为了研究hsa-miR-663在肿瘤细胞辐射应答通路中的功能, 人工合成其前体并构建入表达载体pcDNATM6.2-GW/EmGFP-miR-663, 进一步通过BP/LR重组反应将hsa-miR-663表达框转移至诱导型表达载体pT-REx-DEST30中。然后将hsa-miR-663的诱导型载体转染构建的四环素操纵子稳定表达细胞系HeLa-TetR, 通过定量反转录PCR及荧光蛋白的表达检测证实了hsa-miR-663在四环素诱导时表达水平升高。本载体的构建为深入研究hsa-miR-663的功能奠定了基础。

To Study the function of hsa-miR-663 in responding to ionizing radiation, the precursor of hsa-miR-663 was synthesized and integrated into pcDNATM6.2-GW/EmGFP-miR. After confirmed by restriction enzyme digestion, the expression cassette was transferred to the inducible vector pT-REx-DEST30 through a BP/LR recombination reaction. Meanwhile the tetracyclin repressor (TetR) expressing cell line HeLa-TetR was established. Finally, HeLa-TetR cells were transfected with pT-REx-DEST30-EmGFP-miR-663 and the expression level of hsa-miR-663 was verified by quantitative RT-PCR (qRT-PCR) as well as the fluorescent microscopic examination for EmGFP expression. In a word, the inducible expression vector for hsa-miR-663 was constructed successfully and this would facilitate the functional study of hsa-miR-663.