Elucidation of circadian clock machinery by development of an in vivo multi-gene silencing technique

我们之前已经用慢病毒载体培育了表达与昼夜节律调节相关的候选基因shRNA的基因敲除小鼠。在这项研究中，我们进一步发展了一种在体内表达两种不同shRNA的技术，并成功地产生了双基因敲除小鼠。然而，这些小鼠在基因敲除效率和沉默效果方面在世代之间表现出显著的个体差异。因此，我们接下来研究了双基因敲除或特定细胞类型的基因敲除小鼠。我们的数据显示了不同基因敲除小鼠品系的异常昼夜行为节律，并提示新的候选基因参与了昼夜节律时钟机制。

We have previously generated knockdown mice expressing shRNA of candidate genes involved in circadian rhythm regulation using a lentiviral vector. In this study, we further developed a technique to express two different shRNAs in vivo and successfully generated double knockdown mice. However, these mice exhibited significant individual differences in the knockdown efficiency and silencing effects among generations. Therefore, we next examined double knockout or cell type-specific knockout mice. Our data demonstrated abnormal circadian behavioral rhythms in different knockout mice strains and suggested involvements of novel candidate genes in the circadian clock machinery.