Kanegae Y, Takamori K, Sato Y, Lee G, Nakai M, Saito I
Laboratory of Molecular Genetics, University of Tokyo, Japan.
Gene. 1996 Nov 28;181(1-2):207-12. doi: 10.1016/s0378-1119(96)00516-1.
To develop a method for activating genes located on cell chromosomes, an on/off switching unit regulated by the site-specific recombinase Cre was constructed. The switching unit was designed to express firstly the neo gene and secondly the reporter lacZ gene by Cre-mediated excisional deletion of the neo gene. CV1 cell lines bearing the switching unit on a cell chromosome were isolated and activation of the lacZ gene was examined after infection with a Cre-producing recombinant adenovirus. In one cell line virtually 100% of the cells stably expressed the lacZ gene, whereas in another cell line lacZ-expressing cell populations reached only to about 90% and decreased after cell divisions. The Southern blot analyses showed that the latter type of cells contained a head-to-tail array of the switching units, and that consequently the lacZ-expressing units were excised from a cell chromosome and present as extrachromosomal circular DNAs. These results showed that the system offers efficient activation of genes introduced into cell chromosomes and that the organization of the reporter units are important for efficiency and duration of the activated gene expression.
为了开发一种激活位于细胞染色体上基因的方法，构建了一个由位点特异性重组酶Cre调控的开/关切换单元。该切换单元设计为首先表达新霉素基因（neo基因），然后通过Cre介导的neo基因切除缺失来表达报告基因lacZ基因。分离出在细胞染色体上携带该切换单元的CV1细胞系，并在用产生Cre的重组腺病毒感染后检测lacZ基因的激活情况。在一个细胞系中，几乎100%的细胞稳定表达lacZ基因，而在另一个细胞系中，表达lacZ的细胞群体仅达到约90%，并且在细胞分裂后减少。Southern印迹分析表明，后一种类型的细胞包含切换单元的头对头排列，因此表达lacZ的单元从细胞染色体上切除并以染色体外环状DNA的形式存在。这些结果表明，该系统能够有效激活导入细胞染色体的基因，并且报告单元的组织对于激活基因表达的效率和持续时间很重要。
