Pmx vector

The RNA used to clone hRANKL cDNA was extracted from MG63 cells (ATCC ® CRL-1427 ™ ) expressing RANKL. The quality of the extracted RNA was veri ed by agarose gel electrophoresis. The cDNA was prepared using the AccuPower RT PreMix Kit (Bioneer, Daejeon, Korea), according to the manufacturer's instructions. Ampli cation and cloning of the hRANKL fragment were carried out in a reaction mixture comprising KOD polymerase buffer, 10 mM dNTPs, 25 mM magnesium chloride (MgCl 2 ), 10 μM primers (hRANKL-BclI: 5'-TGATCAAAGCTTGAAGCTCAGCCTTTTGC-3' and hRANKL-XhoI: 5'-CTCGAGATCTATATCTCGAACTTTAAAAGCCCC-3'), 2.5 U of KOD DNA polymerase (EMD Millipore, Billerica, MA, USA) and 2 μL of the RANKL gene construct (template). The thermal cycling conditions were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 30 s and extension at 70°C for 30 s. The polymerase chain reaction (PCR) product was cloned into the BamH1/XhoI sites of a pMX vector (CELL BIOLABS, USA). Sequence analyses were carried out using programs in Vector NTI Advance 9.1.0 (Invitrogen, Carlsbad, CA, USA).