU; the recombination rate was 100%. The fragment size of the recombinantinserted into the cDNA was 0.1-5.8 kb; the average fragment size was about 1.7 kb. These findings indicate that a cDNA library can be successfully constructed by non-radiolabeling technology as well, which has adequate quality and avoid the radiation exposure.