Gene regulation of targeted cells and organelle by laser microinjection

在这里，我的目标是通过使用我们的激光注射方法(激光辅助热膨胀微注射；LTM)来控制靶细胞和细胞器中的基因表达。我特别关注通过使用一种独特的植物四叶草来下调靶基因的表达，这种植物的胚囊从胚珠组织中伸出。我们通过将吗啡反义寡聚体(MOS)注射到突出的胚囊中，成功地下调了靶基因的表达。当我们下调LURE1或LURE2基因，这两个基因是在助细胞中特异表达的花粉管吸引基因的候选基因，那么花粉管吸引作用就会减弱。这些结果导致了对真正引诱剂分子诱饵的识别。研究表明，MOS对植物细胞和原生多头绒泡菌均有效。激光注射和MOS的结合被证明是下调真核细胞的一种强有力的技术。

Here I aimed to control gene expression in targeted cells and organelle by using our laser injection method (Laser-assisted Thermal-expansion Microinjection ; LTM). I especially focused on down-regulation of targeted genes by using a unique plant Torenia fournieri, of which embryo sac is protruding from the ovule tissue. We succeeded in down-regulation of targeted genes by injecting morpholino antisense oligomers (MOs) into the protruding embryo sac. When we down-regulated LURE1 or LURE2 genes, which were candidates of pollen tube attractant genes specifically expressed in the synergid cell, pollen tube attraction was impaired. These results lead to identification of true attractant molecules LUREs. MOs were shown to be effective in plant cells as well as a protist Physarum polycephalum. Combination of laser injection with MOs was shown to be a powerful technique for down-regulation of eukaryotic cells.