Niwa H, Araki K, Kimura S, Taniguchi S, Wakasugi S, Yamamura K
Department of Developmental Genetics, Kumamoto University School of Medicine.
J Biochem. 1993 Mar;113(3):343-9. doi: 10.1093/oxfordjournals.jbchem.a124049.
New trap vectors (U1 and U2) have been developed to trap genes in murine embryonic stem (ES) cells. The polyA addition signal of the neomycin phosphotransferase II (neo) gene was removed from these vectors so that they needed to trap an endogenous polyA signal for expression of the neo gene. The frequency of gene-trap events of these vectors was about five times higher than with the vector containing the polyA signal, and only one copy of the trap vector was integrated in most cases. Four out of five 5'-flanking regions of the integrated vector in ES cell lines were found to be novel endogenous promoters, suggesting that this method is efficient for trapping genes in ES cells. In two cases analyzed, large deletions or rearrangements spanning more than 10 kb were found in the 3'-flanking region of the trap vector introduced by electroporation. This result suggests that phenotypes observed in homozygotes with a mutated allele could be due to the disruption of a gene adjacent to the trapped gene, but not of the trapped gene.
已开发出新型捕获载体（U1和U2）用于在小鼠胚胎干细胞（ES细胞）中捕获基因。从这些载体中去除了新霉素磷酸转移酶II（neo）基因的聚腺苷酸添加信号，以便它们需要捕获内源性聚腺苷酸信号来表达neo基因。这些载体的基因捕获事件频率比含有聚腺苷酸信号的载体高约五倍，并且在大多数情况下仅整合了一份捕获载体。在ES细胞系中，整合载体的五个5'侧翼区域中有四个被发现是新的内源性启动子，这表明该方法在ES细胞中捕获基因是有效的。在分析的两个案例中，电穿孔引入的捕获载体的3'侧翼区域发现了超过10 kb的大片段缺失或重排。该结果表明，在具有突变等位基因的纯合子中观察到的表型可能是由于捕获基因附近的基因被破坏，而不是捕获基因本身。
