Abstract:

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.