HeLa/GFP-Plk1/Cherry-H2B稳定细胞系的构建简

目的：构建可研究Polo样激酶1（Plkl）定位的HeLa细胞系。方法：用PCR方法扩增Plkl基因，定向克隆到pRex-EGFP-IRES-Hygm载体中，构建pRex-EGFP-Plkl-IRES-Hygro表达载体；利用逆转录病毒感染的方法，向HeLa细胞系中依次转染pRex-EGFP-Plkl-IRES-Hygro、pRex-Cherry-H2B-IRES-Hygro，构建Hela/GFP-Plkl/Chef．ry-H2B稳定细胞系；激光共聚焦显微镜观察Hela/GFP-Plkl/Cherry-H2B稳定细胞系在不同有丝细胞分裂期时Plkl的定位。结果：质粒酶切及测序证明pRex-EGFP-Plkl-IRES-Hygro载体构建正确；在Hela/GFP-Plkl/Cherry-H2B稳定细胞系有丝分裂中期和末期时，观察到Plkl分别定位于着丝粒和中间体上。结论：构建了Hela/GFP-Plkl/Cherry-H2B稳定细胞系，为研究Plkl在有丝分裂不同时期的调控机制提供了细胞模型。

Construction of HeLa/GFP-Plkl/Cherry-H2B Cells

Objective： To construct HeLa cells available for study of localization of Polo-like kinasel （Plkl）. Methods： Plkl gene was amplified by PCR and then inserted into vector pRex-EGFP-IRES-Hygro to construct pRex-EGFP-Plkl-IRES-Hygro plasmid. Through retroviral infection, the pRex-EGFP-Plkl-IRES-Hygro and pRex Cherry-H2B-IRES-Hygro plasmids were transfected into HeLa cells in turn to construct HeLa/GFP-Plkl/Cberry H2B stable cells. The localization of Plkl in different mitotic phase was observed under confocal laser scanning microscope. Results： The vector pRex-EGFP-PIkl-IRES-Hygro was verified by enzyme digestion and sequencing. In constructed HeLa/GFP-Plkl/Cherry-H2B stable cells, Plkl was observed in kinetochores and midbody respective- ly in metaphase and telophase. Conclusion： The constructed HeLa/GFP-Plkl/Cherry-H2B stable cells will benefit the further research on the regulation mechanism of Plkl in mitosis.