Takeda Kosuke, Saito Tamao, Tanaka Tomoyuki, Morio Takahiro, Maeda Mineko, Tanaka Yoshimasa, Ochiai Hiroshi
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060-0810, Hokkaido, Japan.
Gene. 2003 Jul 17;312:321-33. doi: 10.1016/s0378-1119(03)00558-4.
We describe a novel restriction enzyme-mediated integration (REMI) method for gene trapping in Dictyostelium based on the use of a terminator-deficient vector. The vector has a blasticidin deaminase (bsr) gene as a selectable marker but lacks a terminator containing a poly(A) addition signal (AATAAA). Thus, the vector was expected to integrate into the coding region of a gene to create a fusion transcript flanked by the 3' proximal region of the trapped gene. The trapped gene can be identified by simply amplifying the fusion transcript by 3' rapid amplification of cDNA ends (3'-RACE). In the analysis of 35 integration events into known genes, the vectors were found to be integrated 20 times in close proximity to the 3' ends of the genes and in the direction of transcription. This strictly localized insertion seemed to be mediated by negative selection via the surveillance system referred to nonsense-mediated mRNA decay. In contrast, in 15 events the vector integrated in the opposite direction to transcription and at random positions throughout the coding sequence. Analysis of the trapped 3' sequences showed that the transcription of the fusion gene terminated prematurely without the apparent use of an endogenous terminator; nevertheless the transcript did exhibit a poly(A) tail. Based on these results, we designated the method terminator-REMI. Using this method, we have generated a library of tagged Dictyostelium clones from which we have thus far isolated 242 developmental mutants.
我们描述了一种基于使用终止子缺陷型载体在盘基网柄菌中进行基因捕获的新型限制酶介导整合（REMI）方法。该载体具有杀稻瘟菌素脱氨酶（bsr）基因作为选择标记，但缺乏包含多聚腺苷酸添加信号（AATAAA）的终止子。因此，预期该载体可整合到基因的编码区，以产生由捕获基因的3'近端区域侧翼的融合转录本。通过简单地通过cDNA末端的3'快速扩增（3'-RACE）扩增融合转录本，即可鉴定捕获的基因。在对35个整合到已知基因中的事件进行分析时，发现载体在基因的3'末端附近并沿转录方向整合了20次。这种严格定位的插入似乎是由通过称为无义介导的mRNA衰变的监测系统进行的负选择介导的。相比之下，在15个事件中，载体以与转录相反的方向并在整个编码序列的随机位置整合。对捕获的3'序列的分析表明，融合基因的转录在未明显使用内源性终止子的情况下过早终止；然而，转录本确实表现出多聚腺苷酸尾巴。基于这些结果，我们将该方法命名为终止子-REMI。使用这种方法，我们生成了一个标记的盘基网柄菌克隆文库，到目前为止，我们已经从中分离出242个发育突变体。
