Vesicle trafficking in phosphoinositide phosphatase-knockdown cells

克隆了一系列能够稳定表达针对磷酸肌苷磷酸酶同工酶的shRNA的RAW264.7巨噬细胞。目标酶为8种3′-磷酸酶（PTEN、Synaptojanin2、Sac1、Sac3、MTM1、MTMR2、MTMR9、MTMR14）， 1种4′-磷酸酶（Inpp4a）和6种5′-磷酸酶（SHIP1、SHIP2、Inpp5e、SKIP、ocl、Inpp5b）。为每种同工酶制备了表达不同靶向序列shRNA的两种细胞系。研究人员检测了这些被敲除的细胞吞噬igg粒子的能力。在吞噬体形成和成熟过程中，还监测了磷酸肌苷衍生物PtdIns(3,4,5)P3、PtdIns(3,4)P2和PtdIns(3)P的数量和定位。结果提示了磷酸酶同工酶的几种特殊功能。

A series of RAW264.7 macrophages was cloned that stably express the shRNA targeting the specific one of the phosphoinositide phosphatase isozymes. The targeted enzymes were eight 3'-phosphatases (PTEN, Synaptojanin2, Sac1, Sac3, MTM1, MTMR2, MTMR9, MTMR14), one 4'-phosphatase (Inpp4a), and six 5'-phosphatases (SHIP1, SHIP2, Inpp5e, SKIP, OCRL, Inpp5b). Two lines of cells that express shRNA with different targeting sequence were prepared for each isozyme. The knockdown cells were examined for their ability to engulf IgG-opsonized particles. The amount and localization of phosphoinositide derivatives, PtdIns(3,4,5)P3, PtdIns(3,4)P2, and PtdIns(3)P, during the phagosome formation and maturation were also monitored. The results suggested several specific functions of the phosphatase isozymes.